The Journal of Immunology

A Mouse Model of Clonal CD8+ T Lymphocyte-Mediated Progressing to Alopecia Universalis

Rajshekhar Alli,* Phuong Nguyen,* Kelli Boyd,*,† John P. Sundberg,‡ and Terrence L. Geiger*

Alopecia areata is among the most prevalent autoimmune diseases, yet compared with other autoimmune conditions, it is not well studied. This in part results from limitations in the C3H/HeJ mouse and DEBR rat model systems most commonly used to study the disease, which display a low frequency and late onset. We describe a novel high-incidence model for spontaneous alopecia areata. The 1MOG244 T cell expresses dual TCRA chains, one of which, when combined with the single TCRB present, promotes the development of CD8+ T cells with specificity for follicles. Retroviral transgenic mice expressing this TCR develop spontaneous alopecia areata at nearly 100% incidence. Disease initially follows a reticular pattern, with regionally cyclic episodes of and regrowth, and ultimately progresses to alopecia universalis. Alopecia development is associated with CD8+ T cell activation, migration into the intrafollicular region, and destruction. The disease may be adoptively transferred with T lympho- cytes and is class I and not class II MHC-dependent. Pathologic T cells primarily express IFNG and IL-17 early in disease, with dramatic increases in cytokine production and recruitment of IL-4 and IL-10 production with disease progression. Inhibition of individual cytokines did not significantly alter disease incidence, potentially indicating redundancy in cytokine responses. These results therefore characterize a new high-incidence model for alopecia areata in C57BL/6J mice, the first to our knowledge to apply a monoclonal TCR, and indicate that class I MHC-restricted CD8+ T lymphocytes can independently mediate the pathologic response. The Journal of Immunology, 2012, 188: 477–486.

