Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs

Shino Ohshima; Tatsuya Matsubara; Asuka Miyamoto; Atsuko Shigenari; Noriaki Imaeda; Masaki Takasu; Masafumi Tanaka; Takashi Shiina; Shingo Suzuki; Noriaki Hirayama; Hitoshi Kitagawa; Jerzy K Kulski; Asako Ando; Yoshie Kametani

doi:10.1371/journal.pone.0242572

. 2020 Nov 25;15(11):e0242572. doi: 10.1371/journal.pone.0242572

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs

Shino Ohshima ¹, Tatsuya Matsubara ², Asuka Miyamoto ¹, Atsuko Shigenari ¹, Noriaki Imaeda ², Masaki Takasu ², Masafumi Tanaka ¹, Takashi Shiina ¹, Shingo Suzuki ¹, Noriaki Hirayama ³, Hitoshi Kitagawa ⁴, Jerzy K Kulski ^1,⁵, Asako Ando ¹, Yoshie Kametani ^1,^3,^*

Editor: Karen L Elkins⁶

PMCID: PMC7688132 PMID: 33237936

Abstract

Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.

Introduction

Cluster of differentiation 4 (CD4), a glycoprotein expressed on immune competent cells such as lymphocytes, monocytes, macrophages and dendritic cells, is widely used as a marker of T cell subsets for the functional analysis of the immune response. CD4 possesses four extracellular immunoglobulin-A subdomains (D1 to D4) [1, 2] and an intracellular tyrosine kinase Lck binding domain that enhance TCR signals [3, 4]. The extracellular domain of CD4 is thought to function as a coreceptor of TCR-MHC cognate interaction [5]. The extracellular domain contains a subdomain called the D1 domain, which binds to a non-polymorphic class II MHC β2 domain [1]. However, there are reports that CD4 also binds directly to the class II MHC molecule with low affinity in a TCR independent manner [6, 7] suggesting that CD4 might function as an adhesion molecule to induce signal transduction [8].

The CD4 receptor is polymorphic in a number of different species [9–13]. In humans, CD4 polymorphisms were correlated to some infectious diseases such as HIV infection [14] and chronic diseases such as type-1 diabetes [15]. Although Microminipigs and NIH minipigs have at least two CD4 isotypes, they could not be distinguished by conventional anti-CD4 antibodies [16, 17]. By sequencing the Microminipig CD4 variant genes, we found that a CD4 allele named CD4.B had 10 amino-acid substitutions in the CD4 domain 1 region in comparison with the swine CD4 amino-acid reference sequence [Gen-Bank: NP_001001908] [18], whereas CD4.B was not recognized by three conventional monoclonal antibodies (mAbs) named 74-12-4, MIL17, and PT90A [17]. The 10 amino-acid substitutions in CD4.2 NIH minipigs were also found in the same positions as those in CD4.B [18]. These regions of amino-acid substitution overlapped with the site of class II MHC recognition by CD4 indicating that the allelic polymorphism may affect the acquired immune response. The percentage frequency of the Microminipigs with the CD4.B allele, not recognized by the available mAbs, was at most 51.1%. No obvious difference of immune cell ratio or severe immunodeficiency was observed in the Microminipig herd [17]. Also, Blanc et al. reported that a high genetic variability of CD4 in the melanoblastoma-bearing Libechov minipig (MeLiM) strain was associated with the immune response against the skin cancer [9].

Microminipigs are novel, extra-small miniature pigs developed for biomedical research in Japan [19, 20]. Recently, we assigned different swine leukocyte antigen (SLA) class I and class II haplotypes to our herd of Microminipigs [21]. We found that the in vitro administration of toxic shock syndrome toxin-1 (TSST-1) activated polyclonal T cells and increased class I SLA transcription showing that TSST-1 and the SLA together are useful for evaluating the in vitro activation level of swine T cells [22, 23].

In this study, we developed two mAbs which recognize Microminipig CD4.B, one specific to CD4.B and the other to CD4.A and CD4.B in order to analyze CD4.A and CD4.B protein expression and function. We used these two antibodies to evaluate (1) the level of CD4 protein expression and T cell activation, and (2) the association of T cell activation with class I SLA protein expression levels in response to in vitro T cell stimulation with theTSST-1.

Materials and methods

Animals and tissues

Experiments using mice were approved by the Institutional Committee for Animal Care of Tokai University and were performed following the University guidelines (The approval numbers are #152030, #170426). Swine study was approved by the Animal Care and Use Committee of Gifu University (The approval number is #17042, May 26, 2017). Eight-week-old female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan) and kept under specific pathogen-free conditions. Adult swine peripheral blood was provided by Fuji Micra Inc and Gifu University. The sex, date of birth, CD4 type and MHC haplotypes of the 14 Microminipigs used in this study are presented in S1 Table. A marmoset blood sample was supplied from Central Institute of Experimental Animals (Kawasaki, Japan). A human blood sample was purchased from Cosmo Bio Co. Ltd (Tokyo Japan). This study was carried out according to the laboratory animal guidelines of Gifu University, Tokai University and Central Institute for Experimental Animals.

SLA genotyping

Eight SLA haplotypes were assigned to a herd of Microminipigs with extremely small body sizes by nucleotide sequence determination of reverse transcriptase-polymerase chain reaction (RT-PCR) products and low-resolution SLA genotyping using PCR-sequence-specific primers (SSP) of the three classical class I SLA genes, SLA-1, SLA-2 and SLA-3, and class II genes, DRB1 and DQB1 [21]. In this study, SLA-1, SLA-2, SLA-3, DRB1 and DQB1 alleles in Microminipigs were assigned by low-resolution SLA genotyping using the aforementioned PCR-SSP method as described previously [21, 24, 25]. The primers are shown in S2 Table. Eight types of SLA haplotypes were deduced from an analysis of the inheritance and segregation of alleles of the three class I and two class II SLA genes in descendants of the Microminipig population. PBMC of Microminipigs with two kinds of homozygous low-resolution class I and class II SLA haplotypes, Hp-35.23 and Hp-43.37, were used for comparison of class I SLA genes expression on T cells with different CD4 isotypes.

CD4 genotyping

We assigned CD4 genotypes in the Microminipig herd using a PCR-RFLP method [18]. Briefly, PCR amplification was performed on genomic DNA to amplify CD4 exon 3 using primers shown in S3 Table, and the PCR products were digested with a restriction enzyme BseRI (New England Biolabs Inc., Ipswich, MA). The digested samples were electrophoresed in 2% agarose gel to assign the genotypes.

Transfection

The transfected cDNA sequences of the swine CD4.A and CD4.B genes were based on the Sus scrofa CD4.A and CD4.B mRNA sequences LC064059 and LC064060, respectively. The primers used to generate the cDNA sequences are shown in S3 Table. RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). The modified S/MAR (scaffold/matrix attachment region) episomal vectors [26] carrying CD4.A or CD4.B were used for the transfection. Transgene-positive cell ratio was determined according to the fluorescent intensity of the co-expressed mVenus protein [27].

HEK293 cells, a cell line derived from human embryonic kidney cells, were cultured in D-MEM (GE Healthcare, Buckingham, UK)-10% fetal calf serum (FCS) (bisera, Kansas City, MO, USA) medium. A20 cells, a BALB/c B lymphoma cell line derived from a spontaneous reticulum cell neoplasm, were cultured in RPMI 1640 (Nissui Pharmaceutical co. Ltd. Tokyo, Japan)-10% FCS medium. The cells were transfected with the cDNA sequence inserted in the modified S/MAR episomal vector using the Invitrogen Neon transfection system (HEK293 cells: 1100 V, 10 ms, 3 pulses; A20 cells: 1500 V, 10 ms, 3 pulses) and cultured in 5% CO₂ at 37°C for 18 ~24 hrs added with G418 (Roche Diagnostics, Indianapolis, USA). Cultured cells were collected and the expression of the mVenus reporter gene was measured by flow cytometry (Becton Dickinson, NJ, USA).

Preparation of the peripheral blood mononuclear cells (PBMCs)

Peripheral blood samples from Microminipigs (S1 Table) were collected into separate heparinized tubes and centrifuged on Lymphoprep (Axis-Shield, Oslo, Norway) at 670 x g for 30 min. The peripheral blood mononuclear cells (PBMCs) were collected and washed with 30 ml of 1% (w/v) bovine serum albumin (BSA) containing phosphate-buffered saline (PBS). The remaining erythrocytes were lysed osmotically. The PBMCs were washed with PBS and used for further experiments. The single human PBMC sample was purchased from Cosmo Bio Co. Ltd (Tokyo Japan), and the mouse and marmoset PBMCs were collected using Lymphoprep, as described above.

Monoclonal antibody preparation

A summary of the protocol used for immunization of the BALB/c mice with swine CD4.A and CD4.B proteins prior to fusion of the mouse splenocytes with the mouse myeloma cell line is outlined in S1 Fig. We initially immunized BALB/c mice with mitomycin C (MMC, Kyowahakko-Kirin, Tokyo Japan)-treated swine CD4.A or CD4.B homozygous PBMCs (1 to 3 ×10⁶ cells/animal). For booster treatments, mice were injected biweekly with MMC-treated A20 transfectants (CD4A⁺/A20 and / or CD4B⁺/A20) for up to 5 times with 1×10⁶ cells/animal. Prior to the injections, the MMC (final, 0.04 mg/ml) was added to the A20 transfectant cultures and incubated at 37°C for 30 min in 5% CO₂. MMC-treated A20 transfectants (1x10⁶) were used for the last booster. The mice were anesthetized with 20% isoflurane and the blood was collected from the orbit. The antibody reactivity of immunized mouse blood plasma was checked by flow cytometry using CD4.A or CD4.B cDNA-transfected HEK293 cells as a source of antigen. After 4 days of the final boost, mice were anesthetized with 20% isoflurane and sacrificed by blood removal; extracted splenocytes were fused with the mouse myeloma cell line P3-X63-Ag8-U1 (P3X) purchased from Riken (Saitama, Japan) according to a standard procedure with Polyethylene glycol (PEG) purchased from Merk Japan co. Ltd (Tokyo, Japan) [28]. RPMI 1640 medium and supplements were purchased from Nissui co. Ltd (Tokyo, Japan) (RPMI 1640), Sigma-Aldrich (HAT) and Thermofisher Scientific K.K. (Tokyo, Japan) (HT). Positive clones were identified by flow cytometry or using an Imaging Analyzer (Array Scan, Thermo scientific, MA, USA). Briefly, CD4.A or CD4.B transfected HEK293 cells were mixed with non-transfected HEK293 cells and plated in the wells of 96-well plates. Culture supernatants were added to each well, incubated for 15 min and washed twice. APC-labeled (APC: allophycocyanin) anti-mouse IgG polyclonal antibody (Poly4053; Bio Legend, San Diego, US) was added and incubated for 15 min. Plates were washed and stained with Hoechst 33342 (Invitrogen, Oregon, USA) for 30 min at room temperature and analyzed using the Imaging Analyzer. Positive cells were picked according to the fluorescence intensity of APC and the co-expressed mVenus fluorescent protein. The same analysis was repeated to select the stable positive clones that were then isolated, expanded and stocked. The mAb isotype was determined using a mouse monoclonal antibody isotyping kit (Iso Strip, Roche, Basel Schweiz). The sequence of each monoclonal antibody was determined by Repertoire Genesis Inc. (Osaka Japan).

