Hyper‐reactive cloned mice generated by direct nuclear transfer of antigen‐specific CD4⁺ T cells

Abstract

T‐cell receptor (TCR)‐transgenic mice have been employed for evaluating antigen‐response mechanisms, but their non‐endogenous TCR might induce immune response differently than the physiologically expressed TCR. Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen‐specific CD4⁺ T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre‐rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic‐type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter‐regulated antigen‐specific TCR are a unique animal model with allergic predisposition for investigating CD4⁺ T‐cell‐mediated pathogenesis and cellular commitment in immune diseases.

Introduction

CD4⁺ T cells drive various immune cascades and are involved in the development of various diseases. The generation of several T‐cell receptor (TCR)‐transgenic (Tg) mouse lines 1, 2 has improved existing knowledge about CD4⁺ T‐cell functions in the immune system and diseases. However, TCR expression in TCR‐Tg T cells is regulated by extrinsic promoters/enhancers, and the TCR expression machinery is integrated into non‐homologous genomic regions; therefore, the expression levels and kinetics of Tg‐TCR differ from those of intrinsic TCR that is normally expressed by T cells. It is unknown whether the antigen‐induced responses of TCR‐Tg CD4⁺ T cells exactly reflect those of physiologically generated antigen‐specific T cells.

Advances in somatic cell nuclear transfer (SCNT) techniques have enabled the generation of cloned mice from several T‐cell lineages. The first monoclonal mice derived from lymphoid T cells had rearranged TCRs 3 identified as MHC class I‐restricted 4. The same strategy was employed to generate cloned mice using freshly isolated effector CD8⁺ T cells specific for Toxoplasma gondii 5. A two‐step cloning procedure was followed in which embryonic stem (ES) cells were established from cloned blastocysts and injected into tetraploid blastocysts because the direct transfer of cloned blastocysts from lymphocytes to the uteri of recipient mice was unsuccessful. We previously found that the developmental potential of a characteristic T‐cell population, natural killer T (NKT) cells, was unexpectedly high and that live fetuses could be obtained, even using a single‐step nuclear transfer (NT) technique 6. More recent technical improvements, including the usage of histone deacetylase inhibitors to treat reconstructed embryos, have enabled cloning from peripheral blood lymphocytes by single‐step NT 7. However, cloned mice with predefined TCR specificities from MHC class II‐restricted CD4⁺ T cells have not been generated using single‐ or two‐step SCNT.

The preparation of antigen‐specific CD4⁺ T cells appropriate for SCNT is difficult. Donor cells should be at the G1/G0 phase to synchronize donor nuclei and recipient ooplasm. It is necessary to culture and grow T cells prepared from mice immunized with a specific antigen in vitro to obtain a sufficient quantity of CD4⁺ T cells expressing antigen‐reactive TCR. However, the cell cycles of CD4⁺ T cells progress normally, and only a small population is expected to be at G1/G0, which may decrease the developmental efficiency of cloned embryos.

We successfully generated cloned mice from antigen‐reactive CD4⁺ T cells prepared using an in vitro culture process by single‐step direct NT (Fig 1A). CD4⁺ T cells that were reactive to several antigens were prepared by serial stimulations and resting culture conditions to obtain live fetuses with a high birth rate. CD4⁺ T cells from these mice exhibited antigen reactivity that was dependent on endogenously pre‐rearranged TCRα (rTα) and TCRβ (rTβ). Both rearranged TCRs are required for antigen reactivity in vitro, whereas hyper‐reactivity to corresponding antigens was surprisingly observed in mice expressing either rearranged TCR alone, especially rTβ, in vivo.

Figure 1. Antigen reactivity and detection of genes and epitopes of rearranged TCR in cloned mice.

1. Schematic representation of the strategy.
2. Proliferative response of CD4⁺ T cells of Df#1 and Df#2 cultured with APC plus Der f.
3. Proliferative response of white blood cells of OVA#6 cultured with APC plus whole OVA protein or OVA326‐339 peptide.
4. Proliferative response of white blood cells of Dp#7, Dp#8, Dp#9, Dp#10, and Dp#11 cultured with APC plus Der p1.
5. Proliferative response of white blood cells cultured with APC plus Der f and the rTα(V6D‐4) and rTβ(V2) genotypes in Df#1‐N1 mice.
6. Proliferative response of CD4⁺ T cells of the Df#1 and Df#2 cultured with APC plus Der f, Der f‐b, Der p, and Der p‐b.
7. Proliferative response of CD4⁺ T cells of the Dp#7 cultured with APC plus Der p1, different parts of Der p1‐GST fusion proteins, and Der p1‐derived synthetic peptides.
8. Cytokine production in the culture supernatants of splenic CD4⁺ T cells of Der f‐stimulated Df#1 and Df#2, OVA peptide‐stimulated OVA#6, and Der p1‐stimulated Dp#7.

Data information: Data are expressed as the mean or mean ± SEM (n = 2–4). Statistical significance was evaluated in (B, E, and G) using one‐way ANOVA followed by Dunnett's multiple comparison tests. *P < 0.05, **P < 0.01, compared with antigen‐less control (B and G) or wTα/wTβ‐expressing mice (#2, E). Some data in (C, D, and F) are the mean of duplicate cultures.

Results and Discussion

Antigen‐reactive CD4⁺ T‐cell‐derived cloned mice may provide a basis for understanding physiologically expressed TCR‐derived immune responses and diseases. Therefore, we performed single‐step direct NT to generate a new mouse model using antigen‐specific CD4⁺ T cells. To obtain live fetuses, we prepared donor CD4⁺ T cells under a combination of stimulating and resting culture conditions. Because donor cells from mice of mixed backgrounds are much more efficient than those from inbred mice for SCNT 8, 9 and MHC class I and II haplotypes in BALB/c are identical to those in DBA2 mice, CD4⁺ T cells from antigen‐immunized male (BALB/c × DBA/2)F1 (CDF1) mice were cultured with the immunized antigens and irradiated CD4⁻ splenocytes as antigen‐presenting cells (APCs). After promotion of antigen‐recognized cell growth by addition of IL‐2, IL‐2 was removed to terminate the cell cycle (Fig EV1A). Over 90% of CD4⁺ T cells were arrested in the G1/G0 phase; without IL‐2 removal, only 72% were in G1/G0 (Fig EV1B).