lopecia areata (AA) presents as a typically patchy, non- melanocytes preceded damage to hair follicle keratinocytes led to , although it may progress to total hair the suggestion that melanocytes are initial targets of autoreactive A loss (alopecia universalis) (1). Lifetime incidence is lymphocytes (1, 9). The absence of widespread melanocyte destruc- greater than 1%. Murine models and human skin transplanted into tion during AA, however, suggests a more complex specificity re- NOD/SCID mice support an autoimmune, T cell-dependent cause lationship. Indeed, although AA is in rare cases associated with in which a breakdown of immune privilege is followed by de- vitiligo, it is also associated with other autoimmune diseases struction of hair follicles (2, 3). Indeed, a case report has described and most commonly presents as an isolated condition (10, 11). the cure of a person with AA by allogeneic hematopoietic stem Furthermore, in the C3H/HeJ mouse model of disease, white , cell transplantation, supporting an immunologic origin (4). During created by localized freeze branding, were not spared (12). AA, class I and class II MHC are upregulated, and CD8+ and The C3H/HeJ mouse is the primary animal model of spontaneous CD4+ lymphocytic infiltrates surround and penetrate affected fol- AA (2). Female mice greater than 6 mo of age have a low frequency licles (5). CD8+ T cells predominate within the follicular epithe- (,1%) of a spontaneous AA-like disease, though this can reach lium during active disease (6–8). It has been suggested, although .20% by 12 mo of age (13, 14). Older DEBR rats similarly develop not proved, that CD8+ T lymphocytes are primarily responsible AA, although at a higher frequency (15). In each case, CD4+ and for the follicular damage, whereas CD4+ T cells provide help CD8+ T cells are required for disease, although the ultimate effec- for the CD8+ response (1). tors of follicular damage have not been conclusively identified. Low The Ag(s) responsible for AA are unknown. Preservation of frequency and late disease onset in these systems are significant amelanotic hairs in AA and a report that damage to hair bulb impediments to studies of disease cause and pathogenesis. We describe a new model for AA in which clonal C57BL/6J- derived CD8+ T lymphocytes independently mediate follicular *Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN destruction. While evaluating theTCRexpressedbyamyelin- 38105; †Department of Pathology, Vanderbilt University, Nashville, TN 37232; and ‡The Jackson Laboratory, Bar Harbor, ME 04609 specific T cell, we identified a dual TCR, one of which guided Received for publication March 8, 2011. Accepted for publication October 22, 2011. specificity against a myelin Ag. Surprisingly, the second directed CD8+ T cells not against myelin, but selectively against hair fol- This work was supported by National Institutes of Health Grants R01 AI056153 (to tm1Mom T.L.G.) and R01 AR056635 (to J.P.S.), the American Lebanese Syrian Associated licles. Retroviral transgenic (retrogenic) mice on a Rag1 Charities/St. Jude Children’s Research Hospital (to R.A., P.N., K.B., and T.L.G.), and (Rag12/2) background, in which T cells exclusively express this the North American Hair Research Society (to K.B.). TCR, develop alopecia at ∼6–7 wk, with a nearly 100% incidence Address correspondence and reprint requests to Dr. Terrence L. Geiger, Department by 4 mo. Alopecia mimics that seen in patients with reticular AA of Pathology, St. Jude Children’s Research Hospital, 262 St. Jude Place, D-4047, Memphis, TN 38105. E-mail address: [email protected] and ultimately progresses to alopecia universalis. The online version of this article contains supplemental material. Abbreviations used in this article: AA, alopecia areata; B6, C57BL/6J; CFP, cyan Materials and Methods fluorescent protein; HPC, hematopoietic progenitor cell; LN, lymph node; m, mouse; Mice MSCV, murine stem cell virus. C57BL/6J (B6), B6.129P2-B2mtm1Unc/J (b2m2/2), and B6.129S7- 2 2 Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 Rag1tm1Mom/J (Rag1 / ) mice were obtained from The Jackson Laboratory www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100657 478 RETROGENIC MODEL OF ALOPECIA and bred under specific pathogen-free conditions, including all detectable Cell proliferation Helicobacter spp. B6.129S-H2dlAb1-Ea (Ab2/2)micewereprovidedby Spleen or lymph node (LN) cells were isolated from retrogenic or B6 mice, Dr. P. Doherty (St. Jude Children’s Hospital, Memphis, TN). Experiments + were performed in accordance with institutional animal care and use and CD8 T cells were purified using PE anti-CD8 Ab (clone 53-6.7) and procedures. anti-PE–coated microbeads (MACS; Miltenyi Biotec). Cells were cultured at 5 3 104 per well in 96-well plates with 3 3 105 irradiated syngeneic Media, reagents, Abs, and flow cytometry splenic APCs and the indicated concentrations of anti-CD3 and 1 mg/ml anti-CD28, pulsed with 1 mCi [3H]thymidine after 72 h, and then harvested Cells were cultured in Eagle’s–Hank’s Amino Acid Medium (BioSource) for scintillation counting. Samples were analyzed in triplicate. supplemented with 10% heat-inactivated Premium FCS (BioWhittaker), penicillin G (100 U/ml), streptomycin (100 mg/ml), L-glutamine 292 Cytokine analysis mg/ml (Invitrogen Life Technologies), and 50 mM 2-mercaptoethanol + 3 4 (Fisher Biotech). mAbs specific for mouse CD4 (clone L3T4), CD8 Primary CD8 T cells (5 10 ) were isolated and stimulated as described (clone 53-6.7), CD25 (clone 7D4), TCRB (clone H57-597), CD69 (clone earlier. Culture supernatants were collected at 24 or 48 h and analyzed for H1.2F3), CD45RB (clone 16A), CD44 (clone IM7), CD3 (clone 145- IL-2, IL-4, IL-10, IFNG, and IL-17 by Bio-Plex assay (Bio-Rad). 