Flow cytometry

Cells were incubated with appropriately diluted, fluorescently-labeled primary mAb for 15 min at 4°C and washed with 1% (w/v) BSA-containing PBS. In some cases, cells were re-incubated with labeled secondary antibodies. The mAb x1E10 was biotinylated by Biotin Labeling Kit (Cosmo Bio co., Ltd, Tokyo Japan) according to manufacturer’s protocol. The mAbs that were used in our analyses are shown in S4 Table. The cells were analyzed using FACS Verse (BD Bioscience, Franklin Lakes, NJ). For each sample, the live gate with white blood cells or lymphocytes was analyzed by FlowJo (TOMY Digital Biology).

Primary sequence and structure analysis of mAb

Total RNA was extracted from specific hybridomas, and the cDNA was checked for the amplification of immunoglobulin heavy- and light-chain-specific genes. Sequence reactions were performed with GenomeLab DTCS Quick Start Kit (Beckman Coulter, Brea, CA, USA) and analyzed using a CEQ8000 Genetic Analysis System (Beckman Coulter). The software Genetyx (GENETYX co. Ltd, Tokyo, Japan) was used for sequence prediction.

Model building of the 3D structure of CD4 molecules

The 3D models of the CD4.A and CD4.B molecules translated from sequenced genes [18] were constructed using the homology modeling method [29] implemented in the software system Molecular Operating Environment (MOE) (2019.01; Chemical Computing Group ULC, 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2019). The protein structure (PDB ID, 3JWD) with the highest sequence identity to CD4.A and CD4.B was selected from the Protein Data Bank(PDB) [30] and used as a template for homology modeling. The amino acid sequence of CD4.A and CD4.B are compared in Fig 1.

Fig 1 — A. Amino acid sequence alignments of two alleles of the CD4 locus (CD4.A, upper line; CD4.B, lower line) [18]. Amino acid substitutions between CD4.A and CD4.B are shown in purple color. Identical amino acid sequences between CD4.A and CD4.B but different from mouse and human are shown in red color. Other sequences indicate identical amino acids among human, mouse and swine. a) the region N;27-N;210 aa corresponding to the C chain of the reference tertiary structure 3JWD obtained from PDB [32], b) the predicted epitope sites for recognizing CD4.A and CD4.B. B. The tertiary structures of the CD4.A (left panel), and CD4.B (right panel) N;27-N;210 aa constructed based on the 3D structure of 3JWD [32]. Purple (CD4.A) and red (CD4.B) colors represent the amino acid sequences (N;54-N;80) that are predicted to be different epitope-candidate sites. As the molecular surfaces of the 3D models are significantly different, their antibody binding affinities should be different.

Histochemical analysis

Swine frozen tissues were micro-sectioned (5 μm) and fixed with 4% buffered paraformaldehyde for 20 min (Wako Pure Chemical Industries, Ltd), then washed and endogenous peroxidase was blocked for 10 min at room temperature. The sections were blocked with goat serum for 30 min, washed and then the primary monoclonal antibody was added. Subsequent incubation with peroxidase-labeled anti-mouse Ig was performed according to the manufacturer’s protocol. The list of antibodies are shown in the S4 Table.

Stimulation of PBMCs

Swine peripheral blood samples were collected into heparinized tubes and centrifuged on Lymphocepar (IBL Co. Fujioka, Japan) at 670 x g for 30 min. PBMCs were isolated and washed with 30 ml of 1% (w/v) BSA-containing PBS (PBSA) by centrifuging at 350 x g for 5 min. The remaining erythrocytes were lysed osmotically. The PBMCs were washed and cultured (3x10⁶/well) in RPMI 1640 medium containing 10% FCS in the presence of the TSST-1 (Toxin Tec. Sarasota, USA) at 1 μg/mL at 37˚C and 5% CO₂. After 72 hrs, the cells were collected, washed with PBS andanalyzed by flow cytometry (FCM). The TSST-1/mAb inhibition assay was performed by adding the purified mAb (x1E10) to the culture medium with TSST-1 at 1 μg/mL. The cells were analyzed at 24, 48 and 72 hrs to detect the surface expression of CD4, CD8 and class I SLA by FCM.

Statistical analysis

Regression lines and regression equation for age and CD4+ cell correlation analysis, and the student’s t-test for the comparison of class I SLA expression on the TSST-1-stimulated and non-stimulated cells were performed by Microsoft EXCEL (Microsoft Office 2016, Microsoft Corporation, Redmond, WA).

Results

Simulation of swine CD4 isotype epitope

Because CD4 possesses four Ig domains in the extracellular domain, we first analyzed the similarity of CD4.A and CD4.B amino acid sequences (Fig 1A) archived at NCBI [18]. In a previous study we found that the CD4.A protein was recognized by available mAbs, whereas the CD4.B protein was not recognized by these mAbs [17]. Based on the primary amino acid sequences of CD4.A and CD4.B [18], we predicted the spatial position of the diverged sequence and the putative epitope sites (Fig 1B). The human CD4 was predicted to bind to class II MHC at the site N;99–226 [31]. On the other hand, the diverged amino acid sequences in CD4.A and CD4.B of Microminpigs were located on N;54–80, which is a little upstream of the predicted sites for human CD4 and class II MHC binding. However, there are two amino acid variants also in the MHC-CD4 binding region (Fig 1A) that might influence TCR-class II MHC interactions. Therefore, we concluded that it would be necessary to prepare specific mAbs against the CD4 isotypes in order to identify and compare the activated CD4.A and CD4.B T cells and confirm whether these T cells can be activated similarly or differently by TCR stimulation.

To examine whether the diverged sequence within the swine CD4 might contain a predicted and unique epitope for recognition by a mAb, we selected a human CD4 reference sequence, which interacts to HIV-1 gp120 (PDB ID: 3JWD) [32, 33] as the protein with the greatest amino acid similarity to the porcine CD4 (<70% identity) for a sequence comparison of possible epitope motifs. As shown in Fig 1A (a), the identities between the human C chain of the protein reference sequence 3JWD (CD4 region) and the 27–210 amino acid sequences of the swine CD4.A or CD4.B chains were 56.5% and 55.4%, respectively. The tertiary structure corresponding to the N-terminal 26 residues and the residues after 211^th residue were not present in the 3JWD C chain. Therefore, the tertiary structure of the N;27 -N;210 amino acid residue domain (N-domain) was constructed by homology modeling [29] using the structure of 3JWD corresponding to the N-domain. We predicted the site of diverged amino acid sequence on the 3D model that was shown in Fig 1B. Each of the N;54-N;80 amino acids (predicted as different epitope sites) were colored in purple and red (Fig 1A) with putative epitope sites were exposed on the surface of both molecules (Fig 1B). Moreover, the five amino acid replacements (T61I, G65R, G66S, G68R and T73K) have a difference in polarity and charge and the tertiary structure of the two molecules is different in our homology modeling (Fig 1B). These molecular differences suggest that the antigenicity of the Microminipig CD4.A and CD4.B is modified by the sequence diversity and that each protein might have unique antibody epitopes that could be distinguished by mAbs.

Preparation of mAbs recognizing swine CD4 isotypes

We prepared two specific mAbs, one that recognized only CD4.B and the other that recognized both CD4.A and CD4.B molecules. In the preliminary analyses, MMC-treated transfectants (CD4A⁺/A20 or CD4B⁺/A20) were injected multiple times to booster the immunization of BALB/c mice (S1A Fig). In the analyses, the reactivity of the antiserum to HEK293 cells expressing either CD4.A or CD4.B after the second booster (S1B Fig) was confirmed by flow cytometry. Therefore, the mice with high fluorescence intensity were selected and used for the monoclonal antibody preparation (S1B Fig).

BALB/c mice were immunized with CD4.B homo-type (CD4.B (+/+)) PBMC twice and boosted with CD4.B transfected A20 cells. In these mice, both the CD4.B and the CD4.A specific antibodies were produced (S1B Fig). The spleen cells of immunized mice with the highest antisera reactivity to both CD4.A and CD4.B gene products were selected and fused with the mouse myeloma cell line P3X. The supernatants of hybridomas were screened for staining only CD4.B or both CD4.A and CD4.B using CD4.A and CD4.B gene-transfected HEK293 cell lines with Array Scan.

The first clone (b1D7) that we obtained for further analysis recognized only the CD4.B protein and not the CD4.A protein (Fig 2A and 2B). This mAb did not react with human, mouse and common marmoset (CM) PBMCs, but it recognized swine PBMCs (Fig 2B) suggesting species specificity. The mRNA was extracted from the hybridoma and next-generation sequencing revealed that the antibody possessed heavy chain, IGHV3-2_x_IGHJ3_IGHG1_CARQGYGDYDPFPYW, and two light chains IGKV6-20_IGKJ2_CGQGYSYPYTF and IGKV4-55_IGKJ2_CQQWSSYPYTF (S2 Fig). These light chains were constitutively expressed even after cloning suggesting that the two B cells were fused with one myeloma cell.