Figure EV1. Preparation of antigen‐specific CD4⁺ T cells.

1. Experimental protocol for preparing antigen‐specific resting CD4⁺ T cells from Der p1‐immunized mice.
2. G1/G0, G2/M, and S‐phase populations determined by flow cytometry with 7‐AAD and Edu staining (lower panels) in CD4⁺ T cells with or without resting treatment by IL‐2 removal (upper panels).

Data information: Representative data for three independent experiments are shown.

From these expanded and arrested cells, a substantial number of cloned fetuses were obtained by single‐step direct NT with a relatively high efficiency (3.74 ± 1.66%) (Table 1 and Fig 1A). This efficiency was comparable to the efficiencies using other somatic cells 8. Among 18 fetuses, 14 were born alive, 11 of which were healthy. Among them, eight mice exhibited antigen reactivity. CD4⁺ T cells of two mice, Df#1 and Df#2, generated from crude Dermatophagoides farinae (Der f)‐immunized and ‐stimulated CD4⁺ T cells, proliferated in the presence of Der f plus APC (Fig 1B), whereas three cloned mice showed no reactivity for their antigen. White blood cells of cloned mice derived from ovalbumin (OVA)326‐339 peptide‐ (Fig 1C) and D. pteronyssinus (Der p1)‐reactive (Fig 1D) CD4⁺ T cells proliferated in response to the corresponding antigens. These were the first cloned mice originating from MHC class II‐restricted CD4⁺ T cells. By employing a two‐step NT procedure, several groups have established cloned mice from MHC class I‐restricted CD8⁺ T cells in 6 weeks from the isolation of lymphocytes to the birth of chimeric mice 3, 5. Our protocol with single‐step direct NT shortened the period necessary to obtain various cloned mice exhibiting distinct antigen specificity.

Table 1.

Development of cloned embryos from antigen‐specific CD4⁺ T cells

Trial	Antigen	Cultured embryos	Embryos after 24‐h culture				Transferred embryos (TE)	Implantation (%/TE)	Fetus (%/TE)	Weaned mice (%/TE)
			Fragmented	Non‐fragmented	1‐cell	2‐cell (%/non‐fragmented)
1	Der f	100	50	50	2	48 (96.0)	48	27 (56.3)	1 (2.1)	1 (2.1)
2	Der f	241	49	192	34	158 (82.3)	158	66 (41.8)	4 (2.5)	3 (1.9)
3	Der f1 peptide	140	26	114	9	105 (92.1)	91	31 (34.1)	0 (0.0)	0 (0.0)
4	Der f1 peptide	192	43	149	10	139 (93.3)	138	54 (39.1)	1 (0.7)	0 (0.0)
5	Der f	91	55	36	1	35 (97.2)	35	23 (65.7)	1 (2.9)	1 (2.9)
6	Der f	35	20	15	1	14 (93.3)	14	8 (57.1)	2 (14.3)	2 (14.3)
7	OVA peptide	153	37	116	18	98 (84.5)	98	56 (57.1)	1 (1.0)	1 (1.0)
8	Der p1	148	13	135	10	125 (92.6)	125	66 (52.8)	8 (6.4)	6 (4.8)
Total (mean ± SEM)		1,100	293	807	85	722 (91.4 ± 1.9)	707	331 (50.5 ± 3.85)	18 (3.74 ± 1.66)	14 (3.38 ± 1.66)

Nuclear transfer was performed using antigen‐reactive CD4⁺ T cells of CDF1 mice immunized with several antigens. Numbers of cultured and transferred embryos, implantations, obtained fetuses, and weaned mice and total numbers and/or means ± SEM of 8 trials are shown.

To evaluate the transmission of antigen reactivity and the causal TCR, the rTα and rTβ genes in antigen‐reactive cloned mice were characterized. Total RNA was extracted from antigen‐stimulated and grown CD4⁺ T cells of cloned mice, and rTα and rTβ were screened by RT–PCR with primer pairs harboring a series of TCRα/β V‐ and C‐regions (Appendix Tables S1 and S2). One or two positive bands obtained for rTα and rTβ of each cloned mice were subcloned into the cloning vector (Fig EV2A). The sequences of inserted PCR products were determined and analyzed using the international ImMunoGeneTics Information System^® (http://www.imgt.org), and the rTα/β pairs in the eight antigen‐reactive clones were identified (Appendix Table S3). The rTα/β structures in Dp#7, Dp#9, and Dp#11 were identical, suggesting that they developed from single‐cell‐originated sibling cells.

Figure EV2. Cloning and characterization of rearranged TCR genes in cloned mice.

1. mRNA for rTα and rTβ in peripheral white blood cells of cloned mice cultured with APCs plus the corresponding antigen for 5 days, amplified by RT–PCR with primer pairs harboring a series of TCRα/β V‐ and C‐regions and subjected to agarose gel electrophoresis.
2. Proliferative response of splenic CD4⁺ T cells of the Df#2 line with rTα(V10) and/or rTβ(V5) cultured with APCs plus Der f.
3. The expression of TCRVβ13‐1 on splenocytes of Df#1, OVA#6, Dp#7, and Dp#8 mice analyzed by flow cytometry upon staining with anti‐TCRVβ13‐1 plus anti‐CD4 or anti‐CD8.

Data information: Representative data from Df#1 mice are shown in (A). The number of V‐regions for each TCR is indicated. M, marker. Representative data for two independent experiments are shown in (B and C). Data in (B) are expressed as the mean or mean ± SEM (n = 2–4).

All cloned mice survived normally in specific pathogen‐free conditions. All lines were successfully backcrossed to BALB/c mice, except Dp#8 and Dp#10, which exhibited infertility caused by azoospermia. In contrast to previously generated cloned mice from T cells 10, frequent lymphomagenesis was not observed in our cloned mouse lines.

