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. Author manuscript; available in PMC: 2013 Mar 1.
Published in final edited form as: J Invest Dermatol. 2011 Nov 17;132(3 Pt 1):677–686. doi: 10.1038/jid.2011.347

Soluble peptide treatment reverses CD8 T cell-induced disease in a mouse model of spontaneous tissue-selective autoimmunity

So Yeon Paek 1,2, Fumi Miyagawa 1, Hong Zhang 1, Jay Linton 1, Shelley Hoover 3, R Mark Simpson 3, Stephen I Katz 1
PMCID: PMC3395362  NIHMSID: NIHMS328418  PMID: 22089830

Abstract

Transgenic (Tg) mouse models of autoimmunity have been utilized to express model antigens that can be recognized by T cells or by autoantibodies. To identify mechanisms of CD8-mediated tissue-specific autoimmune reactions and to identify potential treatments, we generated a double transgenic (DTg) murine model of autoimmunity by crossing K14-sOVA mice, which express soluble chicken ovalbumin (OVA) predominantly in external ear skin, with OT-I mice whose CD8 T cells express Vα2/Vβ5 regions of the T cell receptor and are specific for SIINFEKL peptide (OVA 257-264) in association with class I MHC. The K14-sOVA/OT-I DTg mice develop a destructive process selectively targeting the external ear pinnae in the first 6 days of life. The ear bud area develops an intense inflammatory infiltrate of OT-I cells. Administration of the SIINFEKL peptide i.v. to pregnant F1 mice and subsequently i.p. to newborn pups resulted in normal external ear development. Treatment with this self-peptide markedly reduced OT-I cell numbers as well as down-regulated the CD8 co-receptor. This model can be useful in studying localized, tissue-specific, immune-mediated skin disease and inform us about potential therapies for autoimmune diseases in which specific molecular targets are known.

INTRODUCTION

Autoimmunity results when the immune system fails to control autoregulatory processes and cannot differentiate self from foreign antigens. Autoimmune diseases may be T-cell or B-cell-mediated and may target self antigens or produce autoantibodies targeting specific tissues. Identifying molecular target antigens has been a major investigative challenge in many autoimmune disorders, and for those diseases with known antigens, such as pemphigus vulgaris or bullous pemphigoid (Goldberg et al., 1984), the difficulty remains in the development of antigen-specific therapies (Caspi, 2008).

Autoreactive CD8 T cells play a critical pathogenic role in organ-specific autoimmune diseases (Liblau et al., 2002; Lohr et al., 2005). Their mechanism of action has been studied in various diseases affecting the skin, including alopecia areata, psoriasis vulgaris, vitiligo, and cutaneous lupus erythematosus (Walter and Santamaria, 2005). To study mechanisms of autoreactive CD8 T cells against skin, we previously established three strains of K14-sOVA mice which express different levels of OVA: K14-sOVA (#5), (#15), and (#17) (Miyagawa et al., 2010). K14-sOVA (#15) mice express the highest levels of OVA mRNA in multiple organs, while #5 and #17 mice express much lower levels of OVA mRNA than #15 mice. When K14-sOVA (#15) mice were mated with OT-I mice, 83% of the K14-sOVA/OT-I F1 pups died due to multi-organ inflammation (Gutermuth et al., 2009).

We then hypothesized that we could generate a localized skin disease model by crossing K14-sOVA Tg mice expressing lower levels of OVA with OT-I mice. In the current study, we evaluate the K14-sOVA/OT-I (#5) and K14-sOVA/OT-I (#17) double transgenic (DTg) mice and investigate the role of antigen-specific therapy in modifying the disease in these mice. Except for the finding that the #17 model demonstrated slightly higher levels of OVA mRNA, the K14-sOVA/OT-I (#5 and #17) strains can be considered identical. Both (#5 and #17) Dtg mice demonstrate selective tissue destruction of the external pinnae due to inflammatory infiltration of CD8+ T-cells during the first few days of life.