The second clone that we obtained for further analysis (x1E10) recognized both the CD4.A and the CD4.B proteins. The antibody secreted by the clone did not recognize human, mouse or common marmoset PBMCs, but reacted specifically with swine PBMCs (Fig 3A and 3B). Next generation sequencing of the mRNA extracted from the hybridoma showed that the antibody possessed a heavy chain, IGHV3-1_IGHD2-9 and IGHD2-4_IGHJ4_CARRTYYDYDYYGMDYW_IGHG2A and a light chain, IGKV1-117_IGKJ1_CFQGSHVPWTF (S3 Fig). This antibody also reacted with swine splenocytes in an immunohistochemical analysis of CD4.A (+/+) or CD4.B (+/+) spleen tissue sections revealing weakly colored small cells scattered in periarteriolar lymphoid sheaths (PALS) and strongly colored larger cells scattered around the white pulp (Fig 3C). The strongly colored larger cells scattered around the white pulp in the negative-control suggests a non-specific binding reaction due to the secondary antibodies. Collectively, these results confirmed the specificity and usefulness of the newly developed mAbs against CD4.A and CD4.B of swine T cells.

Fig 3 — A. HEK293 and the transfectant of *CD4*.A and *CD4*.B were stained with x1E10 (1C3-1E8) clone supernatant and anti-mouse IgG-APC and analyzed by FCM. The numbers in the panels show the percentage of mVenus and APC double-positive cells. B. Swine PBMCs (CD4.A (+/+) and CD4.B (+/+)) were stained with the purified mAb and anti-mouse IgG2a-FITC and analyzed by FCM. The species specificity was examined using CD4.A (+/+) and CD4.B (+/+) swine, human, CM and mouse PBMCs. Lymphocyte gate (orbits) was used for the analysis (left panels). Solid lines and shaded peaks represent the x1E10-stained patterns (right panels). Broken lines represent the isotype-control-stained patterns. The bars in the histograms indicate the positions of x1E10 positive cell peaks together with the percentage of positive cells. C. CD4.A (+/+) (upper panels) and CD4.B (+/+) (lower panels) swine spleen tissue sections stained with x1E10 mAb (right panels) and negative controls (no x1E10 mAb) (left panels). Brack arrow points to PALS and central arteries. Red arrows point to highly cross-reactive cells.

Comparison of CD4 and CD8 expression in activation-induced T cells with different CD4 isotypes

To evaluate the utility of our new antibody that interacted with CD4.A and CD4.B, we first analyzed the expression of CD4 and CD8 molecules on the swine PBMCs using the anti-CD4AB mAb (x1E10) that we prepared in the present study and a commercially available anti-CD8 mAb, respectively (S4 Table). As shown in Fig 4A, four samples of CD4.A (+/+) and four samples of CD4.B (+/+) swine PBMCs showed similar patterns of CD4 and CD8 expression. In the swine PBMCs, not only CD4 single-positive (CD4SP) and CD8 single-positive (CD8SP) cells, but also the CD4/CD8 double-positive (DP) cells were detected. The range of total CD4+ T cell ratio was 21–42% for CD4.A (+/+) and 25–35% for CD4.B (+/+). We verified a previous report [34] that the younger swine PBMCs contained fewer DP cells, and we also found that the relationship between the age of swine and DP cell ratio was positively correlated (r² = 0.6083 for CD4.A (+/+) and r² = 0.7474 for CD4.B (+/+)). No significant bias was observed between CD4.A (+/+) and CD4.B (+/+) PBMCs, although the regression coefficient slightly varied between the DP cells (Fig 4B). Collectively, no significant difference in cellular proportion was observed between CD4.A (+/+) and CD4.B (+/+) PBMCs.

Fig 4 — A. Swine PBMCs (CD4.A (+/+)) were stained with the mAb (x1E10) and anti-CD8 mAb. Small lymphocyte-gated cells were analyzed. Individual identification numbers and age in months are shown on the top of each upper panel. The lower quadrant panel shows the CD4/CD8 expression of the PBMCs. The percentages of CD4SP and DP cells respectively, are shown in the panels. B. Correlation between age in months and CD4SP cell subsets (% in lymphocyte-gated cells) (left panel) and DP cell subsets (% in lymphocyte-gated cells) (right panel) in swine PBMCs. Solid line; CD4.A (+/+) swine, broken line; CD4.B (+/+) swine.

In the next experiment, we stimulated the T cells with superantigen [35] to analyze the activation level of swine CD4 T cells. Because class I SLA expression is enhanced by toxic shock syndrome toxin-1 (TSST-1) stimulation [22, 35], we used the molecule as an activation marker. Also, since SLA polymorphism may affect the reactivity of the anti-class I SLA mAb (X2F6) [23] we selected samples of CD4.A (+/+) and CD4.B (+/+) to analyze the CD4 T cell activation levels in Microminipigs with the homozygous haplotypes Hp-35.23 (CD4.A (+/+); n = 1, CD4.B (+/+); n = 2) and Hp-43.37 (CD4.B (+/+); n = 2). The PBMCs of these homozygous haplotype samples were collected and stimulated in vitro with TSST-1; CD4 expression was detected with the prepared mAbs by flow cytometry. To obtain a double-staining pattern, the mAb x1E10 was labeled with biotin (S4 Fig).

After the stimulation of PBMCs with TSST-1, the lymphocyte gate for the activated lymphocytes was enlarged and a cluster of CD4-high cells appeared in the DP cell fraction followed by a decrease of the CD4SP cell ratio (Fig 5 and S5 Fig). In addition, a significant decreases of CD4/CD8 double negative (DN) and CD4SP cells and significant increases of CD8SP and DP cells were observed for the CD4.A (+/+) and the CD4.B (+/+) swine groups (Fig 5B). When the two haplotype groups were examined, both the Hp-35.23 and the Hp-43.37 swine showed a significant increase of the CD4-high fraction after TSST-1 stimulation. Similarly, both the CD4.A (+/+) PBMCs and the CD4.B (+/+) PBMCs showed a significant increase of CD4-high fractions (Fig 5C). Also, the MFI of CD4 on DP cells was increased in the CD4 fraction of the Hp-35.23 haplotype cells (Fig 5D).

Fig 5 — Swine PBMC samples were stained with x1E10 and anti-CD8 mAb and analyzed by FCM. A. The left panel shows the representative pattern of CD4 and CD8 expression in the small and large lymphocyte-gated cells of the DP cells that were divided into CD4-high and CD4-low cells, respectively. The right panels show the histograms of the CD4 expression of the lymphocyte-gated cells. Left panel, 0 hr; middle panel, 72 hrs without TSST-1 stimulation; right panel, 72 hrs with TSST-1 stimulation. B. Four samples of CD4.B (+/+) and one sample of CD4.A (+/+) PBMCs were analyzed for changes in the ratio of T cell subsets with/without TSST-1 stimulation. Statistical analysis was performed on the four samples of the CD4.B (+/+) PBMCs. Open bars, TSST-1 (-); closed bars, TSST-1 (+). C. The changes in the ratio of CD4-low and CD4-high fractions with/without TSST-1 stimulation were compared between Hp-35.23 and Hp-43.37 haplotypes (left two panels) and between the CD4.B (+/+) and CD4.A (+/+) T cells (right two panels). D. MFI of DP cells was compared between samples of TSST-1 (+) and TSST-1 (-) cells. Left panel, three samples of Hp-35.23; right panel, two samples of Hp-43.37. Student’s t-test was performed for significance in the Hp-35.23 group with the analysis of three samples. * p<0.05. **p<0.01. Open bars; TSST-1 (-), closed bars; TSST-1 (+).

We then analyzed the class I SLA expression level of these DP cells. TSST-1 stimulation induced the enhancement of class I SLA protein on a fraction of the CD4+ cells after 72 hrs (S6A Fig). The CD4-high fraction showed a higher expression of class I SLA (S6B Fig), and no difference was observed between the CD4.A (+/+) and CD4.B (+/+) alleles. Similarly, no significant difference was observed between Hp-35.23 and Hp-43.37 (S6C Fig).

To determine whether anti-CD4 mAb treatment could block the CD4 T cell activation, two different samples of PBMCs (#1021 and #3343) were stimulated with TSST-1 in the presence of x1E10 for 72 hrs. As shown in Fig 6 and S7 Fig, the increase in the ratio of CD4-high DP cells in the presence of x1E10 was markedly less than the increase in the ratio of cells cultured in the absence of x1E10 during the 72 hrs for both swine samples. In comparison to the CD4- high DP cells, the CD4-low DP cells expressed not only CD4 but also class I SLA at lower levels and the cell size was not enlarged with x1E10 treatment. These results suggest that the CD4-low DP cells did not involve activated DP cells and that the CD4-low DP cells could not enhance the surface expression of CD4 because of the anti-CD4 mAb binding and CD4 internalization. However, the ratio of CD4-high DP T cell still increased linearly during 72 hrs incubation, so that the blocking effect of anti-CD4 mAb was only to slightly delay or lower the T cell stimulation by TSST-1 (Fig 6B).

Fig 6 — Two swine PBMC samples (upper panels; #1021, lower panels; #3343) were stained with x1E10, anti-CD8 mAb and X2F6, then analyzed through FCM. A. Left panels show the pattern of CD4 and CD8 expression. The DP cells were divided into CD4-high and CD4-low cells. Center panels show the histograms of the class I SLA expression of the CD4-high (left) and CD4-low (right) DP cells. Right panels show the FSC/SSC gates of CD4-high (left) and CD4-low (right) DP cells. Cells were collected at 0 hr (upper panels) and after 72 hrs for negative controls (center panels) and x1E10 (1 μg/mL) mAb treatment (lower panels). B. Kinetics of CD4-high DP cell fraction ratio/DP cell fraction in two TSST-1 stimulated PBMC samples in the presence of x1E10 (1 μg/mL) (solid lines) and absence of x1E10 (broken lines) as a negative control. Left panel, #1021; Right panel, #3343.

These results with the x1E10 mAb show that both CD4.A and CD4.B bearing T cells can be activated and enhance the expression of CD4 and class I SLA molecules on the surface by superantigen stimulation. Therefore, both CD4.A and CD4.B are functional molecules of the Microminipig. The mAb x1E10 also was shown to inhibit the kinetics of CD4 T cell activation induced by TSST-1.

Discussion

Although several anti-swine CD4 mAbs were already available commercially, the CD4.B antigenic variant present in the Microminipig herd did not react with them [17]. The two novel and specific mAbs that we prepared in this study recognized both CD4 isotypes, CD4.A and CD4.B. One of them, b1D7, recognized only CD4.B, whereas the other mAb, x1E10, recognized both of the isotypes. The mAb that recognized both CD4.A and CD4.B was useful for measuring total CD4 expression by FCM and/or immunohistochemistry. These mAbs did not recognize the PBMC of other species when examined by flow cytometry suggesting that they were swine-specific.