2c11), and CD28 (clone 37.51) were obtained from BD Biosciences. mAbs against mouse IL-10R (clone 1B1.3A), IFNG (clone XMG1.2), Real-time PCR TNFA (clone XT3.11), and polyclonal rat IgG (cat. no. BE0094) were Skin specimens were taken from 1MOG244.1 mice with alopecia or control purchased from BioXCell (West Lebanon, NH). Anti-mouse IL-17 B6 mice. Hair was shaved prior to specimen removal. For 1MOG244.1 mice (clone 50104) was purchased from R&D Systems (Minneapolis, MN). without alopecia universalis, specimens were taken at the edges of active Flow cytometry was performed on a FACSCalibur (BD Biosciences) and lesions. Skin samples were flash frozen in liquid nitrogen and then ho- flow cytometric sorting on a MoFlo high-speed cell sorter (DakoCyto- mogenized in TRIzol (Invitrogen) using a 19-gauge needle. RNA was ex- mation). tracted using Phase Lock gel (Eppendorf) and the RNAeasy Mini Kit (Qiagen) following the manufacturers’ protocols. First-strand cDNA was TCR cloning synthesized using the Superscript III RT-PCR kit (Invitrogen). IL-2, IL-10, The 1MOG244.1 TCR was cloned from the 1MOG244 hybridoma as de- IL-17, IFNG, and TNFA were quantified as described after 45 cycles of scribed(16).TCRAandTCRBcDNAseparatedbya2Asequencewas PCR (19). As a positive control, RNA was isolated from B6 splenocytes cloned into a variant of the murine stem cell virus (MSCV)-driven MSCV- activated with ConA and 10 U/ml rhIL-2 for 48 h. Cytokine Ct data were I-GFP retroviral vector that replaces GFP with cyan fluorescent protein compared with GAPDH as an internal control, and this was normalized to Ct data from simultaneously analyzed activated splenocytes using the (CFP) (17). The OT-1 TCR was provided by Dr. D. Vignali (St. Jude 2DDCt Children’s Research Hospital, Memphis, TN). comparative Ct approach (2 ). Retroviral transduction of TCR Ab treatment m Retrovirus was produced as described (16, 18). Briefly, 10 mg each of Abs (250 g/mouse) against mouse (m)IL-10R, mIFNG, mTNFA, mIL-17, retroviral receptor and helper DNA constructs were cotransfected into 293 and polyclonal rat IgG were administered i.p. to five to eight mice per T cells using calcium phosphate precipitation, and the cells were incubated group beginning 7 wk after HPC transplantation and prior to the onset of in DMEM with 10% FCS for 48 h. Supernatant was then collected twice disease symptoms. Administration was biweekly for 5 wk. The treatment daily and used to infect GP+E86 retroviral producer cells in the presence conditions with mIFNG and mTNFA Abs were validated by demonstrating of 8 mg/ml polybrene. Transduced GP+E86 cells were flow-cytometrically that serum from treated mice (3 d postdosing) could fully deplete the re- sorted for the presence of CFP and cell-free supernatant used for trans- spective cytokine in vitro after protein G clearance. For mIL-10R Ab, duction. dosing was validated by demonstrating that serum acquired from similarly treated mice could specifically stain mouse macrophage at a 1:50 dilution. Generation of retrogenic mice The mIL-17 Ab was dosed based on conditions previously described (20– 22). Retrogenic mice were generated as described (18). Briefly, bone marrow cells were harvested from the femurs of Rag12/2 mice 48 h after the administration of 0.15 mg 5-fluorouracil per gram of body weight. The Results pooled cells were cultured in complete Eagle’s–Hank’s Amino Acid Me- Production of 1MOG244.1 retrogenic mice dium containing 20% FCS supplemented with IL-3 (20 ng/ml), IL-6 (50 ng/ml), and stem cell factor (50 ng/ml) for 48 h at 37˚C in 5% CO2. The We identified two productively rearranged TCRA chains within the cells were then cocultured for an additional 48 h with 1200 rad irradiated 1MOG244 T cell hybridoma. One TCRA, when transduced with GP+E86 retroviral producer cells. The transduced hematopoietic progen- + + itor cells (HPCs) were harvested, washed with PBS, analyzed by flow the single rearranged TRBV13-2 TRBJ2-7 TCRB into T cells, cytometry for CFP, and injected into sublethally irradiated (450 rad) re- conferred specificity for the immunizing Ag, myelin oligoden- cipient Rag12/2 mice at a ratio of two recipient mice per bone marrow drocyte glycoprotein (16). The second, a TRAV8D-2+ TRAJ37+ donor. Mice were analyzed at 4–8 wk after HPC transplantation for TCR TCRA, did not. TCRA chain allelic exclusion is not robust, and engraftment. dual TCR expression has been hypothesized to facilitate T cell Alopecia areata escape from tolerance (23). To analyze functionally TCR expres- sing the second TCRA chain, we generated retrogenic mice selec- Cohorts of retrogenic mice were followed for 6 mo or as indicated, and clinical signs of alopecia and body weight were measured three times per tively expressing it. The TCR A and B chains, denoted as week. Hair loss was monitored and scored as follows: 0, ,5% hair loss 1MOG244.1, were introduced into a retroviral vector separated by from total body surface area; 1, 5–20%; 2, 20–40%; 3, 40–60%; 4, 60– the Thosea asigna virus 2A sequence (Fig. 1). The 2A allows for + 80%; 5, .80%. For adoptive transfer disease, CD8 T cells were isolated stoichiometric translation of both TCR chains from a single cis- by magnetic bead selection (MACS; Miltenyi Biotec), and the indicated 2/2 2/2 tron (24). The construct was transduced into B6 Rag1 HPCs, number of cells were transferred intravenously into unmanipulated Rag1 2/2 mice, 450 rad-irradiated B6 mice, or unirradiated B6 mice. Alternatively, which were then transplanted into Rag1 mice. 10 3 106 total splenocytes (∼1.5 3 106 T lymphocytes) were transferred 2 2 2 2 Development of alopecia in 1MOG244.1 mice into 450 rad-irradiated B6 b2m / or Ab / mice. 1MOG244.1 mice showed no gross abnormalities until ∼6–7 wk Histology after HPC transplant, when individuals began experiencing hair Tissues were harvested and fixed overnight in Fekete’s solution, processed loss. At 2 mo, greater than 50% of mice developed alopecia, and routinely, embedded in paraffin, and sectioned at 4 mm. Gram stains and by 4 mo virtually all had developed alopecia (Fig. 2A). Hair loss periodic acid–Schiff stains were performed to rule out bacterial and fungal infection, respectively. Immunohistochemical labeling was performed on did not result from barbering, as a similar incidence of disease was a Lab Vision autostainer. Complete necropsies were performed on two observed in mice housed individually (data not shown). Neither mice from each group to identify concurrent lesions. was the alopecia a nonspecific consequence of the retrogenesis. The Journal of Immunology 479