Previous attempts have been made to 1) study how defective T-cell receptor (TCR) surface expression or signaling correlate with CD8+ T-cell tolerance (Dubois et al., 1998) and to 2) utilize self-peptides in the induction of antigen-specific tolerance (Aichele et al., 1997; Brocke et al., 1996; Liblau et al., 1997). Using a diabetic mouse model, Bercovici et al (2000) found that administration of an agonist peptide in the early neonatal period disrupted the progression of autoimmune diabetes due to down-regulation of autoreactive CD8+ T cells through apoptosis-induced cell death (AICD). Injection of soluble hemagglutinin peptide into DTg autoimmune diabetic mice on days 3-5 after birth resulted in prolongation of survival of the mice via a CD8-mediated process.

We previously reported that administration of soluble OVA peptide resulted in a dose-dependent increase in survival of the K14-sOVA/OT-I (#15) DTg mice (Gutermuth 2009). The aim of the current study was to elucidate the mechanism(s) of selective tissue destruction and to attempt to prevent that inflammation and subsequent loss of pinnae. Administration of self-peptide into K14-sOVA (#5) and (#17) DTg mice at critical time points in the prenatal and neonatal period eliminated the inflammation-producing OT-I cells and induced CD8 co-receptor downregulation, allowing for normal development of ears in the DTg mice.

RESULTS

K14-sOVA/OT-I (#5 and #17) F1 double transgenic pups develop normally, except for the bilateral loss of the pinnae

The K14-sOVA (#5 and #17) strains were generated similarly to the Tg mice described in Shibaki et al, but without the PDGF-receptor transmembrane domain (Shibaki et al., 2004). When these mice were crossed with OT-I mice, the progeny developed normally but underwent a destructive process selectively targeting the outer ear (Figure 1). At day 2, there was hyperemia of dermal vasculature, followed on day 3 by epidermal necrosis and intraepidermal pustule formation. This progressed to involve both cranial and lateral pinnal epidermis, and the underlying facial epidermis, over the following 2-3 days. By day 6 or 7, an inflammatory cleft had formed between the lateral surface of the pinna and adjacent facial skin that adhered distally on days 7-8 and closed by dermal adhesion on day 9. The cranial epidermal surfaces of pinnae regenerated on days 7-9, while the accompanying inflammatory dermal infiltrate resulted in dysplastic chondroplasia of auricular cartilage precursors and permanent disfigurement of the external ear. Histology of tongue, esophagus, and trunk skin of K14-sOVA/OT-I (#5 and #17) mice on days 4, 8, and 14 after birth was normal, indicating that the inflammatory process was limited to the ears (Figure S1).

Figure 1. Development of K14-sOVA/OT-I (#5 and #17) DTg mouse ear on days 1-9 after birth.

Figure 1

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A. DTg mice developed normally until birth, after which the pinnae (external ear auricles) underwent degeneration, with bullous lesions at the dermal-epidermal junction associated with epidermal necrosis and leukocyte infiltration. This degenerative process progressed over days 3-6, resulting in loss of epidermis on the pinnal surface, and eventually, loss of external ears by day 10. Photomicrographs are of K14-sOVA/OT-I (#17) mice, oriented with the mouse’s crown to the right side of each image and ventral (anterior) aspect to the left, with one representative ear shown for each day of age. Day 1 was defined as the first day after birth. H&E stain. (Bars = 100 μm). B. DTg mice ears displayed at higher resolution from mouse #17 at 3 days of age (top and middle panels), and mouse #5 at 4 days of age (bottom panel). The changes include evolving bullae at the dermal-epidermal junction associated with epidermal necrosis and leukocyte infiltration. H&E stain. Bars = 25 micrometers.