The CD4 protein is well-known as one of the receptors that bind to the HIV virus, and the interaction has been detailed at the structural level [36–38]. The molecular structure of CD4 was analyzed in various animal species and comparative analyses revealed that CD4 possesses various isotypes in most of the examined species [9–11, 13, 39]. In the MeLiM minipig strain, the site of mutation and the amino acid substitutions among the CD4 isotypes appear to affect the MHC response [9]. While a report suggested that a human CD4 SNP (C868T) affected the infection of HIV virus, another group reported that no significant difference was observed [13]. Thus, the significance of the effects of CD4 polymorphism on the immune reaction is still unclear.

The CD4 protein is thought to recruit Lck in the intracellular domain when the TCR of T cells and the class II MHC of antigen-presenting cells interact and amplify the activation signal [3]. However, the CD4 molecule may also possess a weak binding affinity to class II MHC in the absence of TCR involvement. Consequently, a hypothesis was proposed that CD4 adheres to the class II MHC-expressing cells and that the CD4 D1 and D2 domains play an important role in the interaction with class II MHC [6]. However, the difference of affinity to class II MHC among the CD4 isotypes has not yet been clarified. To examine the kinetics of protein-protein interaction of CD4 isotypes, mAbs that recognize each allele and/or both alleles are essential. Antibodies that distinguish each isotype CD4 separately may enable the evaluation of the CD4 and class II MHC and/or peptide interaction at the protein level.

Using the new mAbs prepared for this study and the anti-class I SLA mAb that we had already established [23], together with the swine PBMC haplotypes that match the class I SLA allele-specific antibody, we found that the CD4.A and CD4.B proteins are expressed on swine T cells at a similar level. A subset of the CD4 T cells expressed CD8 simultaneously. Stimulation with superantigen TSST-1 induced activation of both CD4.A and CD4.B-expressing T cells, enhanced the expression of both CD8 and CD4, and was followed by the enhancement of specific class I MHC. In our study, the CD4-low DP cells expressed lower levels of class I SLA showing that most of the small memory DP cells are not activated further.

We previously reported that TSST-1 stimulation enhanced the expression of class I SLA on CD4 T cells [22, 23]. In this study, we used this phenomenon to detect the degree of T cell activation. As a result, we observed the increase of CD4, CD8 and class I SLA in TSST-1 stimulated CD4 T cells. Swine PBMCs are reported to involve significant amounts of CD4+CD8+ double-positive (DP) T cells [34]. These DP cells are derived from CD4 single-positive (SP) cells and have a memory phenotype. The reason why these T cells express CD8a has not been clarified yet. However, the newly expressed CD8 may recognize class I MHC together with the TCR to enhance the binding ability of T cells to antigen-presenting cells (APCs), which express both class I and class II MHCs. Moreover, these activated cells enhance CD4 expression. Therefore, the activated Th cells expressing higher level of CD4 and CD8 might induce stronger signals compared to CD4 SP and CD8 SP cells. Our results show that the activated DP positive cells enhance CD4 and class I SLA expression, which may enhance the TCR signals among the Th cells. Therefore, in this regard, the older animals in swine populations possess such DP cells probably to better support the immune response even if the newly provided immune cells were decreased.

Surprisingly, the anti-CD4 antibody x1E10 suppressed the TSST-1 activation of CD4 T cells and the increase in cell size and class I SLA expression. Because superantigens crosslink TCRβ and MHCβ directly, and CD4 also bind to MHCβ, it might be difficult for the CD4 molecule to be involved in the TSST-1 response. However, the binding of CD4 with the antibody may reduce the number of CD4 molecules engaging in the TCR-MHC interaction. Otherwise, if CD4 functions as an adhesion molecule that directly adheres to the MHC molecules before the cognate interaction, and the interaction is mandatory to construct the cognate interaction, the activation of CD4 T cells might be reduced by inhibiting the CD4-MHC interaction [6–8] with the anti-CD4 antibody.

Taken together, our results suggest that the mAbs prepared in this study have specific advantages because the two new antibodies recognize the CD4.B protein that could not be detected by conventional mAbs. Moreover, the quantitative analysis comparing CD4.A and CD4.B expression is possible because the antibody x1E10 recognizes both CD4.A and CD4.B isotypes. Using both of these novel mAbs and the SLA-stabilized and CD4 isotype-identified Microminipigs in our experiments, we found that CD4.B is expressed at about the same level as CD4.A, which was detected previously using the conventional mAb (PT85A). However, more data need to be collected to determine whether the CD4.A and CD4.B molecules have some functional differences that so far have been undetected. Also, the suppression of the TSST-1 stimulatory effect on T-cell activation by one of our antibodies suggests that taken together both of these new antibodies are promising reagents to support porcine T cell activation studies in future.

Supporting information

S1 Fig. Immunization of BALB/c mice with swine CD4 isotype proteins.

A. Protocol for immunization of the BALB/c mice with swine CD4 antigen. Swine PBMC or CD4 isotype transfectants (CD4A⁺/A20 or CD4B⁺/A20) were immunized biweekly. One week after 3^rd and 6^th immunization, peripheral blood (PB) was collected and antibody titers were examined. B. Antibody reactivity to CD4.A and CD4.B was checked by FCM. The fluorescent intensity of mVenus shows the transfection efficiency. Mouse antisera were used for the first antibody for staining the cells. The numbers shown in the panels are the percentage of the positive cells. Left panels show the CD4.B specific mAb preparation. Right panels show the CD4.A and CD4.B specific mAb preparation.

(TIF)

Click here for additional data file.^{(206.1KB, tif)}

S2 Fig. IgH and Igk characterization of b1D7.

The repertoire analysis was performed by Repertoire Genesis (Osaka, Japan). The types of heavy chain and light chains and the sequences are shown.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

S3 Fig. IgH and Igk characterization of x1E10.

The repertoire analysis was performed by Repertoire Genesis (Osaka, Japan). The type of heavy chain and light chains and the sequences are shown.

(TIF)

Click here for additional data file.^{(996.5KB, tif)}

S4 Fig. Evaluation of Biotinylated x1E10 mAb.

The x1E10 mAb was labeled with biotin and HEK293 transfected with CD4.A or CD4.B gene was stained with biotinylated x1E10 and streptavidin PE-cy7. The reactivity was evaluated by FCM showing that the reactivity of labeled antibody against both alleles is almost the same.

(TIF)

Click here for additional data file.^{(357.3KB, tif)}

S5 Fig. Appearance of CD4 high fractions after the stimulation of Hp-35.23 swine PBMC.

Swine PBMCs (CD4.A(+/+) and CD4.B(+/+)) were stimulated and stained with the mAb (x1E10) followed by anti-mouse IgG-PE, and anti-CD8 mAb and analyzed by FCM as mentioned in Fig 5. Left panels; CD4.A (+/+) swine. The same swine collected at a different time (#3617–1 and #3617–2). Right panels; CD4.B (+/+) swine. Two different swine (#2858, #3686) are shown. The ratio of CD4SP and DP cells in the lymphocyte gated cells are shown in the panels. The number shown above each panel represents the sum of CD4SP and DP cell percentages.

(TIF)

Click here for additional data file.^{(436.2KB, tif)}

S6 Fig. Class I SLA expression is increased in the TSST-1 stimulated CD4+ cells.

Swine PBMCs were stained with x1E10 and anti-CD8 mAb and analyzed by FCM. A. The left two panels show the representative pattern of FSC/SSC after 72 hrs of culture with/without TSST-1 stimulation. As the large cells are increased by TSST-1 stimulation, a small and large lymphocyte gate was used for the analysis. CD4/CD8 expression is shown in middle panels. Upper panel shows the CD4/CD8 pattern of Hp-35.23 and lower panels; Hp-43.37. Right panels show the histograms of the class I SLA expression of the lymphocyte-gated T cell subsets after TSST-1 stimulation. X2F6 was used for Hp-35.23 and PT85A was used for Hp-43.37. B. The left panel shows the gate of each CD4+ fraction analyzed for the expression of class I SLA. The CD4/CD8 DP cells were divided into CD4 high and CD4 low groups to examine the expression levels of class I SLA and the MFI data are shown in the right table. The middle panels show the overlay pattern of class I SLA expression in each group of Hp-35.23 and Hp-43.37 swine. The groups were further divided into CD4.A(+/+) and CD4.B(+/+) groups that are shown in the panels. C. Class I SLA expression on T cells after TSST-1 stimulation. Left panel; The MFIs of class I SLA expression on DP T cells of Hp-35.23 swine with/without stimulation of TSST-1. Right panel; class I SLA expression on DP T cells of Hp-43.37. Open bars; 72 hrs culture without TSST-1, Black bars; 72 hrs culture with TSST-1.

(TIF)

Click here for additional data file.^{(355.1KB, tif)}

S7 Fig. Appearance of CD4 high fractions after the stimulation of swine PBMC.

Swine PBMCs (#1021 and #3343) were stimulated in the absence and presence of x1E10. Samples were stained with the mAb (x1E10) followed by anti-mouse IgG-PE. Then, the cells were stained with anti-CD8 mAb and analyzed by FCM as described in Fig 5. Left 4 panels, without TSST-1 stimulation; Right 4 panels, with TSST-1 stimulation. Control groups are stained without x1E10. The x1E10 groups were cultured in the presence of x1E10. The samples were collected at 24, 48 and 72 hrs after the stimulation. The cell size and shape were measured by forward scatter (FSC) and side scatter (SSC). The expressions of CD4 and CD8 were detected in the lymphocyte-gated cells. The data presented in the Figure was used for the Fig 6B.

(TIF)

Click here for additional data file.^{(441.1KB, tif)}

S1 Table

(PDF)

Click here for additional data file.^{(32.3KB, pdf)}

S2 Table

(PDF)

Click here for additional data file.^{(16.4KB, pdf)}

S3 Table

(PDF)

Click here for additional data file.^{(81.5KB, pdf)}

S4 Table

(PDF)

Click here for additional data file.^{(103.3KB, pdf)}

Acknowledgments

We thank Fuji Micra Inc. (Fujinomiya, Japan) for providing blood samples of Microminipigs. We thank the members of the Teaching and Research Support Center in the Tokai University School of Medicine for their technical skills.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The authors received no specific funding for this work.