Primer pairs to detect genomic rTα and rTβ in each cloned mouse were synthesized (Appendix Table S4), and the rearranged TCR genes in each line were analyzed by PCR. rTα(V6D‐4) and rTβ(V2) were randomly transmitted to N1 mice of Df#1 backcrossed to BALB/c according to Mendel's law. Only mice expressing both rTβ and rTα displayed responsiveness to Der f (Fig 1E). The requirement of rTα/β for antigen reactivity was also confirmed in Df#2 (Fig EV2B). TCRVβ13‐1 expressed in OVA#6 and Dp#8 (Appendix Table S3) was detectable on CD4⁺ and CD8⁺ T cells by flow cytometry using a specific antibody (Ab) (Fig EV2C), suggesting that the rearranged TCR was expressed and functioned on the surface of cloned mouse T cells.

The antigen specificity of Df#1 and Df#2 was confirmed by the responsiveness to Der f‐body extract (Der f‐b) and lack of responsiveness to Der p‐derived antigens (Fig 1F). By screening with Der p1‐derived glutathione S‐transferase (GST)‐fusion proteins and synthetic peptides, amino acids 189–208 of Der p1 were identified as the TCR epitope on Dp#7 CD4⁺ T cells (Fig 1G).

The phenotypes of the Df#1 and Df#2 lines were analyzed with respect to rTα/β. Body, spleen, and thymus weights were not affected by rTα and/or rTβ expression (Table 2 and Appendix Table S5). Several common and specific features were observed in the splenic and thymic cell populations (Table 2, Fig EV3 and Appendix Figs S1 and S2). The proportion of splenic CD3⁺ cells increased in both Df#1 and Df#2 with rTα/β, although the increase was only significant for Df#2 (Dunnett's method, P = 0.00012). This is consistent with previous results obtained using transnuclear mice from antigen‐specific 5 and presumptive 4 CD8⁺ T cells. Although increased CD4⁺ T cells and decreased CD8⁺ T cells were seen in rTα/β‐expressing Df#1, the pattern of significantly reduced CD4⁺ and CD8⁺ T cells in rTα/β‐expressing Df#2 was different from the pattern observed in CD8‐transnuclear mice, in which the proportion of peripheral CD8⁺ and CD4⁺ cells increased and decreased, respectively 5. These findings indicate that the peripheral DN T‐cell population was enlarged in rTα/β‐expressing Df#2 but not in Df#1. A comparative analysis between Df#1 and Df#2 would provide insight into the developmental mechanisms and pathogenic role of peripheral DN cells.

Table 2.

Profiles of Df#1 and Df#2 cloned mouse lines

	Df#1				Df#2
	wTα/wTβ	wTα/rTβ(V2)	rTα(V6D‐4)/wTβ	rTα(V6D‐4)/ rTβ(V2)	wTα/wTβ	wTα/rTβ(V5)	rTα(V10)/wTβ	rTα(V10)/rTβ(V5)
Body weight (g)	31.5 ± 2.4	26.6 ± 2.0	29.9 ± 2.1	31.8 ± 2.0	29.9 ± 1.7	28.7 ± 1.6	29.3 ± 1.9	28.0 ± 0.8
Spleen weight (mg)	143 ± 14	146 ± 22	117 ± 3	112 ± 9	108 ± 3	138 ± 11	116 ± 10	108 ± 7
Thymus weight (mg)	14.2 ± 3.2	13.8 ± 2.3	11.6 ± 1.0	12.2 ± 1.9	12.4 ± 1.8	12.1 ± 2.3	11.9 ± 1.4	10.4 ± 1.4
Spleen (%)
CD3+	20.5 ± 2.5	25.3 ± 4.0	20.1 ± 2.3	28.7 ± 1.5	26.1 ± 0.7	24.7 ± 1.0	21.5 ± 1.6	34.9 ± 0.9***
CD4+/CD3+	57.7 ± 4.4	64.8 ± 4.4	49.0 ± 4.0	74.5 ± 2.5**	69.3 ± 1.9	75.1 ± 1.1	63.9 ± 3.5	29.1 ± 1.4***
CD8+/CD3+	35.2 ± 4.6	30.1 ± 4.1	31.2 ± 3.1	20.4 ± 2.5*	26.3 ± 1.7	21.9 ± 1.1	23.1 ± 2.1	1.8 ± 0.4***
CD4−CD8−/CD3+	6.1 ± 0.7	4.3 ± 1.0	19.0 ± 1.5***	4.4 ± 0.4	3.4 ± 0.5	2.5 ± 0.2	12.0 ± 2.0***	69.0 ± 1.6***
CD19+	58.4 ± 2.4	48.5 ± 5.6	59.7 ± 4.5	56.3 ± 1.9	59.0 ± 1.1	55.5 ± 2.1	60.1 ± 2.1	50.0 ± 1.1**
CD3+γδTCR+	0.52 ± 0.12	0.12 ± 0.02*	1.03 ± 0.14**	0.11 ± 0.02**	0.54 ± 0.09	0.12 ± 0.04*	0.90 ± 0.15*	0.11 ± 0.03*
TCRβ+α‐GarCer/CD1d+	0.520 ± 0.094	0.072 ± 0.024***	0.218 ± 0.049**	0.044 ± 0.009***	0.455 ± 0.076	0.057 ± 0.012***	0.317 ± 0.091	0.065 ± 0.021***
CD4+CD62L+CD25−	32.7 ± 4.7	52.9 ± 4.0*	37.1 ± 0.9	52.9 ± 4.3**	41.9 ± 5.8	50.1 ± 6.6	42.1 ± 3.2	50.4 ± 4.0
CD4+Foxp3+CD25+	8.4 ± 2.1	5.6 ± 0.6	9.7 ± 1.5	4.7 ± 0.5	10.1 ± 1.2	8.2 ± 0.6	11.9 ± 0.8	7.9 ± 0.6
CD11c+Siglec‐F−	2.50 ± 0.37	2.51 ± 0.16	2.46 ± 0.16	3.01± 0.35	2.69 ± 0.10	2.83 ± 0.31	2.76 ± 0.23	2.61 ± 0.23
CD11c−Siglec‐F+	0.81 ± 0.25	1.07 ± 0.57	0.61 ± 0.08	0.51 ± 0.08	0.61 ± 0.02	0.93 ± 0.19	0.84 ± 0.13	0.63 ± 0.18
DX5+CD123−	4.75 ± 0.89	4.96 ± 0.99	4.64 ± 0.43	4.41 ± 0.47	4.10 ± 0.22	5.67 ± 1.25	4.50 ± 0.30	4.12 ± 0.33
DX5+CD123+	0.104 ± 0.020	0.114 ± 0.024	0.090 ± 0.011	0.090 ± 0.016	0.256 ± 0.129	0.354 ± 0.252	0.249 ± 0.115	0.193 ± 0.067
CD11bhighGr‐1high	1.98 ± 0.93	3.20 ± 1.62	1.50 ± 0.82	0.76 ± 0.21	0.76 ± 0.23	2.00 ± 1.10	1.02 ± 0.42	1.15 ± 0.42
CD11blowGr‐1−	3.54 ± 0.43	3.53 ± 0.70	3.57 ± 0.86	3.53 ± 0.38	3.44 ± 0.37	4.37 ± 0.34	3.41 ± 0.22	3.45 ± 0.31
Thymus (%)
CD4+	15.9 ± 6.0	20.7 ± 3.4	12.4 ± 2.7	25.4 ± 2.8	12.5 ± 1.6	16.9 ± 2.7	14.4 ± 1.2	20.2 ± 2.8
CD8+	4.1 ± 1.2	4.3 ± 0.5	7.1 ± 1.4	4.1 ± 0.6	3.7 ± 0.6	3.2 ± 0.2	11.9 ± 2.3*	8.4 ± 2.8
CD4+CD8+	71.5 ± 7.9	70.4 ± 3.7	54.1 ± 6.9	53.1 ± 5.1	75.1 ± 2.9	72.7 ± 2.0	48.8 ± 3.2**	29.8 ± 6.7***
Foxp3+CD25+CD4+	3.06 ± 0.46	2.22 ± 0.24	4.91 ± 0.79*	2.18 ± 0.24	4.92 ± 0.50	3.42 ± 0.97	5.86 ± 0.93	8.73 ± 1.78