Ear skin of K14-sOVA (#5 and #17) mice expressed high levels of OVA mRNA

To determine why the inflammation was limited to the ears in our mouse model, the OVA mRNA expression in different tissues of K14-sOVA single Tg mice were assessed. Cell suspensions were prepared from ear skin, back skin, thymus, tongue, esophagus, and liver of wild-type C57BL/6 and K14-sOVA (#5 and #17) single Tg mice. OVA transgene mRNA expression levels were quantified by real-time PCR with mGAPDH as the housekeeping gene (Figure 2). Ear skin exhibited higher levels of OVA mRNA expression compared to other tissues examined. Thus, the presence of immune cell mediated inflammation and the resulting earless phenotype in the sOVA/OT-I (#5 and #17) mice corresponded with the level of mRNA OVA expression in the single Tg mice.

Figure 2. OVA mRNA expression levels in different tissues from C57BL/6 and K14-sOVA (#5 and #17) adult single Tg mice.

Figure 2

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The OVA transgene mRNA expression levels were quantified by real-time PCR with mGAPDH as the housekeeping gene. The average of two mice from each group is shown.

Inflammatory infiltrates in K14-sOVA/OT-I DTg ear buds were comprised of CD8+ T-cells and were present before birth

To determine whether inflammation in the ear was caused by endogenous OT-I cells, cryosections taken from ear buds of DTg pups on days 1, 2, and 3 after birth were stained for CD8 (Figure 3a). Immunofluorescence microscopy exhibited a collection of CD8-positive cells in the dermis, closely abutting the epidermais, on all three days. FACS analysis of cell suspensions from ear buds of day 5 K14-sOVA/OT-I DTg pups demonstrated that these CD8-positive cells were also Vα2/Vβ5-positive (10.8%) when gated on the total lymphocyte population, indicating again that the OT-I cells infiltrated the ear buds (Figure 3b). Immunostaining for CD4+ T cells was negative (data not shown).

Figure 3. Inflammatory infiltrates in ears of K14-sOVA/OT-I (#5 and #17) mice.

Figure 3

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(a) Immunofluorescence of ear bud frozen tissue sections from DTg mice at age day 1-3. Anti-CD8 labeling (red) demonstrated minimal T cell trafficking in the dermis of DTg mice treated with OVA peptide, as compared to more florid dermal infiltration of CD8+ T-cells observed in DTg control mice with NP peptide treatment. Tissues are oriented with the epidermis to the right portion of the photomicrograph. Anti-CD8 immunofluorescence, DAPI nuclear counterstain (blue) shown in overlay photomicrographs. 40X original magnification. (b) FACS analysis of cell suspensions from ears of day 5 DTg pups digested with liberase CI prior to staining and analysis. (c) Ear histology of DTg mice at day E19 (embryonic day 19). Arrows represent zone of epidermal keratinocyte programmed cell death; open space at left arrow is precursor to external auditory canal. (Bars = 50 μm) (d) FACS data of spleens of DTg mice at day E18 with Vα2/Vβ5-positive TCR (range 11.4-24.7%) and CD8+ T-cells (range 10.8-23.9%). (e) Histology of ears of DTg mice treated with CD8 T-cell depleting antibody showing normal development of the pinnae in the immediate postnatal period. (Bars = 100 μm)

Since inflammatory infiltrates were present in ear buds as early as day 1, we further evaluated K14-sOVA/OT-I (#5 and #17) mice on days E18 (embryonic day 18) and E19, immediately prior to birth, to determine if the process occurred in utero. External ear taken during embryonic development day 19 (E19) revealed morphologic evidence of a zone of coordinate, epidermal keratinocyte programmed cell death (Figure 3c, middle and right arrows) that eventually led to the opening of the external auditory canal (Figure 3c, open space at left arrow) and the ultimate creation of lateral pinnal and facial skin surfaces. These surfaces re-epithelialized, freeing the pinna as an appendage from the rest of the head. The epidermal structure was in the same topographical region of epidermolysis manifest at 3 days in K14-sOVA/OT-I mice. Older mice expressed resolved re-epithelialized skin over misshapen chondrified cartilage and dilated external auditory canals (not shown). FACS analysis of spleens of K14-sOVA/OT-I (#5 and #17) pups at day E18 demonstrated that cells were positive for Vα2 and Vβ5 (11.4 to 24.7%), among which cells were also positive for CD8 (Figure 3d). These Vα2/Vβ5+CD8+ (OT-I) cells are likely responsible for the eventual tissue destruction.