References

1.Wang J, Meijers R, Xiong Y, Liu J, Sakihama T, Zhang R, et al. Crystal structure of the human CD4 N-terminal two-domain fragment complexed to a class II MHC molecule. Proc Natl Acad Sci U S A. 2001;98(19):10799–804. 10.1073/pnas.191124098 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Yin Y, Wang X, Mariuzza R. Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4. Proc Natl Acad Sci U S A. 2012;109(14):5045–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Veillette A, Bookman M, Horak E, Samelson L, Bolen J. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature. 1989;338(6212):257–9. 10.1038/338257a0 [DOI] [PubMed] [Google Scholar]
4.Veillette A, Sleckman B, Ratnofsky S, Bolen J, Burakoff S. The cytoplasmic domain of CD4 is required for stable association with the lymphocyte-specific tyrosine protein kinase p56lck. Eur J Immunol. 1990;20(6):1397–400. 10.1002/eji.1830200628 [DOI] [PubMed] [Google Scholar]
5.König R, Huang L, Germain R. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. 1992. 1992;356(6392):796–8. [DOI] [PubMed] [Google Scholar]
6.Doyle C, Strominger J. Interaction between CD4 and class II MHC molecules mediates cell adhesion. Nature. 1987;330(6145):256–9. 10.1038/330256a0 [DOI] [PubMed] [Google Scholar]
7.Kinch M, Strominger J, Doyle C. Cell adhesion mediated by CD4 and MHC class II proteins requires active cellular processes. J Immunol. 1993;151(9):4552–61. [PubMed] [Google Scholar]
8.Glatzová D, Cebecauer M. Dual Role of CD4 in Peripheral T Lymphocytes. Front Immunol. 2019;10:618 10.3389/fimmu.2019.00618 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Blanc F, Créchet F, Bruneau N, Piton G, Leplat J, Andréoletti F, et al. Impact of a CD4 gene haplotype on the immune response in minipigs. Immnunogenetics. 2018;70(4):209–22. 10.1007/s00251-017-1037-z [DOI] [PubMed] [Google Scholar]
10.Boscariol R, Pleasance J, Piedrafita D, Raadsma H, Spithill T. Identification of two allelic forms of ovine CD4 exhibiting a Ser183/Pro183 polymorphism in the coding sequence of domain 3. Vet Immunol Immunopathol. 2006;13(3–4):305–12. 10.1016/j.vetimm.2006.05.015 [DOI] [PubMed] [Google Scholar]
11.Eguchi-Ogawa T, Matsubara T, Toki D, Okumura N, Ando A, Kitagawa H, et al. Distribution of the CD4 Alleles in Sus scrofa Demonstrates the Genetic Profiles of Western Breeds and Miniature Pigs. Anim Biotechnol. 2018;29(3):227–33. Epub 2017/10/17. 10.1080/10495398.2017.1367691 . [DOI] [PubMed] [Google Scholar]
12.Wang L, Fan J, Yu M, Zheng S, Zhao Y. Association of goat (Capra hircus) CD4 gene exon 6 polymorphisms with ability of sperm internalizing exogenous DNA. Mol Biol Rep. 2011;38(3):1621–8. 10.1007/s11033-010-0272-2 [DOI] [PubMed] [Google Scholar]
13.Lu Y, Wu J, Qin X, Xie L, Ma L, Huang X, et al. The CD4 C868T Polymorphism and Its Correlation with HIV-1 Infection in a Chinese Population. AIDS Res Hum Retroviruses. 2015;31(5):525–30. Epub 2015/01/23. 10.1089/AID.2014.0303 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Oyugi J, Vouriot F, Judie Alimonti J, Wayne S, Luo M, Land A, et al. A Common CD4 Gene Variant Is Associated with an Increased Risk of HIV-1 Infection in Kenyan Female Commercial Sex Workers. The Journal of Infectious Diseases. 2009;199(9):1327–34. 10.1086/597616 [DOI] [PubMed] [Google Scholar]
15.Kristiansen O, Karlsen A, Larsen Z, Johannesen J, Pociot F, Mandrup‐Poulsen T. Identification of a Type 1 Diabetes‐Associated CD4 Promoter Haplotype with High Constitutive Activity Scand J Immunol. 2004;59(6):582–91. 10.1111/j.1365-3083.2004.01444.x [DOI] [PubMed] [Google Scholar]
16.Tr Sundt, LeGuern C, Germana S, Smith C, Nakajima K, Lunney J, et al. Characterization of a polymorphism of CD4 in miniature swine. J Immunol. 1992;148(10):3195–201. [PubMed] [Google Scholar]
17.Matsubara T, Nishii N, Takashima S, Takasu M, Imaeda N, Aiki-Oshimo K, et al. Identification of a CD4 variant in Microminipigs not detectable with available anti-CD4 monoclonal antibodies. Vet Immunol Immunopathol. 2015;168(3–4):176–83. 10.1016/j.vetimm.2015.09.008 [DOI] [PubMed] [Google Scholar]
18.Matsubara T, Nishii N, Takashima S, Takasu M, Imaeda N, Aiki-Oshimo K, et al. Identification and characterization of two CD4 alleles in Microminipigs. BMC Veterinary Research. 2016;12(1):222 10.1186/s12917-016-0856-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Kaneko N, Itoh K, Sugiyama A, Izumi Y. Microminipig, a non-rodent experimental animal optimized for life science research: preface. J Pharmacol Sci. 2011;115(2):112–4. [DOI] [PubMed] [Google Scholar]
20.Takasu M, Tsuji E, Imaeda N, Matsubara T, Maeda M, Ito Y, et al. Body and major organ sizes of young mature microminipigs determined by computed tomography. Lab Anim. 2015;49(1):65–70. 10.1177/0023677214557169 [DOI] [PubMed] [Google Scholar]
21.Ando A, Imaeda N, Ohshima S, Miyamoto A, Kaneko N, Takasu M, et al. Characterization of swine leucocyte antigen alleles and haplotypes on a novel miniature pig line, Microminipig. Animal Genetics. 2014;45:791–8. 10.1111/age.12199 [DOI] [PubMed] [Google Scholar]
22.Kametani Y, Ohshima S, Kita Y, Shimada S, Kamiguchi H, Shiina T, et al. Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells. Vet Immunol Immunopathol. 2012;148:252–9. 10.1016/j.vetimm.2012.05.005 [DOI] [PubMed] [Google Scholar]
23.Kametani Y, Ohshima S, Miyamoto A, Shigenari A, Takasu M, Imaeda N, et al. Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs. PLoS One. 2016;11(10):e0164995 10.1371/journal.pone.0164995 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Ho C, Lunney J, Franzo-Romain M, Martens G, Lee Y, Lee J, et al. Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations. Animal Genetics. 2009;40:468–78. 10.1111/j.1365-2052.2009.01860.x [DOI] [PubMed] [Google Scholar]
25.Ho C, Lunney J, Lee J, Franzo-Romain M, Martens G, Rowland R, et al. Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations. Anim Genet. 2010;41(4):428–32. 10.1111/j.1365-2052.2010.02019.x [DOI] [PubMed] [Google Scholar]
26.Mizutani A, Kikkawa E, Matsuno A, Shigenari A, Okinaga H, Murakami M, et al. Modified S/MAR episomal vectors for stably expressing fluorescent-protein tagged transgenes with small cell-to-cell fluctuations. Anal Biochem. 2013;443(1):113–6. 10.1016/j.ab.2013.08.009 [DOI] [PubMed] [Google Scholar]
27.Kremers G-J, Goedhart J, van Munster E, W J Gadella Jr T. Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry. 2006;45:6540–80. [DOI] [PubMed] [Google Scholar]
28.Ohshima S, Mori S, Shigenari A, Miyamoto A, Takasu M, Imaeda N, et al. Differentiation ability of multipotent hematopoietic stem/progenitor cells detected by a porcine specific anti-CD117 monoclonal antibody. Biosci Trends. 2014;8:308–15. 10.5582/bst.2014.01084 [DOI] [PubMed] [Google Scholar]
29.Levitt M. Accurate modeling of protein conformation by automatic segment matching. J Mol Biol. 1992;226(2):507–33. 10.1016/0022-2836(92)90964-l [DOI] [PubMed] [Google Scholar]
30.Bernstein F, Koetzle T, Williams G, Meyer E Jr., Brice M, Rodgers J, et al. The Protein Data Bank: A Computer-based Archival File For Macromolecular Structures. J Mol Biol. 1977;112:535 10.1016/s0022-2836(77)80200-3 [DOI] [PubMed] [Google Scholar]
31.Lamarre D, Ashkenazi A, Fleury S, Smith D, Sekaly R, Capon D. The MHC-binding and gp120-binding functions of CD4 are separable. Science. 1989;245(4919):743–6. 10.1126/science.2549633 [DOI] [PubMed] [Google Scholar]
32.Pancera M, Majeed S, Ban Y, Chen L, Huang C, Kong L, et al. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility. Proc Natl Acad Sci U S A. 2010;107(3):1166–71. 10.1073/pnas.0911004107 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Kwong P, Ryu S, Hendrickson W, Axel R, Sweet R, Folena-Wasserman G, et al. Molecular characteristics of recombinant human CD4 as deduced from polymorphic crystals. Proc Natl Acad Sci U S A. 1990;87(16):6423–7. 10.1073/pnas.87.16.6423 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Zuckermann F, Husmann R. Functional and phenotypic analysis of porcine peripheral blood CD4/CD8 double-positive T cells. Immunology. 1996;87:500–12. [PMC free article] [PubMed] [Google Scholar]
35.Tuffs S, Haeryfar S, McCormick J. Manipulation of Innate and Adaptive Immunity by Staphylococcal Superantigens. Pathogens. 2018;7(2):53 10.3390/pathogens7020053 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Andrianov A. Spatial structure model of the CD4 receptor-binding site of the HIV envelope protein gp120. J Biomol Struct Dyn. 1998;15:1101–13. 10.1080/07391102.1998.10509004 [DOI] [PubMed] [Google Scholar]
37.Singh M, Singh P, Gaudray G, Musumeci L, Thielen C, Vaira D, et al. An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγnull mice allows HIV replication and development of anti-HIV immune responses. PLoS ONE. 2012;7(6):e38491 10.1371/journal.pone.0038491 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Gawron M, Duval M, Carbone C, Jaiswal S, Wallace A, Martin Jr, et al. Human Anti-HIV-1 gp120 Monoclonal Antibodies with Neutralizing Activity Cloned from Humanized Mice Infected with HIV-1. J Immunol 2019;202(3):799–804. 10.4049/jimmunol.1801085 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Burstein R, Henry NJ, Collison ML, Marczak LB, Sligar A, Watson S, et al. Mapping 123 million neonatal, infant and child deaths between 2000 and 2017. Nature. 2019;574(7778):353–8. Epub 2019/10/18. 10.1038/s41586-019-1545-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0242572.r001

Decision Letter 0

Karen L Elkins

22 Jun 2020

PONE-D-20-14895

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs

PLOS ONE

Dear Dr. Kametani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As you will see, the reviewers expressed generally favorable views of the manuscript in the context of PLOS ONE publication criteria, but both also suggested important revisions. I ask that you particularly focus on the recommendations to further support the utility of the new anti-CD4 monoclonal antibodies in characterizing the superantigen responses (Fig 5); revisit the proposed supplemental figures with a view toward selecting only those that enhance the results in the main body of the manuscript; and, remembering that PLOS ONE does not copyedit manuscripts, please edit the text carefully throughout for clarity and brevity (the reviewers have provided some specific editorial suggestions for your consideration).