FIGURE 1. 1MOG244.1 retroviral vector. The 1MOG244.1 TCR A and B chain cDNA were linked with a T. asigna 2A sequence as previously described (16). CDR3 amino acid and nucleotide sequences are shown. Invariant sequence for the TRAV8D-2 TRAJ37 TCRA and TRBV13-2 TRBJ2-7 TCRB is accessible through the ImMunoGeneTics resource (http://imgt. cines.fr). The MSCV retroviral vector incorporates an IRES-linked CFP.

This phenotype has not been observed with other class I- or class developed complete hair loss or died, progressed to alopecia uni- II-restricted TCRs produced in our or other laboratories (16, 25– versalis. A median of three cycles of hair loss and regrowth, as de- 27). Further, mice retrogenic for the OVA-specific, class I MHC- fined by a change in overall disease score $1, was seen (Table I). restricted OT-1 TCR (28) produced simultaneously and co-housed Therefore, 1MOG244.1 retrogenic mice develop a disease that with a cohort of 1MOG244.1 mice did not develop alopecia (Fig. resembles reticular AA progressing to alopecia universalis. 2A). The hair loss additionally did not accompany other clinical Spontaneous T cell activation in 1MOG244.1 mice manifestations of illness. The weight of 1MOG244.1 and control OT-1 mice did not significantly differ over a .120-d observation We next examined T lymphocyte engraftment in 1MOG244.1 mice. + + period (Fig. 2B), and no abnormalities were detected other than T cells were predominantly CD8 , with few CD4 or coreceptor those affecting the skin in 1MOG244.1 mice by necropsy and his- negative cells (Fig. 4A). Good splenic engraftment was seen in tologic analysis (data not shown). Therefore, the 1MOG244.1 mice prior to the onset of disease, and T cell numbers were in- TCR transgene provokes alopecia in the normally AA-resistant B6 creased in age-matched (18.7 6 1.6 wk posttransplant, score 0–1; mouse strain. Disease is spontaneous in that it does not require any 18.5 6 1.6 wk, score 3–5) alopecic mice with more advanced 6 intervention in the 1MOG244.1 mice for its initiation. disease (Fig. 4B; 3.8 6 1.5 3 10 cells, score 0–1; 6.9 6 2.2 3 6 1MOG244.1 retrogenic hair loss was patchy, most typically with 10 cells, score 3–5). An increase in T cell blasts, determined as a clear delineation of alopecic and normal areas. Alopecia was the percent of cells with increased forward and side scatter mea- not linearly progressive; measurements of the extent of hair loss surements,wasalsoseenin1MOG244.1micewithmoread- in individual mice demonstrated a and waning pattern of vanced disease compared with those with no or early disease and disease in most animals (Fig. 3A). Furthermore, similar to the with simultaneously produced OT-1 mice (Fig. 4C,16.56 5.9%, reticular pattern of AA in humans, different hair patches experi- score 0–1; 50.1 6 9.7%, score 3–5; Supplemental Fig. 1A). enced hair loss and regrowth independently (Fig. 3B); simulta- 1MOG244.1 AA was associated with manifest adenopathy and neous loss and regrowth in different regions was often apparent. splenomegaly compared with control OT-1 or C57BL/6 mice (Fig. This suggests that local skin factors, rather than a more general- ized state of immune responsiveness, was responsible for the re- gional periodicity. All mice in a cohort of 20, followed until they

FIGURE 2. Spontaneous alopecia in 1MOG244.1 mice. Rag12/2 mice were transplanted with Rag12/2 HPCs transduced with retrovirus encod- ing the 1MOG244.1 or control OT-1 TCR. Mice were observed longitu- FIGURE 3. Disease patterns in individual 1MOG244.1 mice. A, Plots dinally. A, Kaplan–Meier analysis of disease-free survival for 1MOG244.1 demonstrate disease scores of four representative individual 1MOG244.1 (n = 63) and OT-1 (n = 10) TCR retrogenic mice. The 1MOG244.1 data are mice. Scores were calculated based on total body alopecia as described in pooled from several independently prepared cohorts of mice. A score $1 Materials and Methods and demonstrate the waxing and waning of disease (.5% surface hair loss) was considered a positive event. B,Average with ultimate progression to alopecia universalis. B, Regional hair loss weight of groups of simultaneously produced 1MOG244.1 (n = 20) and from four representative mice is plotted. The presence of alopecia within OT-1 (n = 10) TCR retrogenic mice is plotted. No significant difference is the neck, left or right flanks, and back at an individual evaluation point are seen. indicated by the ♦, 4, d, and N symbols, respectively. 480 RETROGENIC MODEL OF ALOPECIA

Table I. Development of AA in 1MOG244.1 mice

Time to Score $1(d) Time to Score = 5 (d) Number of Relapses (n)

Maximal Retrogenic TCR Mice (n) Mean Median Range Mean Median Range Mean Median Range Score 1MOG244.1 20 58 6 16 53 38–118 139 6 4 140 132–143 2.7 6 1.1 3 0–5 5 6 0 OT-1 10 NA NA NA NA NA NA NA NA NA 0 6 0 The indicated numbers of 1MOG244.1 or control OT-1 TCR retrogenic mice were monitored for clinical signs of alopecia. Time to initial disease, to alopecia universalis, and the number of disease relapses, indicated by a change in disease score $1, are listed. AA was not observed in the OT-1 mice. NA, not applicable.