Administration of CD8-depleting antibody in utero resulted in normal ear development

To definitively prove that the CD8+OT-I cells caused the destruction of the pinna and that the destructive process in K14-sOVA/OT-I (#5 and #17) DTg mice began before birth, three doses of the CD8-depleting antibody (clone YTS169.4) were administered i.p. to pregnant K14-sOVA (#5 and #17) dams on days E14, E16, and E18 of gestation. All pups subsequently born to these treated mothers exhibited normal ear development and lacked the inflammatory infiltrates that were present in untreated K14-sOVA/OT-I pups (Figure 3e, compare with Figure 1). This result indicates that CD8+ T cells are responsible for tissue destruction of the ear.

SIINFEKL peptide treatment rescued the ear phenotype in K14-sOVA/OT-I (#5 and #17) mice

Treatment of K14-sOVA (#5 and #17) mothers with CD8-depleting antibody was effective in preventing the destructive process. However, it is a nonspecific treatment and affects all CD8 T cells. Thus, we determined whether similar results could be obtained using the TCR-recognized SIINFEKL peptide (OVAp) to target OT-I cells specifically and leave the remainder of the immune system intact. Initial i.p. injections of pregnant K14-sOVA (#5 and #17) mice with 100 μg of peptide had no effect on ear inflammation and destruction, but repeated larger doses (300 μg to 1 mg) resulted in some pups being born with partial or full ears (Table 1). Since i.p.-injected peptide may have a variable distribution, we administered i.v. injections of 200 μg peptide to pregnant dams on days E14, E16, and E18 and obtained a larger percentage of pups with intact ears (Table 1). The inconsistency of phenotype reversal appeared to be due to the persistence of selective ear tissue destruction after birth. Therefore, in addition to the i.v. dosing of the pregnant dams, we continued to administer SIINFEKL peptide to the newborn pups at 50 μg i.p. after birth. 100% of pups whose pregnant mothers received SIINFEKL peptide i.v. in utero and who then received peptide i.p.after birth developed normal-looking ears (Table 1). Complete prevention of ear tissue destruction in K14-sOVA/OT-I (#5 and #17) mice was obtained with the optimal injection schedule shown in Figure S2: i.v. 200 μg SIINFEKL peptide on days E14, E16, and E18, followed by i.p. 50 μg peptide on days 2 and 4 after birth. These peptide-treated mice did not experience ear loss as adults, indicating that peptide treatment at a critical time period permanently reversed the ear destruction (data not shown). Mice treated with control influenza NP peptide (ILRGSVAHK) in the same concentration and same dosing regimen did not develop ears. By immunofluorescence microscopy many fewer CD8+ T cells infiltrated the dermis. (Figure 3a.)

Table 1. SIINFEKL peptide treatment of K14-sOVA/OT-I (#5 and #17) mice.

DTg pups treated with self-peptide only in utero inconsistently developed ears, whereas pups whose mothers were treated both i.v.or i.p.and were then treated i.p. after birth with OVAp developed normal-looking ears 100% of the time

Method Total pups Full/partial ears No ears
i.p. in utero (100 μg-1mg) 62 14 (23%) 48 (77%)
i.v. in utero (200 μg) 36 14 (39%) 22 (61%)
i.v. in utero (200 μg) +i.p. after birth (50 μg) 39 39 (100%) 0 (0%)
i.v. in utero (200 μg) +i.p. after birth (50 μg)1 8 0 (0%) 8 (100%)
i.p. after birth (50 μg) 30 16 (53%) 14 (47%)
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Abbreviations: i.p., intraperitoneal; i.v., intravenous; K14-sOVA, keratin-14-soluble chicken ovalbumin.