Weights of the body, spleen, and thymus, and cellular populations in the spleen and thymus of the Df#1 line with rTα(V6D‐4) and/or rTβ(V2) and the Df#2 line with rTα(V10) and/or rTβ(V5) were analyzed. Data are expressed as the mean ± SEM (n = 3–7).

*P < 0.05, **P < 0.01, ***P < 0.001, compared with wTα/wTβ‐expressing mice of each line (Dunnett's method). Flow cytometry graphs are available in Appendix Figs S1 and S2.

Figure EV3. Role of rearranged TCRs in peripheral and thymic development of T cells.

The expression of CD3, CD4, CD8, CD19, γδTCR, α‐GarCer/CD1d‐reactive TCR, CD11c, Siglec‐F, Gr‐1, CD11b, DX5, CD123, CD62L, CD25, and Foxp3 in the spleen and/or thymus of the Df#1 line with rTα(V6D‐4) and/or rTβ(V2) and the Df#2 line with rTα(V10) and/or rTβ(V5) determined by flow cytometry. Representative data from 3 to 6 animals are shown.

Consistent with findings in TN mice from unidentified T cells 4, the proportion of γδ T cells (CD3⁺γδTCR⁺) decreased in wild‐type TCRα (wTα)/rTβ⁺ and rTα⁺/β⁺ mice of both Df#1 and Df#2. In contrast, the proportion of these cells increased in rTα⁺/wild‐type TCRβ (wTβ) mice. Similarly, an extensive reduction in the proportion of NKT cells (TCRβ⁺α‐GarCer/CD1d⁺) was observed in rTβ‐expressing Df#1 and Df#2. Considering the requirement of invariant TCRα(V14/J18) for development of CD1d‐restricted and α‐GarCer‐reactive NKT cells 11, nearly normal and reduced development of NKT cells was seen in rTα(V10/J42)‐expressing Df#2 and rTα(V6D‐4/J18)‐expressing Df#1, respectively, with the wTβ allele. V14 and J18 segments remained in the rTα(V10/J42) allele, but V14 was excluded from the rTα(V6D‐4/J18) allele 12. Therefore, invariant TCRα(V14/J18) could be expressed from both the rTα and wTα alleles in Df#2, but only from the wTα allele in Df#1. Consistent with the previous demonstration that TCRβ in NKT cells is mostly composed of Vβ8 or Vβ7 11 and with the dogma of allelic exclusion, especially in the TCRβ allele, very few NKT cells were detected in rTβ‐expressing Df#1 and Df#2.

Peripheral naïve CD4⁺ T cell (CD4⁺CD62L⁺CD25⁻) and Treg cell (CD4⁺Foxp3⁺CD25⁺) proportions tended to increase and decrease, respectively, in an rTβ‐dependent manner. Other cell types, such as dendritic cells (DC; CD11c⁺Siglec‐F⁻), eosinophils (CD11c⁻Siglec‐F⁺), natural killer (NK) cells (DX5⁺CD123⁻), basophils (DX5⁺CD123⁺), neutrophils (CD11b^highGr‐1^high), and monocytes/macrophages (CD11b^lowGr‐1⁻), were not affected by rearranged TCR expression.

The number of thymic CD4⁺ cells increased in Df#1 and Df#2 in an rTβ‐dependent manner, considering that the thymus weight was not affected by rearranged TCR expression (Table 2, Fig EV3, Appendix Table S5 and Appendix Figs S1 and S2). These results were consistent with reports showing increased numbers of CD8⁺ T cells in cloned mice derived from antigen‐specific 5 and presumptive 4 CD8⁺ T cells. In contrast to the dramatically reduced and unchanged proportions of peripheral CD8⁺ cells in rTα⁺/β⁺ and rTα⁺/wTβ mice, respectively, the percentage of thymic CD8⁺ T cells in Df#2 tended to increase in an rTα‐dependent manner, but were unchanged in rTα⁺/β⁺ Df#1. The percentage of double‐positive (DP) cells was reduced in rTα‐expressing Df#1 and significantly reduced in Df#2. In contrast to the expression of various rTα isoforms at DP stage in wild‐type mice, selective expression of rTα(V10) was induced at double‐negative (DN) stage and down‐regulated at DP stage in Df#2 (Fig EV4A). Although the mechanisms and physiological meaning of transient up‐/down‐regulation of rTα expression in the DN/DP stage remain to be elucidated, these findings suggest a dynamic transition of thymic T‐cell development; after rapidly progressing through the DP stage, most CD8‐preferentially maturated rTα⁺ T cells were probably eliminated by negative selection. In fact, although the peripheral CD8⁺ T‐cell population was dramatically reduced in rTα⁺/β⁺ Df#2 (Table 2, Fig EV3, Appendix Table S5 and Appendix Figs S1 and S2), the proportion of cells expressing TCRα isoform other than the germline transmitted rTα(V10) in CD8⁺ cells of these rTα(V10)/rTβ(V5) mice was equivalent to that of wTα/wTβ mice (Fig EV4B). Probably because the population of thymic DP cells expressing TCRα from the wild‐type allele in rTα/rTβ mice was much smaller than that in wTα/wTβ mice, and cells expressing rTα(V10) rapidly passed through the DP stage, expression of all rTα isoforms tested was not detectable in DP cells of rTα⁺/β⁺ Df#2 by RT–PCR (Fig EV4A).