4D), and moderately diminished TCR expression was frequently T cells in 1MOG244.1 mice (disease scores of 1–2) was also in- apparent on T cells from diseased animals (data not shown). Cumu- creased compared with that in age-matched OT-1 retrogenics (Sup- latively, these results indicate spontaneous activation of T cells plemental Fig. 1B,1C, p , 0.01 for each). Notably, in individual in 1MOG244.1 mice with the development of AA. mice, T cell activation measured by CD69 expression was elevated The association of T cell activation with disease development was in skin-draining LN compared with that in paired spleens, implying confirmed by immunophenotyping. A dramatic increase of splenic that T cell priming is more pronounced at these locations (Fig. 6, T cells expressing the CD25 and CD69 activation markers was seen p , 0.05). Therefore, the 1MOG244.1 TCR guides T cells toward in concert with the progression of alopecia (Fig. 5). Disease was the CD8 lineage. These T cells are spontaneously and specifically likewise associated with markedly increased proportions of cells activated in retrogenic mice and differentiate into memory cells in with a memory/activated phenotype as defined by diminished lev- association with the development of alopecia. els of CD45Rb and CD62L and increased CD44 compared with To determine whether the AA was associated with elevated C57BL/6 T cells. The percentage of memory, CD69+, and CD25+ expression of skin-homing markers, we analyzed expression of CD162 and CCR10. However, CD162 expression was not altered relative to that observed on B6 splenocytes, and CCR10 was similar or modestly diminished (data not shown). This suggests that the spontaneous activation of 1MOG244.1 T cells is not associated with the acquisition of a phenotype biasing the cells toward skin- specific homing. Histopathology of 1MOG244.1 mice Skin sections from mice affected by the cyclical alopecia had classical features of AA. Characteristics of cicatricial alopecia that can normally be found in B6 mice were also seen (29), although the microscopic lesions of AA predominated. Lymphocyte infiltrates were observed in and around anagen and late-catagen stage hair follicles, extending from the bulge downward to just above the hair bulb (Fig. 7A and not shown). Analysis of diseased skin, however, failed to correlate disease presence or magnitude with the hair cycle phase of residual follicles (data not shown). Intra- follicular mononuclear cells expressing CD3 were readily detec- ted, demonstrating that retrogenic T cells had infiltrated the fol- licles (Fig. 7B). In the dermis, just adjacent to the hair follicles, lymphocytic infiltrates were mixed with macrophages and a few neutrophils (Fig. 7 and data not shown). Small, fragmented, con- densed, and membrane-bound nuclear material consistent with apoptosis was a common finding in the affected follicular epithe- lium. Apoptosis was confirmed in these cells by positive labeling with a cleaved caspase 3 Ab (data not shown). Special stains for bacteria and fungi were consistently negative in all sections of skin examined, indicating that the response did not result from sec- ondary infections (data not shown). The progression through multiple hair cycles from patchy alo- pecia to alopecia universalis was correlated to the microscopic findings. In early hair cycles in haired skin adjacent to alopecic areas, swarms of lymphocytes were present in and around intact FIGURE 4. T cell engraftment in 1MOG244.1 mice. A, Flow cytometric hair follicles as described earlier. As the disease and follicular + analysis of splenocytes demonstrates a predominant CD8 T cell engraft- destruction progressed over time, the density of hair follicles de- ment in 1MOG244.1 retrogenic mice compared with B6 controls. B and C, clined, and the absence of intact follicles became more prominent. Total numbers of splenic 1MOG244.1 CD8+ T cells (B) and percent of these cells that are blasted (C), as determined by increased flow cyto- In mice with alopecia universalis, the alopecic areas were devoid of metrically determined forward and side scatter, are plotted. D, Gross photo- follicles with only remnant clusters of hair bulbs in the deep dermis graph of LN and spleens from a representative 1MOG244.1 mouse (score = and few remaining lymphocytes in the dermal tissue. 3), age-matched OT-1 mouse, and C57BL/6 mouse indicates adenop- With lesion progression, superficial fibrous connective tissue or- athy and splenomegaly in diseased 1MOG244.1 animals. ganized parallel to the epidermis at the dermal–epidermal junc- The Journal of Immunology 481

FIGURE 5. Increased 1MOG244.1 T cell acti- vation with progressive alopecia. Flow cytometry plots from mice with the indicated disease score or from control B6 mice. Expression of the acti- vation markers CD25 and CD69 on gated CD8+ T cells demonstrates an increasing percentage of activated T cells with disease progression. Ex- pression of CD62L, CD44, and CD45Rb indicates progressive increases in the percentage of memory T lymphocytes. Representative data are shown.

tion, a sequela of the B6 alopecia and complicating this Lymphopenia may promote T cell survival and homeostatic model. In areas interpreted as containing long-standing injury, expansion. To examine whether the lymphopenia in the Rag12/2 there were stretches of dermal tissue in which hair follicles were recipients played a role in disease transfer, isolated CD8+ T cells absent. Remnant follicular structures consisted of mild linear fi- were transferred into either unirradiated (1 3 106 or 10 3 106 brosis, dermal papilla, and small clusters of melanocytes. In sum- doses) or control sublethally irradiated (1 3 106 dose) C57BL/6 mary, histopathology was consistent with severe AA together with recipients. Whereas the irradiated recipients showed a disease the sequelae of concurrent cicatricial alopecia and dermatitis incidence similar to that observed after transfer into the Rag12/2 previously described in B6 mice (29). mice, the unirradiated recipients did not achieve a minimal score of 1. However, by 20–40 d after transfer, these mice manifested Class I MHC dependence of T cell response substantial hair shedding with routine manipulation, something To establish better the independent role of T cells in alopecia de- not observed in control mice. The incidence and time course of velopment, we determined whether they could transfer disease to this shedding was T-cell dose dependent (Supplemental Fig. 2). + unaffected mice. Splenic CD8 T cells were isolated from mice with Similar to primary disease in 1MOG244.1 mice, shedding was re- 5 intermediate hair loss (disease score 3), and doses of 7.5 3 10 or gional. Therefore, CD8+ T cells can transfer AA into lymphore- 6 1.5 3 10 T cells were transferred i.v. into otherwise unmanipulated plete recipients. However, disease penetrance and severity is 2/2 Rag1 mice (Fig. 8A). All mice developed AA, and time to initial markedly attenuated. disease was dose dependent (low dose, 63 6 6d;highdose,436 Considering that the T cells in 1MOG244.1 mice were pre- 2d;p = 0.001). All mice also eventually progressed to alopecia dominantly CD8+, we hypothesized that the alopecia would be universalis, and time to this was likewise dose dependent (low class I MHC dependent. To test this, we transferred ∼1.5 3 106 dose, 133 6 17 d; high dose, 108 6 2d;p = 0.03). 1MOG244.1 T cells into sublethally irradiated class I MHC-defi- cient b2m2/2 or class II MHC-deficient IAb2/2 mice. Whereas 7of8IAb2/2 mice developed alopecia, 0 of 8 b2m2/2 recip- ients did (Fig. 8B). Therefore, an exclusively class I MHC- restricted response is adequate for alopecia development after transfer; class II MHC is not required. Notably, in humans and C3H/HeJ mice, the T cell infiltrate colocalizes with aberrant up- regulation of class I MHC (30, 31), consistent with these obser- vations. Promiscuous cytokine production by 1MOG244.1 T cells