Please submit your revised manuscript by Aug 06 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Karen L. Elkins

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In their manuscript entitled “Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs” Shino Ohshima et al. describe the development of monoclonal antibodies against gene products of CD4.A and CD4.B alleles in swine. In this manuscript the authors focus on the CD4.B expression. Interestingly the gene product of CD4.B is not recognized by most of the mAb used for the characterization of porcine CD4+ T cells and there is indeed a lack of reagents.

This is an interesting manuscript and the molecular data are highly appreciated. In regard to the functional studies with the superantigen stimulation I have some questions and concerns.

1. Can the authors block their superantigen stimulation with their anti-CD4 mAb?

2. Can one block a superantigen stimulation with anti CD4 mAb at all?

3. Is CD4 at all involved in the superantigen stimulation? I do not think so.

4. To get a more precise information I would strongly recommend to analyze the function of the respective CD4 molecules in an antigen-specific immune response e.g. against a vaccine antigen or ovalbumin.

5. Summerfield et al. (Cellular Immunology 168, 291-296 (1996)) could show a blocking of their PRV-specific T-cell response with mAb 74-12-4. Would the new mAbs do the same?

6. An alternative might be to try blocking of a CD4 response in allogeneic mixed leukocyte cultures with the novel antibodies.

Additional points:

7. Lines 95 -100 these sentences are hard to understand. Why do the authors use anti-MHC-I antibodies?

8. Line 305: introduced

9. Does the novel anti-CD4 mAb against CD4.A (x1E10) recognize another epitope than the existing mAbs e.g. 74-12-4?

10. Discussion line 531- 541: What is the deeper sense to discuss MHC-I expression in a CD4 manuscript? I think this part of the discussion should be deleted.

11. These cells have an activated and memory phenotype. Can the authors give any information about central and effector memory cells?

12. Line 554 – 566: Personally, I do not think that this part of the discussion contributes a lot to the CD4 story.

13. The immunohistochemistry presented in figure 3c is poor. It would have been of advantage to see e.g. in lower magnification in spleen staining of the PALS or in lymph node staining of T-cell areas. The same for thymus. A comparison of the respective staining pattern with staining pattern of existing anti-CD4 mAb would have been helpful.

14. Figure 5: is there a reason why the authors selected after 72h stimulation with the superantigen a lymphocyte gate? Would it not be much better to analyze the stimulated cells in a blast gate as presented in figure 6 and compare their phenotype with the non-stimulated resting cells in the lymphocyte gate. Another way to get the phenotype of activated proliferating cells would be the use of CFSE or violet and a respective gating on proliferating cells. These additional analyses would enhance the quality of this manuscript significantly.

15. Figure 6 should be skipped or moved to supplementary figures

16. Figure S6 should be skipped and the results mentioned in the text

17. The same for figure S7.

Reviewer #2: This manuscript is on generation of novel monoclonal antibody reagents valuable for characterization of CD4 T cell subpopulations including CD4.A and CD4.B alleles in minipigs with different haplotypes. The authors also tried to use two developed antibody clones recognizing CD4.A and CD4.AB specifically in characterizing the up-regulation of CD4 molecule and SLA on T-cells in PBMC stimulated with TSST-1. The analysis using these monoclonal antibodies could define the specific population of CD4.B T cells activated after TSST-1 stimulation. However, a major revision on following aspects should be made before further review and consideration for publication in PLOS One journal.

Major comments:

1. Overall English writing and grammar should improved for clear communication to readers. It is difficult to understand the authors' interpretation and meaning.

2. Lines 372-488 are not clearly written and have many grammatical errors and incomplete sentences. It is difficult to understand authors' meaning. Rewrite the section on comparison of ClassI SLA expression on T cells with different CD4 isotypes. Discussion sentences in this result section should be moved to Discussion part.

3. Lines 459-462 and fig 6C used two different antibodies for two pigs with different haplotypes to analyze the sensitivity to TSST-1 stimulation. How can you interpret whether the difference in sensitivity is due to pig haplotypes or monoclonal antibodies use?

4. Lines 485-486:

Minor comments:

1. Lines 39: mRNA expression needs to be changed into mRNA transcription.

2. Line 202: The sequences of monoclonal antibody hybridoma clones should be submitted to GenBank and submission numbers need to be included in the text.

3. Line 259: Replace 'was' with 'were'.

4. Line 280: Replace 'become' with 'be'.

5. Line 290: Replace 'is' with 'was'.

6. Line 298: Delete 'in Microminipigs each'.

7. Line 306 and many other sentences: Replace 'cross-reactivity' with 'specific reactivity' or 'reactivity'.

8. Line 309: Change a peak of mean into a peak mean.

9. Lines 313-314: 'Produced both---- specific antibodies.' should be changed into 'both the CD4.B and the CD4.A specific antibodies were produced.'

10. Line 333 figure 2 and other figures: 'mVenus' channel needs to be described.

11. Lines 345 and 346: Change 'recognizes' into 'recognized' for use of identical tense in the sentence.

12. Line 346: Delete 'also'.

13. Line 347: Change 'PBMC but only' into PBMC, but recognized only.

14. Line 348: Change 'Accordingly' into 'Therefore'.

15. Lines 387 and 388: Delete 'were this' and 'detected'. move 'also' right after but to make 'but also'.

16. Line 399: 'Individual numbers' should be changed into 'Individual identification numbers'.

17. Line 402: Change 'Correlation of' into 'Correlation between'.

18. Many more English errors need to be fixed.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Armin Saalmüller

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Nov 25;15(11):e0242572. doi: 10.1371/journal.pone.0242572.r002

Author response to Decision Letter 0

10 Sep 2020

This is an interesting manuscript and the molecular data are highly appreciated. In regard to the functional studies with the superantigen stimulation I have some questions and concerns.

1. Can the authors block their superantigen stimulation with their anti-CD4 mAb?

2. Can one block a superantigen stimulation with anti CD4 mAb at all?

3. Is CD4 at all involved in the superantigen stimulation? I do not think so.

5. Summerfield et al. (Cellular Immunology 168, 291-296 (1996)) could show a blocking of their PRV-specific T-cell response with mAb 74-12-4. Would the new mAbs do the same?

6. An alternative might be to try blocking of a CD4 response in allogeneic mixed leukocyte cultures with the novel antibodies.

We appreciate the reviewer to give us important suggestions. Till now, we cannot find any study that describe the blocking of superantigen stimulation with anti-CD4 mAb. Because the superantigen reaction only use TCRβ and MHC class II, CD4 molecules were supposed not to be involved. Therefore, we did not check the function in the previous version. According to the reviewer’s suggestions, we performed an experiment for blocking of TCR signals. Because the vaccine response and allogeneic mixed leukocyte reaction (MLR) are different among SLA-haplotypes because the epitope peptide-presentation or allogeneic MHC recognition depend on the MHC type, we performed TSST-1 stimulation but not vaccine stimulation. As a result, our mAb x1E10 delayed the activation of CD4 T cells when they were added to TSST-1 stimulation. We showed the results in Fig.6. It is not clear why TSST-1 stimulation could be suppressed by anti-CD4 mAb, and the issue will be addressed in near future. While Microminipig herds have not been bred to select SLA homozygous pigs, we will try to select the most reactive combination of the haplotypes and perform the experiments in future.

Additional points:

7. Lines 95 -100 these sentences are hard to understand. Why do the authors use anti-MHC-I antibodies?

According to the reviewer’s comment, we changed the description of Lines 94~99 as follows,

“In this study, we developed two mAbs which recognize Microminipig CD4.B, one for only CD4.B and the other for CD4.A and CD4.B in order to analyze CD4.A and CD4.B protein expression and function. We used these antibodies to evaluate (1) the level of CD4 protein expression and T cell activation, and (2) the association of T cell activation with class I SLA protein expression levels in response to in vitro T cell stimulation with the toxic shock syndrome-1 (TSST-1) enterotoxin.”

8. Line 305: introduced

We changed the description and the word ‘introduced was not used in the sentence of Lines 318~320.

9. Does the novel anti-CD4 mAb against CD4.A (x1E10) recognize another epitope than the existing mAbs e.g. 74-12-4?

We think that the antibody (x1E10) recognizes another epitope, where CD4.A and CD4.B share common sequence, although the epitope of x1E10 has not been defined yet,

10. Discussion line 531- 541: What is the deeper sense to discuss MHC-I expression in a CD4 manuscript? I think this part of the discussion should be deleted.

As reviewer’s suggestion, we deleted the description and concentrated on the CD4 function.

11. These cells have an activated and memory phenotype. Can the authors give any information about central and effector memory cells?

We appreciate the reviewer’s opinion. We don’t have any data supporting the evidences till now. However, as it is very important, we will check the phenotype in future.

12. Line 554 – 566: Personally, I do not think that this part of the discussion contributes a lot to the CD4 story.

According to the reviewer’s suggestion, we deleted the part.

According to the reviewer’s suggestions, we added a lower magnification image (x10), although we could not obtain the thymus tissues. As shown in the Fig 3, PALS and central arteries are clearly shown. Brown signals are involved in the PALS, suggesting the CD4 T cell accumulation.

We already analyzed the activation time and found that after 72 hrs the activation of Th cells are highly detectable (ref 22). During 72 hrs of the activation, cells become enlarged and increase expression. According to the reviewer’s suggestion, we added the time course of CD4 and expression from 0 to 72 hrs in the revised version (Fig 6B, S7 Fig).