Figure EV4. Expression of rearranged TCRα genes in thymus of cloned mice.

1. mRNA for rTα of sorted thymic CD4⁺CD8⁺ or CD4⁻CD8⁻ cells (left panels) amplified by RT–PCR with primer pairs harboring a series of TCRα V‐ and C‐regions and subjected to agarose gel electrophoresis (right panels).
2. The expression of TCRVα2 and Vα11 on peripheral CD8⁺ cells was analyzed by flow cytometry.

Data information: Representative data from 2–3 Df#2 mice with wTα/wTβ and rTα(V10)/rTβ(V5) are shown (A). The number of V‐regions for each TCR is indicated. M, marker. Data in (B) are expressed as the mean ± SEM (n = 5–6).

The distinct patterns of peripheral and thymic lymphocyte development between Df#1 and Df#2 suggest that at least two rearranged TCR sets exhibiting different antigen/MHC‐binding properties were obtained. The regulatory mechanisms of CD4⁺/CD8⁺ T‐cell fate are currently debated; instructive, stochastic, and kinetic models have been proposed 13. The instructive selection model, which suggests the termination of inappropriate coreceptor gene expression by quantitatively and/or qualitatively differential TCR‐MHC signals, is most consistent with the differential CD4/CD8 developmental patterns in cloned mice with distinct pre‐rearranged TCRs observed in this study and previous reports 4, 5.

Consistent with previous reports showing that T cells expressing different TCRs display distinct cytokine production patterns 14, cloned mouse CD4⁺ T cells showed differential cytokine‐producing activity (Fig 1H). Upon stimulation with Der f, Df#1 CD4⁺ T cells produced IL‐2, IFN‐γ, IL‐9, IL‐13, and IL‐17 and exhibited decreased IL‐4 and IL‐5 production, whereas, along with IL‐10, these cytokines were all highly produced by Df#2. OVA#6 CD4⁺ T cells produced excessive Th1 and Th17 cytokines in response to OVA peptide stimulation. IL‐2 and IL‐17 were highly produced in Der p1‐stimulated Dp#7 T cells.

Given the reactivity of their CD4⁺ cells against corresponding antigens, our cloned mice have potential uses for investigation of allergic diseases. We have developed airway inflammation models induced by Der f challenge to wild‐type mice without systemic immunization, although more than 10 challenges and month‐long durations were required to achieve inflammation in C57BL/6 mice in our previous investigation 15. The requirement of 5–7 weeks, with five antigen challenges per week, to induce significant airway inflammation has been reported 16. Consistently, in CDF1 and BALB/c mice, that is, the parental strains, at least 12 intranasal Der f provocations were required to induce weak bronchial hyper‐responsiveness (BHR) (Fig EV5A) and eosinophil accumulation (Fig EV5B). Using Df#1 expressing rTα(V6D‐4)/rTβ(V2) (Appendix Table S3), only four antigen challenges significantly induced BHR (Fig 2A). In contrast to the requirement for both rTα and rTβ for in vitro antigen reactivity (Figs 1E and EV2B), mice with either one of the rearranged TCR chains were hyper‐reactive in the development of BHR, compared to wTα/wTβ mice. Additionally, eosinophil, neutrophil, and lymphocyte infiltration into the lungs after four antigen challenges was observed in rTα⁺/rTβ⁺ mice and to a lesser extent in wTα/rTβ⁺ mice, followed by rTα⁺/wTβ mice (Fig 2B). Apparent inflammatory features with massive lymphocyte and eosinophil accumulation along with epithelial hyperplasia were also observed in the lungs of the rearranged TCR‐expressing Df#1 by Der f challenge, based on a histopathological examination (Fig 2C). Increases in lung IL‐4, IL‐5, and IL‐13 levels (Fig 2D) and serum IgE levels (Fig 2E) were observed in antigen‐challenged rTα⁺/β⁺ mice, suggesting that Th2‐preferential airway inflammation developed in Df#1. Because Df#1 exhibited weak Th2 cytokine production, it may also be interesting to examine Df#2, which had higher Th2 cytokine production, in in vivo studies.

Figure EV5. Antigen‐induced bronchial inflammation in normal mice and nasal inflammation in cloned mice and TCR‐Tg mice.

1. BHR at 72 h after the last antigen challenge in BALB/c and CDF1 mice challenged with 4–12 instillations of Der f at 3‐ to 4‐day intervals.
2. Inflammatory cells in BALF at 72 h after the last antigen challenge in BALB/c and CDF1 mice challenged with 4–12 instillations of Der f at 3‐ to 4‐day intervals.
3. NHR and inflammatory cells in NALF at 6 h after the 3^rd and 5^th antigen challenge in OVA#6 (rTα(V4‐4)/rTβ(V13‐1)), DO11.10, and BALB/c mice transferred with CD4⁺ T cells of OVA#6 (rTα(V4‐4)/rTβ(V13‐1)) challenged with daily instillations of OVA and saline.

Data information: Data are expressed as the mean ± SEM (n = 2–4). Statistical significance was evaluated using one‐way ANOVA followed by Dunnett's multiple comparison tests (C). *P < 0.05, compared with antigen‐challenged DO11.10 mice.

Figure 2. Antigen‐induced bronchial and nasal inflammation in cloned mice.

1. BHR in Df#1 with rTα(V6D‐4) and/or rTβ(V2) 72 h after 4 instillations of Der f or saline.
2. The number of BALF eosinophils, neutrophils, and lymphocytes in the rTα(V6D‐4) and/or rTβ(V2) Df#1.
3. Representative image of the lungs in the rTα(V6D‐4) and/or rTβ(V2) Df#1 after staining with hematoxylin and eosin (H&E) and periodic acid‐Schiff (PAS) for serial sections (several sections were excluded in some cases). Scale bar, 50 μm.
4. Concentration of BALF cytokines in the rTα(V6D‐4) and/or rTβ(V2) Df#1.
5. Concentration of serum IgE in the rTα(V6D‐4) and/or rTβ(V2) Df#1.
6. NHR in OVA#6 with rTα(V4‐4) and/or rTβ(V13‐1) evaluated as histamine‐evoked sneezing 6 h after 5 instillations of OVA or saline.
7. The number of eosinophils, neutrophils, and lymphocytes in the NAL fluid (NALF) of OVA#6 with rTα(V4‐4) and/or rTβ(V13‐1).

Data information: Data are expressed as the mean ± SEM (n = 3–8). Statistical significance was evaluated in (A, B, D–G) using one‐way ANOVA followed by Dunnett's multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared with antigen‐challenged wTα/wTβ‐expressing mice.

The development of nasal allergic responses was evaluated using another line, OVA#6. We have recently shown that allergic rhinitis‐like inflammatory responses with nasal hyper‐responsiveness (NHR) develop after repeated antigen challenges in immunized mice in a CD4⁺ T‐cell‐dependent manner 17. In OVA#6 expressing rTα(V4‐4)/rTβ(V13‐1) (Appendix Table S3), only five daily intranasal administrations of OVA, even without systemic immunization, resulted in significant augmentation of the sneezing response evoked by histamine (Dunnett's method, P = 0.00975, Fig 2F), suggesting that NHR developed in these mice. Equivalent and weaker NHR also developed in wTα/rTβ‐ and rTα/wTβ‐expressing mice, respectively. Almost in parallel, we observed enhanced nasal infiltration of eosinophils, neutrophils, and lymphocytes in rTα⁺/rTβ⁺ and wTα/rTβ⁺ mice and weaker infiltration in rTα⁺/wTβ mice (Fig 2G). Similar but slightly weaker nasal inflammatory responses were induced in OVA‐reactive TCR‐Tg DO11.10 mice, whereas stronger responses were evoked in BALB/c mice by transferring 2 × 10⁷/head CD4⁺ T cells of rTα(V4‐4)/rTβ(V13‐1)‐expressing OVA#6 (Fig EV5C).

Cloned mice that develop substantial allergic lung and nasal inflammation within 1–2 weeks with only 4–5 antigen challenges will be beneficial for investigating allergic diseases. Furthermore, mice expressing either one of the rearranged TCR chains, especially rTβ, displayed hyper‐reactivity to antigens in vivo, in contrast to in vitro reactivity. Zehn and Bevan demonstrated rTβ‐dependent antigen hyper‐reactivity of CD8⁺ T cells in a central and peripheral tolerance detection system employing OT‐I T‐cell‐derived TCRβ‐Tg mice 18. In addition to rTβ, we further elucidated the contribution of rTα to antigen hyper‐reactivity in MHC class II‐restricted CD4⁺ T cells. As a result of the pre‐optimization of either one of the TCR chains, antigen‐reactive TCR heterodimers are probably constructed more efficiently than those occasionally made from randomly rearranged TCRs for wTα/wTβ alleles. Additionally, in contrast to mice expressing both rTα/βs, multiple TCR pairs corresponding to various antigens/epitopes could be configured. The repeated antigen provocation steps might propagate such antigen‐reactive, “half‐optimized” T cells in vivo. Mice expressing either rTα or rTβ derived from our cloned mice are valuable new tools to investigate the pathogenesis of allergic and other immunological diseases.

Numerous studies using TCR‐Tg mice have made extensive progress in elucidating immune mechanisms. However, T‐cell activation and differentiation processes are affected by the affinity and avidity of TCR with antigens/MHC. Cloned mice generated by single‐step direct NT from CD4⁺ T cells and expressing endogenously regulated TCR corresponding to various antigens could be used to re‐evaluate current immunological theories established via TCR‐Tg mouse studies.

Materials and Methods

Animals

Eight‐ to sixteen‐week‐old male CDF1 mice (Charles River Japan, Kanagawa, Japan) were used to prepare donor CD4⁺ T cells. Eight‐ to ten‐week‐old (C57BL/6 × DBA/2)F1 (Japan SLC, Shizuoka, Japan) and ICR (CLEA Japan, Tokyo, Japan) female mice were used to collect recipient oocytes and as embryo transfer recipients, respectively. The mice were maintained under specific pathogen‐free conditions. The experimental protocols were approved by the Animal Use and Care Committee of Tokyo Metropolitan Institute of Medical Science (No. 12‐36, 13057, 14027, and 15035) and that of RIKEN Tsukuba Institute (No. 15‐005).

Preparation of antigen‐reactive CD4⁺ T cells

Mice were immunized with 20 μg of Der f (Cosmo Bio, Tokyo, Japan), 10 μg of Der p1 (Indoor Biotechnologies, Charlottesville, VA, USA), a mixture of 100 nmol synthetic 18–20‐amino acid peptides derived from Der f1 (Der f1 peptide), and 100 nmol synthetic OVA326‐339 peptide emulsified with 100 μl of complete Freund's adjuvant (Nacalai Tesque, Kyoto, Japan) by tail base injection. Seven to 10 days later, 10⁵ CD4⁺ T cells prepared from inguinal lymph nodes by positive selection using CD4 microbeads and a magnetic cell sorting system (Miltenyi, Bergisch Gladbach, Germany) were cultured with 10 μg/ml or 1 μM corresponding antigen plus 4 × 10⁵ X‐ray (4,000 rad)‐irradiated CD4⁻ splenocytes as APC in 100 μl of Dulbecco's modified Eagle's medium (DMEM, Sigma‐Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum. Four days later, 50 μl of 20 U/ml recombinant IL‐2 (Shionogi, Osaka, Japan) was added. After 3 days, cells were recovered, washed two times, and then cultured again without IL‐2. After another 3–4 days, CD4⁺ T cells were purified and subjected to NT. The cell cycle of the resulting CD4⁺ T cells was also investigated by flow cytometry using Click‐iT Edu Flow Cytometry Assay Kits (Thermo Fisher Scientific, Waltham, MA, USA) and an anti‐CD4‐APCeFluor780 Ab (eBioscience, San Diego, CA, USA).

Nuclear and embryo transfer

Nuclear transfer was performed as described previously 19. Briefly, oocytes were transferred to a droplet of HEPES‐buffered potassium‐enriched simplex optimized medium (KSOM) containing 7.5 μg/ml cytochalasin B (CB) in a micromanipulation chamber, and the zona pellucida was perforated by applying several piezo pulses with the tip of an enucleation pipette. The metaphase II chromosome spindle complex was removed by drawing it into the pipette with a small amount of accompanying ooplasm. Before NT, the donor nucleus was collected by drawing it in and out of an injection pipette until the plasma membrane was disrupted. The zona pellucida and the membrane of the oocyte were perforated with a few piezo pulses, and then, the donor nucleus was injected into the ooplasm. The reconstructed oocytes were transferred into a droplet of KSOM and incubated at 37.5°C under 5% CO₂ in humidified air for 1–3 h. The oocytes were activated by incubation for 1 h in Ca²⁺‐free KSOM containing 2.5 mM Sr²⁺, 10 μM latrunculin A (LatA, Sigma‐Aldrich), and 50 nM trichostatin A (TSA, Sigma‐Aldrich). After activation, they were cultured in KSOM containing 10 μM LatA and 50 nM TSA for 7 h. Then, SCNT‐derived embryos were transferred to KSOM and incubated at 37°C under 5% CO₂ in humidified air. After 24 h in culture, the SCNT‐derived embryos that developed to the 2‐cell stage were transferred into the oviducts of pseudopregnant ICR female mice, which had been mated with a vasectomized ICR male mouse, at 0.5 days postcoitum. All recipient females were euthanized at 19.5 days postcoitum and examined for the presence of fetuses. Cloned mice were backcrossed to BALB/c mice, and the randomly selected N1‐N7 offspring were subjected to in vitro and in vivo experiments. The cloned mice have been deposited and are available at the RIKEN BioResource Center (RIKEN BRC; Tsukuba, Japan, http://mus.brc.riken.jp/en/).

Antigen‐reactivity assay

CD4⁺ cells from the peripheral blood or spleen, or peripheral white blood cells (10⁶/ml), were cultured with 10 μg/ml Der f, Der f‐b (Institute of Tokyo Environmental Allergy (ITEA), Tokyo, Japan), crude Der p (ITEA), Der p‐body extract (Der p‐b, ITEA), Der p1, or OVA (Sigma‐Aldrich), or 1 μM synthetic OVA326‐339 peptide plus APC from BALB/c mice (4 × 10⁶/ml). After 4–5 days, the proliferative response and cytokine production in the culture supernatant were analyzed by using WST‐1 cell proliferation reagent (Sigma‐Aldrich) and BD Cytometric Bead Array (BD Bioscience, San Jose, CA, USA), respectively, according to the manufacturers’ protocols. Data for the proliferative response are presented as the mean or mean ± SEM of optical density values measured at 450 nm (OD₄₅₀), from which the values of background wells were subtracted.

In some experiments, glutathione S‐transferase (GST)‐fusion proteins (1–5 μM) of various Der p1 fragments were prepared for CD4⁺ T‐cell stimulation. The FLAG‐tag (MDYKDDDK)‐coding sequence was subcloned into the XhoI site of the pGEX‐4T expression vector (GE Healthcare, Little Chalfont, UK), in which the GST‐coding sequence was located at the N‐terminus. The PCR fragments of cDNA encoding several parts of Der p1 (Acc. No. AAB60215) were subcloned in‐frame into the EcoRI‐SalI site in the resulting pGEX‐4T‐FLAG. The GST‐Der p1‐FLAG fusion protein was expressed in Escherichia coli strain BL21. Briefly, the overnight culture was diluted 1/20 and grown until OD₆₆₀ reached 0.6–0.8. Production of the fusion protein was induced by adding isopropyl β‐d‐thiogalactoside to the growth culture to 0.1 mM, followed by incubation for 3 h at 37°C with vigorous shaking. The cell pellet was collected by centrifugation, suspended in sonication buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride, and sonicated. After addition of Nonidet P‐40 to reach a concentration of 1%, cell debris was removed by centrifugation, and the supernatants were incubated with anti‐FLAG (M2) agarose beads (Sigma‐Aldrich) for 2 h at 4°C. The resulting beads were packed in an open column, washed extensively with wash buffer containing 50 mM Tris (pH 7.5) and 150 mM NaCl, and eluted with 100 μM FLAG peptide (Sigma‐Aldrich) in the same buffer. After the removal of endotoxins using the EndoTrap Blue Endotoxin Removal Kit (Hyglos GmbH, Bernried am Starnberger See, Germany), the purified protein was dialyzed against phosphate‐buffered saline, subjected to ultrafiltration with the Vivaspin 6‐5K (GE Healthcare), and employed for cell stimulation. The precise expression and purity of the protein were confirmed by running the sample on a sodium dodecyl sulfate polyacrylamide gel followed by Coomassie staining and Western blotting with the anti‐FLAG antibody (Sigma‐Aldrich) (data not shown).