FIGURE 6. CD69 expression on skin-draining LN and splenic T cells. To better delineate the effector potential of the spontaneously ac- Paired splenic and skin-draining LN (axial, cervical, and inguinal) CD8+ tivated 1MOG244.1 T cells, we analyzed their proliferative re- T cells from 1MOG244.1 mice with varied disease scores (range 1–5) were sponse to stimulation with CD3- and CD28-specific Abs. Similar assessed for expression of the early T cell activation marker CD69. In- or only mildly diminished proliferative responses compared with creased expression was observed in the LN by paired t test (p , 0.05). those of control B6 CD8+ T cells were seen regardless of disease 482 RETROGENIC MODEL OF ALOPECIA

FIGURE 8. Adoptive transfer of AA by 1MOG244.1 T cells. A, The indicated numbers of CD8+ 1MOG244.1 T cells from mice with alopecia were adoptively transferred i.v. into unmanipulated Rag12/2 mice and recipients monitored for the development of alopecia. A Kaplan–Meier analysis of disease-free survival is shown. n = 5 per group. Data are rep- resentative of two experiments. B, Disease-free survival of semilethally irradiated (450 rad) b2m2/2 and IAb2/2 mice after transfer of splenocytes including ∼1.5 3 106 T cells from 1MOG244.1 mice with alopecia. Data are pooled from two experiments (n = 8 per group).

score 2 disease. The enhanced and promiscuous cytokine pro- duction of the 1MOG244.1 mice was not a consequence of ret- rogenesis. Direct comparison of T cells from score 3 1MOG244.1 and simultaneously produced OT-1 mice demonstrated markedly FIGURE 7. Clinical appearance and histopathology of 1MOG244.1 increased production of all cytokines by the 1MOG244.1 T mice. A, Skin sections (boxes) were isolated from mice with the indicated cells (Supplemental Fig. 3). Therefore, the cytokine profile of alopecia scores and a clinically unaffected control B6 mouse with follic- 1MOG244.1 T cells varies both quantitatively and qualitatively ular dystrophy unrelated to alopecia areata. H&E-stained paraffin sections with disease state, a finding consistent with studies showing dy- of representative fields at low magnification (320) and high magnification namic changes in expression of immune response genes by array (360; enlargement of boxed area) are shown. B, Immunohistochemistry profiling of C3H/HeJ mice during the course of skin graft-induced with T-specific CD3 Ab (brown) of skin sections from mice with the in- AA (32). Early disease in 1MOG244.1 mice is primarily associ- dicated disease scores. Representative data from a total of 24 analyzed ated with IFNG and lesser amounts of IL-17 production by pe- 1MOG244.1 skin sections is shown. ripheral T cells, with progressive increases in IL-17 and Tc2 cyto- kines with advancing disease. score and in the presence or absence of exogenous IL-2 (Fig. 9). To assess for cytokine production in target tissue, RNA was Therefore, disease progression in the 1MOG244.1 mice is not isolated from skin specimens of 1MOG244.1 mice with alopecia or associated with the development of overt anergy or proliferative from control B6 mice. TNFA, IFNG, IL-17, and IL-10 were an- defects among T lymphocytes. alyzed by quantitative PCR and normalized to levels present in In contrast to the proliferative responses, 1MOG244.1 cytokine ConA-stimulated B6 splenocytes (Fig. 10B). Production of single production differed substantially from that of control CD8+ B6 cytokines varied substantially between individual specimens, with T cells. IL-2 and IFNG generation by 1MOG244.1 T cells from often several log differences in quantities. mice without disease was modestly elevated compared with that With the exception of positive testing for IFNG in 1 of 4 B6 skin from control cells (Fig. 10A). Neither 1MOG244.1 nor control samples, none of the cytokines were detectable in the control cells produced detectable quantities of IL-4 or IL-10 (data not specimens. Significant quantities of TNFA, IL-17, and IL-10 were shown). However, moderate quantities of IL-17 were produced by observed in mice with disease scores of 1–3. IL-17 and IL-10 1MOG244.1 T cells, a cytokine not detected from the B6 T cells. production remained elevated in mice with alopecia universalis Therefore, even prior to clinical disease development, different T (score = 5), although TNFA production was diminished, with 2 of effector responses are detected in 1MOG244.1 and control mice. 4 mice showing no detectable cytokine. This is consistent with the In mice with intermediate disease (score = 2), dramatically presence of active inflammation and a regulatory response in the increased cytokine production was observed. An ∼3-fold increase skin of mice with active disease. in IL-2 production was accompanied by an ∼7-fold increase in To determine the role of individual cytokines, a single large IL-17 and .20-fold increase in IFNG production. Further, low but cohort of 1MOG244.1 retrogenic mice was segregated into sub- detectable levels of IL-4 and IL-10 were seen. Progression to groups that were treated with neutralizing Abs against TNFA (n = severe disease (score = 5) was associated with additional dramatic 7), IL-17 (n = 5), or IFNG (n = 7), blocking IL-10R Ab (n = 7), or enhancements in IL-17 (.10-fold), IL-4 (.100-fold), and IL-10 an Ab control (n = 8). Treatment began at 7 wk, at which time (.10-fold) production compared with T cells from mice with alopecia was first detected in the population, and continued for 5 The Journal of Immunology 483