Double-transgenic pups treated with self-peptide only in utero inconsistently developed ears, whereas pups whose mothers were treated both i.v. or i.p. and were then treated i.p. after birth with ovalbumin peptide developed normal-looking ears 100% of the time.

1

These mice were treated identically to the group above except that a control NP influenza peptide was used instead of the SIINFEKL peptide.

Peptide treatment markedly reduced OT-I cell numbers at early time points

To determine the mechanism of phenotype reversal in our mouse model of tissue-specific autoimmunity, we analyzed the effect of peptide treatment on the spleen. Spleens from K14-sOVA/OT-I (#5) untreated and K14-sOVA/OT-I (#5 and #17) pups treated with the optimal peptide dosing schedule were harvested on days E18, 3, 5, and 9 after birth, pooled into single cell suspensions, and stained with anti-Vα2 and Vβ5. Mice evaluated at days 5 and 9 continued to receive i.p. 50 μg of peptide on days 4 and 6.

A decrease in the number of Vα2/Vβ5-positive OT-I cells in spleens of peptide-treated K14-sOVA/OT-I (#5) mice compared to untreated mice on day E18 and day 3 after birth was observed, indicating that peptide injection markedly decreased OT-I cell numbers in the spleen. Interestingly, the percentage of OT-I cells in peptide-treated mice increased on days 5 and 9, whereas it decreased in untreated K14-sOVA/OT-I (#5) mice (Figure 4a). These changes were not associated with differences in total number of splenocytes (data not shown). The increase in OT-I cells at days 5 and 9 may be due to the expansion of peripheral CD8+ T-cells following peptide injection, as observed in the studies of Bercovici et al. However, this proliferation does not result in immune inflammatory changes or ear destruction.

Figure 4. Marked reduction of OT-I cell numbers at earlier time points and down-regulation of the CD8 coreceptor following peptide treatment.

Figure 4

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(a) FACS analysis of spleens of untreated and peptide-treated K14-sOVA/OT-I (#5 and #17) pups harvested on days E18, 3, 5, and 9 and stained for Vα2/Vβ5 (OT-I cells).

(b) Spleens harvested and pooled from untreated K14-sOVA/OT-I (#5) and peptide-treated K14-sOVA/OT-I (#5 and #17) mice on days 3, 5, and 9 were stained for CD8. Mean fluorescence intensity of CD8 in OT-I cells was lower at all time points in peptide-treated mice compared to untreated mice.

Peptide treatment induced down-regulation of the CD8 coreceptor

Since the number of OT-I cells increased at later time points after peptide injection (Figure 4a), elimination of OT-I cells may not be the only mechanism by which the injected peptides function. We next determined if the CD8 expression levels of OT-I cells were changed by peptide injection. Spleens of untreated K14-sOVA/OT-I (#5) and peptide-treated K14-sOVA/OT-I (#5 and #17) mice were harvested on days 3, 5, and 9 and stained for CD8. The mean fluorescence intensity (MFI) of CD8 at all time points in OT-I cells of peptide-treated DTg mice was lower than that in untreated mice, suggesting that CD8 was down-regulated upon peptide injection, and this down-regulation persisted (Figure 4b). These data suggest that CD8 co-receptor downregulation may also be a factor in the peptide-induced prevention of pinna destruction.

K14-sOVA/OT-I (#5 and #17) mice treated with SIINFEKL peptide demonstrated an activated CD8 T-cell phenotype at an earlier time point than untreated mice

To further determine the mechanism of peptide treatment efficacy, OT-I cell activation status in vivo was evaluated by FACS staining of splenocytes for surface activation markers after gating on the OT-I population of cells. OT-I cells from peptide-treated K14-sOVA/OT-I (#5 and #17) mice exhibited a CD25high CD44high CD62Llow CD69high activated phenotype on postnatal day 3, whereas untreated mice exhibited a CD25low CD44low CD62Lhigh CD69low naïve phenotype on day 3 (Figure 5). In peptide-treated mice, OT-I cells rapidly became memory-like cells, down-regulating CD25 and CD69 and up-regulating CD44 and CD62L by postnatal day 5. This suggests that peptide injection activated OT-I cells. In contrast, untreated K14-sOVA/OT-I mice (#5) demonstrated an activated effector CD8 T-cell expression pattern on day 9, with down-regulation of CD62L and moderate up-regulation of CD25, CD44, and CD69.