15. Figure 6 should be skipped or moved to supplementary figures

According to the reviewer’s suggestion, we moved Fig. 6 to supplemental data ( S6 Fig).

16. Figure S6 should be skipped and the results mentioned in the text

17. The same for figure S7.

According to the reviewer’s suggestion, we deleted them.

Major comments:

1. Overall English writing and grammar should improved for clear communication to readers. It is difficult to understand the authors' interpretation and meaning.

According to the reviewer’s comment, we extensively improved the English.

2. Lines 372-488 are not clearly written and have many grammatical errors and incomplete sentences. It is difficult to understand authors' meaning. Rewrite the section on comparison of class I SLA expression on T cells with different CD4 isotypes. Discussion sentences in this result section should be moved to Discussion part.

According to the reviewer’s comment, we rewrote the part with improved the English. The section was named as “Comparison of class I SLA expression on T cells with different CD4 isotypes” and emphasized that class I SLA was used as an activation marker.

As the reviewer pointed out, it was difficult to interpret the difference. In our manuscript, even if the CD4 isotype was different, as shown in S6 Fig B, the CD4 high DP cells showed higher expression of class I SLA (Hp.35.23) compared with CD4 low DP cells. Similar result was obtained using Hp.43.37. However, the case was not enough and it takes time to definitely say that the reaction is same or not. Therefore, we deleted most of the description in the section. Only the increase of class I SLA after the stimulation was mentioned here, which was used to identify the activation of CD4 T cells.

4. Lines 485-486:

Do you mean the sentence “Therefore, both CD4.A and CD4.B are functional molecules of the Microminipig.” The description itself might be OK.

Minor comments:

1. Lines 39: mRNA expression needs to be changed into mRNA transcription.

According to the reviewer’s suggestion, we changed the sentence and deleted the word.

2. Line 202: The sequences of monoclonal antibody hybridoma clones should be submitted to GenBank and submission numbers need to be included in the text.

Because the mAb was submitted for the patent, the sequence will be automatically sent to GenBank.

3. Line 259: Replace 'was' with 'were'.

According to the reviewer’s suggestion, we changed the word (Line 274).

4. Line 280: Replace 'become' with 'be'.

According to the reviewer’s suggestion, we changed the sentence and deleted the word (Lines 295~299).

5. Line 290: Replace 'is' with 'was'.

According to the reviewer’s suggestion, we replaced the word (Line 307).

6. Line 298: Delete 'in Microminipigs each'.

According to the reviewer’s suggestion, we changed the sentence and deleted the word (Lines 312~314).

7. Line 306 and many other sentences: Replace 'cross-reactivity' with 'specific reactivity' or 'reactivity'.

According to the reviewer’s suggestion, we replaced the word (Line 320).

8. Line 309: Change a peak of mean into a peak mean.

According to the reviewer’s suggestion, we changed the sentence and deleted the word (Line 322~323).

9. Lines 313-314: 'Produced both---- specific antibodies.' should be changed into 'both the CD4.B and the CD4.A specific antibodies were produced.'

According to the reviewer’s suggestion, we changed the description (Lines 325 and 326).

10. Line 333 figure 2 and other figures: 'mVenus' channel needs to be described.

We described about mVenus in materials methods Page 10 Line148-149 as “Transgene-positive cell ratio was determined according to the fluorescent intensity of the co-expressed mVenus protein,” and in Fig2 legend Page 21 Line 345 “The numbers in the panels show the percentage of mVenus and APC double positive cells.” And Fig3 legend Page 22 Line 370-371. The numbers in the panels show the percentage of mVenus and APC double positive cells.”

11. Lines 345 and 346: Change 'recognizes' into 'recognized' for use of identical tense in the sentence.

According to the reviewer’s suggestion, we changed the word (Line 353).

12. Line 346: Delete 'also'.

According to the reviewer’s suggestion, we deleted the word and changed the description.

13. Line 347: Change 'PBMC but only' into PBMC, but recognized only.

According to the reviewer’s suggestion, we changed the word and changed the description.

14. Line 348: Change 'Accordingly' into 'Therefore'.

We changed the description largely and the description was deleted. We described the summarized sentence in Line 354-356.

15. Lines 387 and 388: Delete 'were this' and 'detected'. move 'also' right after but to make 'but also'.

According to the reviewer’s suggestion, we changed the word (Lines 390 to 392).

16. Line 399: 'Individual numbers' should be changed into 'Individual identification numbers'.

According to the reviewer’s suggestion, we changed the word (Line 404).

17. Line 402: Change 'Correlation of' into 'Correlation between'.

According to the reviewer’s suggestion, we changed the word (Line 407).

18. Many more English errors need to be fixed.

We checked and corrected the grammar.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(31.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0242572.r003

Decision Letter 1

Karen L Elkins

21 Oct 2020

PONE-D-20-14895R1

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs

PLOS ONE

Dear Dr. Kametani,

Thank you for submitting your revised manuscript to PLOS ONE, which the reviewers find to have satisfactorily addressed most of the original comments. Pending remaining editorial revisions that are outlined by the reviewers, I believe that the manuscript will meet PLOS ONE’s publication criteria. Please provide a revised manuscript that addresses the remaining points raised during the review process, as outlined below, for a final editorial review.

Please submit your revised manuscript by Dec 05 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Karen L. Elkins

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: In my opinion this manuscript is fine now. The authors corrected most of the topics. Therer are still some open points but as the authors ,mentioned these can be analyzed in further studies.

Reviewer #2: This revised manuscript has significantly improved writing overall. However, one more improvement in grammar and writing described below will make the manuscript more accessible to readers.

Major comment:

1. Lines 48-56 are about practical applications of developed monoclonal antibodies. Please try to rewrite this paragraph and ask your peers to read with fresh eyes.

Minor comments:

2. "Using" would be a more reasonable and concise expression than "By using" in many sentences.

3. Line 46: Using these two mAbs would be more readable than "By using both of these mAbs".

4. Line 46: CD4.B allele-specific or unique regions would read better than "CD4.B allele proteins".

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Armin Saalmüller

Reviewer #2: No

PLoS One. 2020 Nov 25;15(11):e0242572. doi: 10.1371/journal.pone.0242572.r004

Author response to Decision Letter 1

30 Oct 2020

On behalf of all authors,

Major comment:

1. Lines 48-56 are about practical applications of developed monoclonal antibodies. Please try to rewrite this paragraph and ask your peers to read with fresh eyes.

We rewrote the lines 48-56 to read as follows:

“Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.”

Minor comments:

2. "Using" would be a more reasonable and concise expression than "By using" in many sentences.

We removed all ‘by’ from ‘by using’.

3. Line 46: Using these two mAbs would be more readable than "By using both of these mAbs".

We corrected according to the reviewer’s suggestion.

4. Line 46: CD4.B allele-specific or unique regions would read better than "CD4.B allele proteins".

We corrected according to the reviewer’s suggestion.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(23.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0242572.r005

Decision Letter 2

Karen L Elkins

5 Nov 2020

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs

PONE-D-20-14895R2

Dear Dr. Kametani,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Karen L. Elkins

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0242572.r006

Acceptance letter

Karen L Elkins

13 Nov 2020

PONE-D-20-14895R2

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs

Dear Dr. Kametani:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Karen L. Elkins

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Immunization of BALB/c mice with swine CD4 isotype proteins.

(TIF)

Click here for additional data file.^{(206.1KB, tif)}

S2 Fig. IgH and Igk characterization of b1D7.

The repertoire analysis was performed by Repertoire Genesis (Osaka, Japan). The types of heavy chain and light chains and the sequences are shown.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

S3 Fig. IgH and Igk characterization of x1E10.

The repertoire analysis was performed by Repertoire Genesis (Osaka, Japan). The type of heavy chain and light chains and the sequences are shown.

(TIF)

Click here for additional data file.^{(996.5KB, tif)}

S4 Fig. Evaluation of Biotinylated x1E10 mAb.

(TIF)

Click here for additional data file.^{(357.3KB, tif)}

S5 Fig. Appearance of CD4 high fractions after the stimulation of Hp-35.23 swine PBMC.

(TIF)

Click here for additional data file.^{(436.2KB, tif)}

S6 Fig. Class I SLA expression is increased in the TSST-1 stimulated CD4+ cells.

(TIF)

Click here for additional data file.^{(355.1KB, tif)}

S7 Fig. Appearance of CD4 high fractions after the stimulation of swine PBMC.

(TIF)

Click here for additional data file.^{(441.1KB, tif)}

S1 Table

(PDF)

Click here for additional data file.^{(32.3KB, pdf)}

S2 Table

(PDF)

Click here for additional data file.^{(16.4KB, pdf)}

S3 Table

(PDF)

Click here for additional data file.^{(81.5KB, pdf)}

S4 Table

(PDF)

Click here for additional data file.^{(103.3KB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(31.6KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(23.6KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0242572.ref001] 1.Wang J, Meijers R, Xiong Y, Liu J, Sakihama T, Zhang R, et al. Crystal structure of the human CD4 N-terminal two-domain fragment complexed to a class II MHC molecule. Proc Natl Acad Sci U S A. 2001;98(19):10799–804. 10.1073/pnas.191124098 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref002] 2.Yin Y, Wang X, Mariuzza R. Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4. Proc Natl Acad Sci U S A. 2012;109(14):5045–10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref003] 3.Veillette A, Bookman M, Horak E, Samelson L, Bolen J. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature. 1989;338(6212):257–9. 10.1038/338257a0 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref004] 4.Veillette A, Sleckman B, Ratnofsky S, Bolen J, Burakoff S. The cytoplasmic domain of CD4 is required for stable association with the lymphocyte-specific tyrosine protein kinase p56lck. Eur J Immunol. 1990;20(6):1397–400. 10.1002/eji.1830200628 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref005] 5.König R, Huang L, Germain R. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. 1992. 1992;356(6392):796–8. [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref006] 6.Doyle C, Strominger J. Interaction between CD4 and class II MHC molecules mediates cell adhesion. Nature. 1987;330(6145):256–9. 10.1038/330256a0 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref007] 7.Kinch M, Strominger J, Doyle C. Cell adhesion mediated by CD4 and MHC class II proteins requires active cellular processes. J Immunol. 1993;151(9):4552–61. [PubMed] [Google Scholar]