FIGURE 9. Proliferative response of 1MOG244.1 T cells. CD8+ T lymphocytes were purified by magnetic bead selection from 1MOG244.1 mice or control B6 mice and identical numbers stimulated with varied concentrations of anti-CD3 and 1 mg/ml anti-CD28 in the presence or absence of 10 U/ml rhIL-2. Proliferation was measured by [3H]thymidine incorporation at 72 h. Data shown are representative from separate ex- periments assessing mice with disease scores of 0 (A), 3 (B), or 5 (C). wk. The index diseased mice used to identify first symptoms in the population were excluded from treatments and further analyses. Mice were monitored for disease incidence and severity in a blinded manner, and mice from the different treatment regimens were co-housed in individual pens to minimize environmental effects. Kaplan–Meier and area-under-the-curve analyses failed to find significant differences between any of the groups (data not shown). This suggests that either these cytokines primarily exert their effects prior to alopecia development or that they do not play strong independent roles in alopecia progression and that redun- dant pathways are available.

Discussion AA is a chronic, highly prevalent disfiguring condition, which an FIGURE 10. Cytokine production by 1MOG244.1 T cells and in 1MOG244.1 skin. A, CD8+ T lymphocytes from 1MOG244.1 or control abundance of data now indicates is primarily mediated by auto- B6 mice were purified and stimulated as in Fig. 9. At 48 h, cell-free cy- reactive T lymphocytes. Dissecting disease pathogenesis has been tokine was collected and concentrations of the indicated cytokines mea- hampered by the limitations in the animal model systems available. sured. B, RT-PCR was performed on skin specimens from 1MOG244.1 Both the C3H/HeJ mouse and DEBR rat model systems are mice with alopecia (scores 1–3 or 5) or B6 controls. Cytokine levels were characterized by low-incidence spontaneous disease first devel- compared with simultaneous quantitative PCR performed on B6 spleno- oping late in life. Although adoptive transfer of lymphocytes or skin cytes stimulated with ConA for 48 h, and relative quantity was calculated grafting can transfer disease, understanding the pathogenesis of using the comparative Ct approach. Circles indicate data from individual incipient spontaneous alopecia requires analysis of de novo disease mice, and bars indicate group averages. *p , 0.05 (by Kruskal–Wallis test development. We characterize a new high-incidence retrogenic using Dunn’s multiple comparison test). model for AA. It is to our knowledge the first described TCR transgenic AA model system and the first in the B6 mouse strain. elucidated. McElwee and colleagues (33) found that s.c. injected In both humans and rodents, AA is associated with the infil- CD8+ T cells from alopecic mice promoted local disease at the tration of both CD4+ and CD8+ T cells in the hair follicle, though injection site at high incidence, whereas CD4+ T cells could pro- CD8+ cells more specifically localize to the intrafollicular region voke a more generalized alopecia although at lower incidence. (6–8). The relative roles of these T cell subsets are not fully This study differed from ours in mouse strain (C3H versus B6) and 484 RETROGENIC MODEL OF ALOPECIA route of cell administration (s.c. versus i.v.), potentially explaining transcriptome study at 5-wk intervals (J.P. Sundberg, unpublished differences in outcome. The localized reaction in that study may data). IL-17 is prominently induced in the 1MOG 244.1 T cells have resulted from immune exhaustion in the transferred cell pop- during alopecia development, and though not well studied in the ulation or a failure to migrate from the skin, effects obviated by context of AA, its significance in other inflammatory autoimmune our transfers into lymphopenic mice, which will promote cell sur- conditions is well recognized. IL-10 is widely considered to play vival and homeostatic expansion, and i.v. transfer, which will pro- a regulatory role in AA and was found to be upregulated in C3H/ mote dissemination of the transferred cells. In contrast, in studies HeJ mice that were resistant to AA through grafting of alopecic of human scalp explants, cooperation between CD4+ and CD8+ skin (40). However, C3H AA lesional skin grafts placed on IL102/2 T cells was required for reproducible hair loss (34). Likewise, mice paradoxically also showed decreased transfer of disease depletion of CD4+ or CD8+ T cells with mAb proved transiently compared with wild-type controls (51). Therefore, these cytokines therapeutic in DEBR rats (35, 36). With development of alopecia, have complex roles in AA, with seemingly mixed pro- and anti- MHC class I and II is upregulated on follicular epithelium, po- inflammatory activities. tentially supporting both CD4+ and CD8+ T cell responses (3). mAb inhibition of TNFA, IFNG, IL-17, and IL-10 in One role for CD4+ T cells is to provide help for the formation of 1MOG244.1 mice did not lead to significant differences in disease pathologic CD8+ T cells (37). A key finding of our analysis is that incidence or severity compared with controls, suggesting that they class I MHC-restricted T cells are capable of independently me- do not play essential roles in the CD8+ T cell AA response. This diating alopecia development and progression. Few CD4+ T cells interpretation must be tempered, however, by the fact that mAb are present in 1MOG244.1 retrogenic mice (Fig. 4A). Further, inhibition was performed for a period of just 5 wk beginning at the purified adoptively transferred CD8+ T cells mediated disease time of anticipated clinical disease development, and it is possible after transfer into Rag12/2 mice but failed to induce alopecia that critical cytokine actions occur earlier than this. It is also in class I MHC-deficient b2m2/2 recipients. In contrast, class II possible that functional redundancy among cytokines in this model MHC was not required for disease development after adoptive permits normal disease development despite blockade of indi- transfer. This implies that class I MHC-restricted 1MOG244.1 vidual cytokines. The generation of 1MOG244.1 retrogenics CD8+ T cells are capable of independently producing effector in Rag2/2 mice bred for select cytokine deficiencies will be im- responses necessary for AA development and full progression. An portant to evaluate further the role of individual cytokines. alternative possibility is that the CD4+ T cells provided help to the Importantly, no Ag has been identified that is recognized by AA- CD8+ cells prior to transfer. We consider this improbable, as it associated T cells. The 1MOG244.1 T cell clone independently would seem unlikely that the very small number of these cells mediates AA and may therefore serve as a tool for Ag screening. present would be effectively stimulated by a class I-restricted The loss of immune privilege in the hair follicle in association autoantigen in the absence of a CD8 coreceptor. More likely, with AA suggests that follicle-associated Ags stimulate autoim- the relatively high numbers of monoclonal 1MOG244.1 CD8+ munity. Melanocytes have also been hypothesized to be a target, T cells bypasses requirements for CD4+ T cell help, as has been and decreased numbers of follicle-associated melanocytes are ob- observed elsewhere (38). Indeed, alopecia was only weakly in- served in cases of AA (52). Likewise, stimulation of T cells with duced after transfer into wild-type recipients, contrasting with the melanocyte-derived peptides enhanced AA in an experimental more severe disease after transfers into Rag12/2 or sublethally model, and of a mouse model of melanoma led to irradiated mice. Disease in these wild-type recipients was regional the development of alopecia (9, 53). However, AA is not intrin- and associated with infiltrates of CD3+ cells. Potentially, the ho- sically associated with melanocyte loss, and though graying was meostatic stimulus of the lymphodeplete microenvironment helps seen in hairs in the 1MOG244.1 model (Fig. 7), this was typically sustain the 1MOG244.1 T cells and compensates for the absence observed only after several rounds of hair loss and regrowth and of CD4+ T cell help. The development of model systems assessing not uniformly seen, particularly in early disease states. We there- monospecific CD4+ T cell responses, similar to that in this study fore consider it more probable that the 1MOG244.1 T cells are with CD8+ cells, will assist in fully evaluating the role of Th cells. specific for Ags that are not melanocyte associated, and that by- Notably, unlike CD8+ T cells, both regulatory and effector func- stander loss of follicular melanocytes with progressive cycles of tions have been ascribed to CD4+ T cells in AA (39, 40). alopecia leads to amelanosis. Keratins have also been implicated The effector pathways used by the 1MOG244.1 T cells remain as immunologic targets in AA (54) and may serve as good can- to be defined. Several cytokines have been implicated in AA patho- didates for screening with the 1MOG244.1 TCR. genesis. TNFA affects skin in multiple ways, and polymorphisms Although alopecia universalis ultimately developed in all mice, in it have been associated with AA (41). Notably, despite being disease was cyclical and highly regional. Alopecic regions were not generally proinflammatory, blockade of TNFA is not only ineffec- defined by dermatomes or other obvious borders and were often tive in AA but also has been associated in case reports with AA irregular in shape and with distinct borders. Histologic studies were development (42–45). Biologically, TNFA has been shown to unable to associate the follicular loss with specific hair follicle block the IFN-mediated MHC upregulation in hair follicles that phases. Further, cyclical disease was regional, and at a single time promotes AA (3, 46). A pathologic role for IFNG is more estab- point distinct patches showed hair loss or regrowth. Patchy baldness lished in model systems, and the effector T cell response in AA is characteristic of the most common forms of alopecia areata in appears primarily Th1/Tc1 (32, 47). Ifng2/2 C3H/HeJ mice were people, too, and the simultaneous growth and loss of hair is ap- found to be resistant to AA (48). IFNG treatment was also asso- parent in the reticular variant of the disease. This seemingly ar- ciated with enhanced disease in one study (49) but not a second bitrary regionalism may indicate that disease is unassociated with (50). It was therefore curious that relatively small amounts of either global cycles of T cell functional potential or with hair cycle IFNG were observed in skin sections in 1MOG244.1 mice (Fig. changes. Alternatively, studies of hair cycle in mice, including B6 10B). However, this may reflect the limited number of T cells mice, have demonstrated complex hair cycle domains in individual present within the skin mass, which may serve as the primary animals (55). Indeed, some mutant mice possess a cyclical alo- source of the cytokine. Indeed, similar findings were present in pecia where hair shafts dissociate from the follicles during speci- a cross-sectional transcriptome study done at 2-wk intervals of fic hair cycle stages, and patchiness is observed in these circum- the C3H/HeJ skin graft model (32) and confirmed in a more recent stances. The Journal of Immunology 485

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