Figure 5. Activation markers on OT-I cells in vivo.

Figure 5

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OT-I cells of untreated and peptide-treated K14-sOVA/OT-I DTg pup spleens were stained for the surface markers CD25, CD44, CD62L, and CD69 on days 3, 5, and 9 after birth. K14-sOVA/OT-I pups treated with SIINFEKL peptide demonstrated an activated phenotype on postnatal day 3 but were CD25low CD44high CD62Lhigh CD69low by days 5 and 9. Legend: red = sOVA/OT-I (#5) untreated; blue = sOVA/OT-I (#5) peptide-treated; green = sOVA/OT-I (#17) peptide-treated.

The results suggest that early after exposure to peptide, the OT-I cells in these newborn DTg mice are not activated, a finding that correlates with the continuing normal pinnal development described above in treated mice. The late (at P9) activation of OT-I cells in these DTg mice is puzzling but may signify that their cytotoxic potential is decreased in fully formed ears. The timing of the reduction of OVA-reactive OT-I cells in peptide-treated DTg mice (Figure 4a) is so well correlated with upregulation of activation markers on OT-I cells at approximately 3 days of age (Figure 5), suggesting that peptide injection eliminates OT-I cells through activation-induced cell death.

DISCUSSION

In this study, the development and evaluation of a localized skin disease model of CD8+ T cell-mediated autoimmunity and the potential for antigen-specific therapy are presented. Destruction of ears of K14-sOVA/OT-I (#5 and #17) double transgenic pups in response to OVA self-antigen mimicry in external ear skin was obviated by self-peptide treatment, which markedly reduced Vα2/Vβ5+CD8+ OT-I cells that participate in tissue destruction, likely by activation-induced cell death, and down-regulated the CD8 co-receptor in the postnatal period.

Although we initially thought that Vα2Vβ5 CD8+ T cells in our double transgenic (DTg) mouse model might be tolerized in utero, we found that spontaneous autoimmunity ensued in these mice. There are examples in the literature that support the findings herein described (Bercovici et al., 2000; Verdaguer et al., 1997). DTg systems are preferred models to study peripheral immune tolerance of activated CD8+ T cells that have escaped thymic deletion and are involved in ongoing disease processes. Mechanisms of peripheral tolerance include clonal anergy, reduced surface expression of TCRs and CD8 T-cell deletion, immune deviation, and T-cell receptor editing (McGargill et al., 2000; Park et al., 2007). In clonal anergy, after encountering antigen, T cells rapidly enter the cell cycle, express early activation markers, and enter a state of hyporesponsiveness with decreased proliferation. CD8-specific T cells can further undergo peripheral clonal deletion after antigen administration, resulting in either complete or partial loss of memory T cells. Autoreactive T cells have been stimulated to undergo deletion mediated by apoptosis when peptide treatment is used in animal models of experimental autoimmune neuritis and encephalomyelitis (Ishigami et al., 1998; Weishaupt et al., 1997). Finally, a shift from Th1 to Th2 response may occur in some tolerance models due to mechanisms of immune deviation (McGargill et al., 2002).

In our peptide-treated DTg mice, a decrease in the percentage of Vα2/Vβ5+ CD8 T-cells and down-regulation of the CD8 co-receptor appear to be mechanisms by which soluble peptide therapy exerts its protective effects. Targeted therapy utilizing soluble peptides against T cell populations has become a topic of interest in immunology (Larche and Wraith, 2005). This transgenic mouse model is informative with regard to peripheral tolerance mechanisms and may be used to study localized CD8-mediated skin diseases where there is intense superficial dermal infiltration by T cells, such as in lichen planus and fixed drug eruptions. The pathogenesis of both these conditions is unclear, but studies have demonstrated involvement of intraepidermal CD8 T-cells (Duarte et al., 2010; Gilhar et al., 1989; Roopashree et al., 2010; Shiohara, 2009). Our model may also provide insight into potential therapeutic vaccines for children at high-risk for development of tissue-selective autoimmune diseases, particularly diabetes.

MATERIALS AND METHODS

Mice

All mice used in this study were maintained on a 12-h light-dark cycle with free access to feed and water in a clean animal facility (mouse hepatitis virus- and pinworm-free). Animals were handled, bred, and used in accordance with institutional policies and with prior approval by the Animal Care and Use Committee of the National Institutes of Health. K14-sOVA Tg mice were generated similarly to the membrane-bound K14-mOVA mice previously described (Shibaki et al., 2004), but without the PDGF-receptor transmembrane domain, myc, and hemagglutinin sequences. The K14-sOVA (#5) and K14-sOVA (#17) were crossed with OT-I mice (OVA257-264-specific, class-I restricted TCR transgenic mice) to generate sOVA/OT-I (#5 and #17) double transgenic mice. OT-I mice were obtained from Dr. J. Kapp (Emory University, Atlanta, GA).

Timed pregnancies

Pregnancies were timed by limiting the period of mating to 24 h (classifed as day E0-E1) between K14-sOVA (#5 or #17) female and OT-I male mice. Mice were separated by gender after that period and observed until first sign of pregnancy, roughly day E14. Birth was considered day 0.

Quantitative real-time PCR

Total RNA was extracted from various tissues using RNeasy Plus Kit (QIGEN, Clarita, CA) according to the manufacturer’s instructions. Q-RT-PCR mixtures were assembled employing iScript One-Step RT-PCR Kit With SYBR Green, and Q-RT-PCR was performed in a Chromo4 RT-PCR machine (Bio-Rad, Hercules, CA). The following primer pairs were used: mGAPDH, forward primer 5’ CGT GTT CCT ACC CCC AAT GT 3’, reverse primer 5’ TGT CAT CAT ACT TGG CAG GTT TCT 3’; OVA, forward primer 5’ GGC ATC AAT GGC TTC TGA GAA 3’, reverse primer 5’ CCA ACA TGC TCA TTG TCC CA 3’.

CD8 T-cell depletion

250 μg of the anti-CD8 monoclonal antibody YTS169.4 (1 mg/mL, Serotec) was injected i.p. into pregnant K14-sOVA (17) mice on days E14 (embryonic day 14), E16, and E18. The 250 μg dose was determined to sufficiently deplete CD8 T-cells.

Peptide treatment

SIINFEKL peptide (monomeric OVAp, composition Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu) was synthesized by PolyPeptide Group (San Diego, CA) and certified as 99.3% pure by HPLC. Initial studies involved i.p. injection of 100μg to 1 mg SIINFEKL peptide in 1× PBS (Life Technologies) into pregnant mice. In later studies, SIINFEKL peptide (1 mg/mL in PBS) was injected i.v. (200 μg) on days E14, E16, and E18 into pregnant K14-sOVA (#5) and K14-sOVA (#17) mice. Pups of these litters were subsequently injected i.p. (50 μg) on postnatal days 2 and 4. Control peptide was used at the same concentrations as the latter for both i.v. and i.p. injections with Influenza NP (266-274), from PolyPeptide Laboratories in France.

The sequence is: Ile-Leu-Arg-Gly-Ser-Val-Ala-His-Lys (ILRGSVAHK)

Immunofluorescence microscopy

Ear buds from sOVA/OT-I (#17) pups were harvested, frozen in Neg 50 medium (Thermo Scientific), and stored in -20° C. Cryosections were cut at 6 μm thickness and mounted onto silane-prep slides (Sigma). Prior to staining, slides were left in room temperature for 30 min to defrost. For immunofluorescence staining, slides were blocked with 3% skim milk-PBS + 5% goat serum, rinsed in PBS, and incubated with any of the following primary antibodies (BD pharmingen): rat anti-mouse CD8a (2 μg/mL), isotype rat IgG2a (2 μg/mL). After three PBS washes, secondary antibody (Alexa Fluor 568 goat anti-rat IgG, Invitrogen) was added. The slides were again washed in PBS × 3 before being mounted with Prolong Gold with Dapi (Invitrogen). Images were viewed with a fluorescence microscope (Zeiss MicroImaging).

Flow cytometry

Single-cell suspensions were prepared from either pooled ear buds or pooled spleens of sOVA/OT-I (#5 and #17) mice and resuspended in RPMI with 5% fetal bovine serum. RBC were lysed using ACK lysing buffer (Invitrogen). Cell counts were obtained manually with trypan blue exclusion. For cell surface staining, FITC-conjugated Vα2 (B20.1.1), PE (phycoerythrin)-conjugated Vβ5 (MR9.1), and APC (allophycocyanin)-conjugated anti-CD8, CD25, CD44, CD62L, CD69, and isotype control antibodies were used (BD Pharmingen). Stained cells were evaluated on a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed using Flo Jo software (Tree Star Inc, Ashland, OR).

Histopathology

The heads from embryonic and neonatal mice were fixed in 10% neutral-buffered formalin. Paraffin-embedded tissues were sectioned and stained with H&E using standard techniques (American Histolabs).

Clinical score

Ear development of sOVA/OT-I (#5 and #17) pups was assessed and put into the following categories by a blinded grader: no ears, partial ears, and fully formed ears. This procedure was standardized for all pups evaluated.

Supplementary Material

Figure S1. Histology of esophagus, tongue, and trunk skin of K14-sOVA/OT-I (#5 and #17) mice.

H&E sections obtained at day 4, 8, and 14 after birth revealed a lack of inflammation. Photomicrographs are of K14-sOVA/OT-I (#5) mice. (Bar = 25 μm)

Figure S2. Optimal schedule of peptide injections into K14-sOVA/OT-I (#5 and #17) DTg mice.

Following a 24 h mating period, females were separated and observed for pregnancy until day E14. Pregnant mice were then injected i.v. with SIINFEKL peptide (200 μg) on days E14, E16, and E18. Pups of these litters were also injected i.p. with SIINFEKL (50 μg) on days 2 and 4 after birth.

Acknowledgments

We thank Harry Schaefer for excellent assistance with digital media.

Abbreviations

Tg

transgenic

DTg

double transgenic

K14

keratin-14

mOVA

membrane-bound chicken ovalbumin

sOVA

soluble chicken ovalbumin

SIINFEKL/OVAp

chicken ovalbumin peptide 257-264

TCR

T-cell receptor

MHC

major histocompatibility complex

E16

embryonic day 16

F1

filial 1, first filial generation of animal offspring from cross mating two parental types

Footnotes

Conflict of Interest

The authors state no conflict of interest.

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Associated Data

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

Supplementary Materials

Figure S1. Histology of esophagus, tongue, and trunk skin of K14-sOVA/OT-I (#5 and #17) mice.

H&E sections obtained at day 4, 8, and 14 after birth revealed a lack of inflammation. Photomicrographs are of K14-sOVA/OT-I (#5) mice. (Bar = 25 μm)

NIHMS328418-supplement-Fig__S1.docx (3.2MB, docx)
Figure S2. Optimal schedule of peptide injections into K14-sOVA/OT-I (#5 and #17) DTg mice.

Following a 24 h mating period, females were separated and observed for pregnancy until day E14. Pregnant mice were then injected i.v. with SIINFEKL peptide (200 μg) on days E14, E16, and E18. Pups of these litters were also injected i.p. with SIINFEKL (50 μg) on days 2 and 4 after birth.

NIHMS328418-supplement-Fig__S2.docx (38.5KB, docx)

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