Cell population assay

Splenocyte and thymocyte cell populations were analyzed by flow cytometry upon staining with anti‐CD3‐PECy7, anti‐CD4‐APC‐eFluor780, anti‐CD8‐Pacific Blue, anti‐CD25‐Alexa488, anti‐CD62L‐Brilliant Violet510 (BioLegend, San Diego, CA, USA), anti‐γδTCR‐APC, anti‐DX5‐APC, anti‐TCRβ‐PE, anti‐Gr‐1‐APC‐eFluor780, anti‐CD123‐PE (eBioscience), anti‐CD19‐PE, anti‐CD11b‐PerCP‐Cy5.5, anti‐Siglec‐F‐PE, anti‐CD11c‐biotin (BD Bioscience) followed by streptavidin‐Pacific Blue (Thermo Fisher Scientific), anti‐F4/80‐FITC (Serotec, Oxford, UK), and α‐GalCer/CD1d tetramer‐APC (ProImmune, Oxford, UK). Expression of Foxp3 was determined with anti‐Foxp3‐PerCP‐Cy5.5 (eBioscience) by the standard intracellular staining procedure. In addition to the detection of CD3⁺, CD4⁺, and CD8⁺ cells, CD3⁻CD19⁺, CD3⁺γδTCR⁺, TCRβ⁺α‐GarCer/CD1d⁺, CD62L⁺CD4⁺CD25⁻, CD4⁺Foxp3⁺CD25⁺, CD11c⁺Siglec‐F⁻, CD11c⁻Siglec‐F⁺, DX5⁺CD123⁻, DX5⁺CD123⁺, CD11b^highGr‐1^high, and CD11b^lowGr‐1⁻ cells were recognized as B cells, γδT cells, NKT cells, naïve CD4⁺ T cells, Treg cells, DC, eosinophils, NK cells, basophils, neutrophils, and monocytes/macrophages, respectively. In some experiments, thymic CD4⁺CD8⁺ and CD4⁻CD8⁻ cells were purified using a FACSAria cell sorting system (BD Biosciences).

Characterization and genotyping of rearranged TCR

Total RNA was extracted from 2 × 10⁵ antigen‐stimulated peripheral T cells and thymic CD4⁺CD8⁺ and CD4⁻CD8⁻ cells. After reverse transcription using random primers (Toyobo, Osaka, Japan) and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) as described previously 20, PCR was performed with primer pairs for a series of V‐ and C‐regions of TCRα (Appendix Table S1) and TCRβ (Appendix Table S2) using the KAPA HiFi PCR Kit (KAPA Biosystems, Wilmington, MA, USA). The PCR cycling conditions were as follows: denaturation at 98°C for 20 s, annealing at 57°C for 20 s, and extension at 72°C for 1 min, repeated 35 times. Following agarose gel electrophoresis, amplified products were recovered with the QIAquick Gel Extraction Kit (QIAGEN, Venlo, the Netherlands) and subcloned into the pCR‐BluntII‐TOPO vector (Thermo Fisher Scientific). The insert products were sequenced using M13 forward and reverse primers.

Genotyping of rearranged TCR was performed by PCR using genomic DNA with primer sets designed to detect the V‐regions of rTα and rTβ (Appendix Table S4) and TaKaRa Ex Taq (Takara Bio, Shiga, Japan). The PCR cycling conditions were as follows: denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 45 s, repeated 35 times.

Expression of rTα and rTβ was also analyzed by flow cytometry in spleen or peripheral blood cells stained with anti‐TCRVα2, anti‐TCRVα11, or anti‐TCRVβ13‐1 plus anti‐CD4 or anti‐CD8 Abs.

Assessment of allergic bronchial inflammation

Mice were challenged with 4–12 intranasal instillations of 20 μl of endotoxin‐removed Der f solution (1 mg/ml saline) at a 3‐ to 4‐day interval. At 72 h after the last antigen challenge, BHR, inflammatory cells in bronchoalveolar lavage fluid (BALF), and histopathological changes in the lungs were evaluated as described previously 20 with minor modifications. For the BHR assessment, mice were anesthetized by intraperitoneal injection of 5 mg/kg pentobarbital, and then, a 19‐gauge metal cannula was inserted into the trachea. Mechanical ventilation was performed under diaphragmatic perforation using a small animal ventilator (FlexiVent; SCIREQ, Quebec, Canada) at a respiratory rate of 150 breaths/min, a tidal volume of 10 ml/kg body weight, and a positive end expiratory pressure of 3 cmH₂O. Progressive changes in respiratory system resistance (Rrs) were estimated following the inhalation of increasing doses of aerosolized methacholine (MCh; Nacalai Tesque, Kyoto, Japan) through an in‐line nebulizer. Inflammatory cells in BALF were classified based on morphological criteria as described previously 20. Lung sections (5 μm thick) were stained with hematoxylin and eosin (H&E) or Periodic acid‐Schiff (PAS) and observed under optical microscopy. The concentrations of BALF cytokines were determined by the Cytometric Bead Array. Serum IgE levels were measured by ELISA using goat anti‐mouse IgE (Southern Biotech, Birmingham, AL, USA), horseradish peroxidase‐conjugated monoclonal anti‐mouse IgE (Serotec), and control mouse IgE (Southern Biotech).

Assessment of antigen‐induced nasal responses

OVA#6 and DO11.10 mice were challenged once a day with an intranasal injection of 5 μl of OVA solution (30 mg/ml in saline) without anesthesia for 3–5 consecutive days. At 24 h after the intravenous injection of splenic CD4⁺ T cells from rTα(V4‐4)/rTβ(V13‐1)‐expressing OVA#6 (2 × 10⁷/head), the same challenge was conducted for BALB/c mice. NHR was assessed 6 h after antigen challenge by counting the number of sneezes for 5 min following the intranasal administration of 10 μl of histamine (100 mM). A nasal lavage (NAL) analysis was performed as described previously 17.

Statistical analysis

The experimenters were blind to the genotype of the tested animals for data collection and analyses. The results are presented as the arithmetic mean or the mean ± SEM. The statistical analysis was performed by one‐way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests using Prism (GraphPad Software Inc., La Jolla, CA, USA). P < 0.05 was considered to indicate statistical significance.

Author contributions

OK and KK conceived and designed the project. OK prepared and analyzed T cells with help from KK, NK, and AM. KI and AO performed NT with help from NO, SK, and MO. KK and OK performed TCR cloning and genotyping. The in vitro antigen‐reactivity assay was conducted by OK, KK, NK, YS, SI, and ST. Flow cytometry was performed by OK. MS, TN, and NK performed in vivo experiments with help from SK. OK and KK analyzed and coordinated the interpretation of the data with help from KK, AO, and KI. MS and TH also contributed to the interpretation of the data. OK wrote the manuscript with help from KK, KI, and AO.

Conflict of interest

The authors declare that they have no conflict of interest.

Supporting information

Appendix

Expanded View Figures PDF

Review Process File

EMBO Reports (2017) 18: 885–893