[pone.0242572.ref008] 8.Glatzová D, Cebecauer M. Dual Role of CD4 in Peripheral T Lymphocytes. Front Immunol. 2019;10:618 10.3389/fimmu.2019.00618 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref009] 9.Blanc F, Créchet F, Bruneau N, Piton G, Leplat J, Andréoletti F, et al. Impact of a CD4 gene haplotype on the immune response in minipigs. Immnunogenetics. 2018;70(4):209–22. 10.1007/s00251-017-1037-z [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref010] 10.Boscariol R, Pleasance J, Piedrafita D, Raadsma H, Spithill T. Identification of two allelic forms of ovine CD4 exhibiting a Ser183/Pro183 polymorphism in the coding sequence of domain 3. Vet Immunol Immunopathol. 2006;13(3–4):305–12. 10.1016/j.vetimm.2006.05.015 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref011] 11.Eguchi-Ogawa T, Matsubara T, Toki D, Okumura N, Ando A, Kitagawa H, et al. Distribution of the CD4 Alleles in Sus scrofa Demonstrates the Genetic Profiles of Western Breeds and Miniature Pigs. Anim Biotechnol. 2018;29(3):227–33. Epub 2017/10/17. 10.1080/10495398.2017.1367691 . [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref012] 12.Wang L, Fan J, Yu M, Zheng S, Zhao Y. Association of goat (Capra hircus) CD4 gene exon 6 polymorphisms with ability of sperm internalizing exogenous DNA. Mol Biol Rep. 2011;38(3):1621–8. 10.1007/s11033-010-0272-2 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref013] 13.Lu Y, Wu J, Qin X, Xie L, Ma L, Huang X, et al. The CD4 C868T Polymorphism and Its Correlation with HIV-1 Infection in a Chinese Population. AIDS Res Hum Retroviruses. 2015;31(5):525–30. Epub 2015/01/23. 10.1089/AID.2014.0303 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref014] 14.Oyugi J, Vouriot F, Judie Alimonti J, Wayne S, Luo M, Land A, et al. A Common CD4 Gene Variant Is Associated with an Increased Risk of HIV-1 Infection in Kenyan Female Commercial Sex Workers. The Journal of Infectious Diseases. 2009;199(9):1327–34. 10.1086/597616 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref015] 15.Kristiansen O, Karlsen A, Larsen Z, Johannesen J, Pociot F, Mandrup‐Poulsen T. Identification of a Type 1 Diabetes‐Associated CD4 Promoter Haplotype with High Constitutive Activity Scand J Immunol. 2004;59(6):582–91. 10.1111/j.1365-3083.2004.01444.x [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref016] 16.Tr Sundt, LeGuern C, Germana S, Smith C, Nakajima K, Lunney J, et al. Characterization of a polymorphism of CD4 in miniature swine. J Immunol. 1992;148(10):3195–201. [PubMed] [Google Scholar]

[pone.0242572.ref017] 17.Matsubara T, Nishii N, Takashima S, Takasu M, Imaeda N, Aiki-Oshimo K, et al. Identification of a CD4 variant in Microminipigs not detectable with available anti-CD4 monoclonal antibodies. Vet Immunol Immunopathol. 2015;168(3–4):176–83. 10.1016/j.vetimm.2015.09.008 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref018] 18.Matsubara T, Nishii N, Takashima S, Takasu M, Imaeda N, Aiki-Oshimo K, et al. Identification and characterization of two CD4 alleles in Microminipigs. BMC Veterinary Research. 2016;12(1):222 10.1186/s12917-016-0856-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref019] 19.Kaneko N, Itoh K, Sugiyama A, Izumi Y. Microminipig, a non-rodent experimental animal optimized for life science research: preface. J Pharmacol Sci. 2011;115(2):112–4. [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref020] 20.Takasu M, Tsuji E, Imaeda N, Matsubara T, Maeda M, Ito Y, et al. Body and major organ sizes of young mature microminipigs determined by computed tomography. Lab Anim. 2015;49(1):65–70. 10.1177/0023677214557169 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref021] 21.Ando A, Imaeda N, Ohshima S, Miyamoto A, Kaneko N, Takasu M, et al. Characterization of swine leucocyte antigen alleles and haplotypes on a novel miniature pig line, Microminipig. Animal Genetics. 2014;45:791–8. 10.1111/age.12199 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref022] 22.Kametani Y, Ohshima S, Kita Y, Shimada S, Kamiguchi H, Shiina T, et al. Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells. Vet Immunol Immunopathol. 2012;148:252–9. 10.1016/j.vetimm.2012.05.005 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref023] 23.Kametani Y, Ohshima S, Miyamoto A, Shigenari A, Takasu M, Imaeda N, et al. Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs. PLoS One. 2016;11(10):e0164995 10.1371/journal.pone.0164995 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref024] 24.Ho C, Lunney J, Franzo-Romain M, Martens G, Lee Y, Lee J, et al. Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations. Animal Genetics. 2009;40:468–78. 10.1111/j.1365-2052.2009.01860.x [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref025] 25.Ho C, Lunney J, Lee J, Franzo-Romain M, Martens G, Rowland R, et al. Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations. Anim Genet. 2010;41(4):428–32. 10.1111/j.1365-2052.2010.02019.x [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref026] 26.Mizutani A, Kikkawa E, Matsuno A, Shigenari A, Okinaga H, Murakami M, et al. Modified S/MAR episomal vectors for stably expressing fluorescent-protein tagged transgenes with small cell-to-cell fluctuations. Anal Biochem. 2013;443(1):113–6. 10.1016/j.ab.2013.08.009 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref027] 27.Kremers G-J, Goedhart J, van Munster E, W J Gadella Jr T. Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry. 2006;45:6540–80. [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref028] 28.Ohshima S, Mori S, Shigenari A, Miyamoto A, Takasu M, Imaeda N, et al. Differentiation ability of multipotent hematopoietic stem/progenitor cells detected by a porcine specific anti-CD117 monoclonal antibody. Biosci Trends. 2014;8:308–15. 10.5582/bst.2014.01084 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref029] 29.Levitt M. Accurate modeling of protein conformation by automatic segment matching. J Mol Biol. 1992;226(2):507–33. 10.1016/0022-2836(92)90964-l [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref030] 30.Bernstein F, Koetzle T, Williams G, Meyer E Jr., Brice M, Rodgers J, et al. The Protein Data Bank: A Computer-based Archival File For Macromolecular Structures. J Mol Biol. 1977;112:535 10.1016/s0022-2836(77)80200-3 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref031] 31.Lamarre D, Ashkenazi A, Fleury S, Smith D, Sekaly R, Capon D. The MHC-binding and gp120-binding functions of CD4 are separable. Science. 1989;245(4919):743–6. 10.1126/science.2549633 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref032] 32.Pancera M, Majeed S, Ban Y, Chen L, Huang C, Kong L, et al. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility. Proc Natl Acad Sci U S A. 2010;107(3):1166–71. 10.1073/pnas.0911004107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref033] 33.Kwong P, Ryu S, Hendrickson W, Axel R, Sweet R, Folena-Wasserman G, et al. Molecular characteristics of recombinant human CD4 as deduced from polymorphic crystals. Proc Natl Acad Sci U S A. 1990;87(16):6423–7. 10.1073/pnas.87.16.6423 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref034] 34.Zuckermann F, Husmann R. Functional and phenotypic analysis of porcine peripheral blood CD4/CD8 double-positive T cells. Immunology. 1996;87:500–12. [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref035] 35.Tuffs S, Haeryfar S, McCormick J. Manipulation of Innate and Adaptive Immunity by Staphylococcal Superantigens. Pathogens. 2018;7(2):53 10.3390/pathogens7020053 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref036] 36.Andrianov A. Spatial structure model of the CD4 receptor-binding site of the HIV envelope protein gp120. J Biomol Struct Dyn. 1998;15:1101–13. 10.1080/07391102.1998.10509004 [DOI] [PubMed] [Google Scholar]

[pone.0242572.ref037] 37.Singh M, Singh P, Gaudray G, Musumeci L, Thielen C, Vaira D, et al. An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγnull mice allows HIV replication and development of anti-HIV immune responses. PLoS ONE. 2012;7(6):e38491 10.1371/journal.pone.0038491 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref038] 38.Gawron M, Duval M, Carbone C, Jaiswal S, Wallace A, Martin Jr, et al. Human Anti-HIV-1 gp120 Monoclonal Antibodies with Neutralizing Activity Cloned from Humanized Mice Infected with HIV-1. J Immunol 2019;202(3):799–804. 10.4049/jimmunol.1801085 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0242572.ref039] 39.Burstein R, Henry NJ, Collison ML, Marczak LB, Sligar A, Watson S, et al. Mapping 123 million neonatal, infant and child deaths between 2000 and 2017. Nature. 2019;574(7778):353–8. Epub 2019/10/18. 10.1038/s41586-019-1545-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs

Shino Ohshima

Tatsuya Matsubara

Asuka Miyamoto

Atsuko Shigenari

Noriaki Imaeda

Masaki Takasu

Masafumi Tanaka

Takashi Shiina

Shingo Suzuki

Noriaki Hirayama

Hitoshi Kitagawa

Jerzy K Kulski

Asako Ando

Yoshie Kametani

Roles

Abstract

Introduction

Materials and methods

Animals and tissues

SLA genotyping

CD4 genotyping

Transfection

Preparation of the peripheral blood mononuclear cells (PBMCs)

Monoclonal antibody preparation

Flow cytometry

Primary sequence and structure analysis of mAb

Model building of the 3D structure of CD4 molecules

Fig 1. Microminipig CD4 protein structure.

Histochemical analysis

Stimulation of PBMCs

Statistical analysis

Results

Simulation of swine CD4 isotype epitope

Preparation of mAbs recognizing swine CD4 isotypes

Fig 2. Characterization of anti-swine CD4.B mAb (b1D7).

Fig 3. Characterization of anti-swine CD4.AB mAb (x1E10).

Comparison of CD4 and CD8 expression in activation-induced T cells with different CD4 isotypes

Fig 4. Surface expression of CD4 and CD8 molecules on the swine T cells.

Fig 5. CD4 expression on DP cells is enhanced by TSST-1 stimulation.

Fig 6. Activation of CD4 T cells by TSST-1 is inhibited by an anti-CD4 mAb x1E10.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Karen L Elkins

Roles

Author response to Decision Letter 0

Decision Letter 1

Karen L Elkins

Roles

Author response to Decision Letter 1

Decision Letter 2

Karen L Elkins

Roles

Acceptance letter

Karen L Elkins

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases