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Progressing to Alopecia UniversalisLymphocyte-Mediated Alopecia Areata

T+A Mouse Model of Clonal CD8

Sundberg and Terrence L. GeigerRajshekhar Alli, Phuong Nguyen, Kelli Boyd, John P.

http://www.jimmunol.org/content/188/1/477doi: 10.4049/jimmunol.1100657November 2011;

2012; 188:477-486; Prepublished online 23J Immunol 

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7.DC1http://www.jimmunol.org/content/suppl/2011/11/23/jimmunol.110065

Referenceshttp://www.jimmunol.org/content/188/1/477.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2011 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

A Mouse Model of Clonal CD8+ T Lymphocyte-MediatedAlopecia Areata 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 hair 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 hair loss

and regrowth, and ultimately progresses to alopecia universalis. Alopecia development is associated with CD8+ T cell activation,

migration into the intrafollicular region, and hair follicle 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.

Alopecia areata (AA) presents as a typically patchy, non-scarring hair loss, although it may progress to total hairloss (alopecia universalis) (1). Lifetime incidence is

greater than 1%. Murine models and human skin transplanted intoNOD/SCID mice support an autoimmune, T cell-dependent causein which a breakdown of immune privilege is followed by de-struction of hair follicles (2, 3). Indeed, a case report has describedthe cure of a person with AA by allogeneic hematopoietic stemcell transplantation, supporting an immunologic origin (4). DuringAA, class I and class II MHC are upregulated, and CD8+ andCD4+ lymphocytic infiltrates surround and penetrate affected fol-licles (5). CD8+ T cells predominate within the follicular epithe-lium during active disease (6–8). It has been suggested, althoughnot proved, that CD8+ T lymphocytes are primarily responsiblefor the follicular damage, whereas CD4+ T cells provide helpfor the CD8+ response (1).The Ag(s) responsible for AA are unknown. Preservation of

amelanotic hairs in AA and a report that damage to hair bulb

melanocytes preceded damage to hair follicle keratinocytes led tothe suggestion that melanocytes are initial targets of autoreactivelymphocytes (1, 9). The absence of widespread melanocyte destruc-tion during AA, however, suggests a more complex specificity re-lationship. Indeed, although AA is in rare cases associated withvitiligo, it is also associated with other autoimmune diseasesand most commonly presents as an isolated condition (10, 11).Furthermore, in the C3H/HeJ mouse model of disease, white hairs,created by localized freeze branding, were not spared (12).The C3H/HeJ mouse is the primary animal model of spontaneous

AA (2). Female mice greater than 6 mo of age have a low frequency(,1%) of a spontaneous AA-like disease, though this can reach.20% by 12 mo of age (13, 14). Older DEBR rats similarly developAA, although at a higher frequency (15). In each case, CD4+ andCD8+ T cells are required for disease, although the ultimate effec-tors of follicular damage have not been conclusively identified. Lowfrequency and late disease onset in these systems are significantimpediments 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 folliculardestruction. While evaluating the TCR expressed by a myelin-specific T cell, we identified a dual TCR, one of which guidedspecificity against a myelin Ag. Surprisingly, the second directedCD8+ T cells not against myelin, but selectively against hair fol-licles. Retroviral transgenic (retrogenic) mice on a Rag1tm1Mom

(Rag12/2) background, in which T cells exclusively express thisTCR, develop alopecia at ∼6–7 wk, with a nearly 100% incidenceby 4 mo. Alopecia mimics that seen in patients with reticular AAand ultimately progresses to alopecia universalis.

Materials and MethodsMice

C57BL/6J (B6), B6.129P2-B2mtm1Unc/J (b2m2/2), and B6.129S7-Rag1tm1Mom/J (Rag12/2) mice were obtained from The Jackson Laboratory

*Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN38105; †Department of Pathology, Vanderbilt University, Nashville, TN 37232; and‡The Jackson Laboratory, Bar Harbor, ME 04609

Received for publication March 8, 2011. Accepted for publication October 22, 2011.

This work was supported by National Institutes of Health Grants R01 AI056153 (toT.L.G.) and R01 AR056635 (to J.P.S.), the American Lebanese Syrian AssociatedCharities/St. Jude Children’s Research Hospital (to R.A., P.N., K.B., and T.L.G.), andthe North American Hair Research Society (to K.B.).

Address correspondence and reprint requests to Dr. Terrence L. Geiger, Departmentof Pathology, St. Jude Children’s Research Hospital, 262 St. Jude Place, D-4047,Memphis, TN 38105. E-mail address: terrence.geiger@stjude.org

The online version of this article contains supplemental material.

Abbreviations used in this article: AA, alopecia areata; B6, C57BL/6J; CFP, cyanfluorescent protein; HPC, hematopoietic progenitor cell; LN, lymph node; m, mouse;MSCV, murine stem cell virus.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100657

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and bred under specific pathogen-free conditions, including all detectableHelicobacter spp. B6.129S-H2dlAb1-Ea (Ab2/2) mice were provided byDr. P. Doherty (St. Jude Children’s Hospital, Memphis, TN). Experimentswere performed in accordance with institutional animal care and useprocedures.

Media, reagents, Abs, and flow cytometry

Cells were cultured in Eagle’s–Hank’s Amino Acid Medium (BioSource)supplemented with 10% heat-inactivated Premium FCS (BioWhittaker),penicillin G (100 U/ml), streptomycin (100 mg/ml), L-glutamine 292mg/ml (Invitrogen Life Technologies), and 50 mM 2-mercaptoethanol(Fisher Biotech). mAbs specific for mouse CD4 (clone L3T4), CD8(clone 53-6.7), CD25 (clone 7D4), TCRB (clone H57-597), CD69 (cloneH1.2F3), CD45RB (clone 16A), CD44 (clone IM7), CD3 (clone 145-2c11), and CD28 (clone 37.51) were obtained from BD Biosciences.mAbs against mouse IL-10R (clone 1B1.3A), IFNG (clone XMG1.2),TNFA (clone XT3.11), and polyclonal rat IgG (cat. no. BE0094) werepurchased from BioXCell (West Lebanon, NH). Anti-mouse IL-17(clone 50104) was purchased from R&D Systems (Minneapolis, MN).Flow cytometry was performed on a FACSCalibur (BD Biosciences) andflow cytometric sorting on a MoFlo high-speed cell sorter (DakoCyto-mation).

TCR cloning

The 1MOG244.1 TCR was cloned from the 1MOG244 hybridoma as de-scribed (16). TCRA and TCRB cDNA separated by a 2A sequence wascloned into a variant of the murine stem cell virus (MSCV)-driven MSCV-I-GFP retroviral vector that replaces GFP with cyan fluorescent protein(CFP) (17). The OT-1 TCR was provided by Dr. D. Vignali (St. JudeChildren’s Research Hospital, Memphis, TN).

Retroviral transduction of TCR

Retrovirus was produced as described (16, 18). Briefly, 10 mg each ofretroviral receptor and helper DNA constructs were cotransfected into 293T cells using calcium phosphate precipitation, and the cells were incubatedin DMEM with 10% FCS for 48 h. Supernatant was then collected twicedaily and used to infect GP+E86 retroviral producer cells in the presenceof 8 mg/ml polybrene. Transduced GP+E86 cells were flow-cytometricallysorted for the presence of CFP and cell-free supernatant used for trans-duction.

Generation of retrogenic mice

Retrogenic mice were generated as described (18). Briefly, bone marrowcells were harvested from the femurs of Rag12/2 mice 48 h after theadministration of 0.15 mg 5-fluorouracil per gram of body weight. Thepooled cells were cultured in complete Eagle’s–Hank’s Amino Acid Me-dium containing 20% FCS supplemented with IL-3 (20 ng/ml), IL-6 (50ng/ml), and stem cell factor (50 ng/ml) for 48 h at 37˚C in 5% CO2. Thecells were then cocultured for an additional 48 h with 1200 rad irradiatedGP+E86 retroviral producer cells. The transduced hematopoietic progen-itor cells (HPCs) were harvested, washed with PBS, analyzed by flowcytometry for CFP, and injected into sublethally irradiated (450 rad) re-cipient Rag12/2 mice at a ratio of two recipient mice per bone marrowdonor. Mice were analyzed at 4–8 wk after HPC transplantation for TCRengraftment.

Alopecia areata

Cohorts of retrogenic mice were followed for 6 mo or as indicated, andclinical signs of alopecia and body weight were measured three times perweek. Hair loss was monitored and scored as follows: 0, ,5% hair lossfrom total body surface area; 1, 5–20%; 2, 20–40%; 3, 40–60%; 4, 60–80%; 5, .80%. For adoptive transfer disease, CD8+ T cells were isolatedby magnetic bead selection (MACS; Miltenyi Biotec), and the indicatednumber of cells were transferred intravenously into unmanipulated Rag12/2

mice, 450 rad-irradiated B6 mice, or unirradiated B6 mice. Alternatively,10 3 106 total splenocytes (∼1.5 3 106 T lymphocytes) were transferredinto 450 rad-irradiated B6 b2m2/2 or Ab2/2 mice.

Histology

Tissues were harvested and fixed overnight in Fekete’s solution, processedroutinely, embedded in paraffin, and sectioned at 4 mm. Gram stains andperiodic acid–Schiff stains were performed to rule out bacterial and fungalinfection, respectively. Immunohistochemical labeling was performed ona Lab Vision autostainer. Complete necropsies were performed on twomice from each group to identify concurrent lesions.

Cell proliferation

Spleen or lymph node (LN) cells were isolated from retrogenic or B6 mice,and CD8+ T cells were purified using PE anti-CD8 Ab (clone 53-6.7) andanti-PE–coated microbeads (MACS; Miltenyi Biotec). Cells were culturedat 5 3 104 per well in 96-well plates with 3 3 105 irradiated syngeneicsplenic APCs and the indicated concentrations of anti-CD3 and 1 mg/mlanti-CD28, pulsed with 1 mCi [3H]thymidine after 72 h, and then harvestedfor scintillation counting. Samples were analyzed in triplicate.

Cytokine analysis

Primary CD8+ T cells (5 3 104) were isolated and stimulated as describedearlier. Culture supernatants were collected at 24 or 48 h and analyzed forIL-2, IL-4, IL-10, IFNG, and IL-17 by Bio-Plex assay (Bio-Rad).

Real-time PCR

Skin specimens were taken from 1MOG244.1 mice with alopecia or controlB6mice. Hair was shaved prior to specimen removal. For 1MOG244.1 micewithout alopecia universalis, specimens were taken at the edges of activelesions. Skin samples were flash frozen in liquid nitrogen and then ho-mogenized in TRIzol (Invitrogen) using a 19-gauge needle. RNA was ex-tracted using Phase Lock gel (Eppendorf) and the RNAeasy Mini Kit(Qiagen) following the manufacturers’ protocols. First-strand cDNA wassynthesized using the Superscript III RT-PCR kit (Invitrogen). IL-2, IL-10,IL-17, IFNG, and TNFA were quantified as described after 45 cycles ofPCR (19). As a positive control, RNA was isolated from B6 splenocytesactivated with ConA and 10 U/ml rhIL-2 for 48 h. Cytokine Ct data werecompared with GAPDH as an internal control, and this was normalized toCt data from simultaneously analyzed activated splenocytes using thecomparative Ct approach (22DDCt).

Ab treatment

Abs (250 mg/mouse) against mouse (m)IL-10R, mIFNG, mTNFA, mIL-17,and polyclonal rat IgG were administered i.p. to five to eight mice pergroup beginning 7 wk after HPC transplantation and prior to the onset ofdisease symptoms. Administration was biweekly for 5 wk. The treatmentconditions with mIFNG and mTNFA Abs were validated by demonstratingthat serum from treated mice (3 d postdosing) could fully deplete the re-spective cytokine in vitro after protein G clearance. For mIL-10R Ab,dosing was validated by demonstrating that serum acquired from similarlytreated mice could specifically stain mouse macrophage at a 1:50 dilution.The mIL-17 Ab was dosed based on conditions previously described (20–22).

ResultsProduction of 1MOG244.1 retrogenic mice

We identified two productively rearranged TCRA chains within the1MOG244 T cell hybridoma. One TCRA, when transduced withthe single rearranged TRBV13-2+ TRBJ2-7+ TCRB into T cells,conferred specificity for the immunizing Ag, myelin oligoden-drocyte glycoprotein (16). The second, a TRAV8D-2+ TRAJ37+

TCRA, did not. TCRA chain allelic exclusion is not robust, anddual TCR expression has been hypothesized to facilitate T cellescape from tolerance (23). To analyze functionally TCR expres-sing the second TCRA chain, we generated retrogenic mice selec-tively expressing it. The TCR A and B chains, denoted as1MOG244.1, were introduced into a retroviral vector separated bythe Thosea asigna virus 2A sequence (Fig. 1). The 2A allows forstoichiometric translation of both TCR chains from a single cis-tron (24). The construct was transduced into B6 Rag12/2 HPCs,which were then transplanted into Rag12/2 mice.

Development of alopecia in 1MOG244.1 mice

1MOG244.1 mice showed no gross abnormalities until ∼6–7 wkafter HPC transplant, when individuals began experiencing hairloss. At 2 mo, greater than 50% of mice developed alopecia, andby 4 mo virtually all had developed alopecia (Fig. 2A). Hair lossdid not result from barbering, as a similar incidence of disease wasobserved in mice housed individually (data not shown). Neitherwas the alopecia a nonspecific consequence of the retrogenesis.

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This phenotype has not been observed with other class I- or classII-restricted TCRs produced in our or other laboratories (16, 25–27). Further, mice retrogenic for the OVA-specific, class I MHC-restricted OT-1 TCR (28) produced simultaneously and co-housedwith a cohort of 1MOG244.1 mice did not develop alopecia (Fig.2A). The hair loss additionally did not accompany other clinicalmanifestations of illness. The weight of 1MOG244.1 and controlOT-1 mice did not significantly differ over a .120-d observationperiod (Fig. 2B), and no abnormalities were detected other thanthose affecting the skin in 1MOG244.1 mice by necropsy and his-tologic analysis (data not shown). Therefore, the 1MOG244.1TCR transgene provokes alopecia in the normally AA-resistant B6mouse strain. Disease is spontaneous in that it does not require anyintervention in the 1MOG244.1 mice for its initiation.1MOG244.1 retrogenic hair loss was patchy, most typically with

a clear delineation of alopecic and normal areas. Alopecia wasnot linearly progressive; measurements of the extent of hair lossin individual mice demonstrated a waxing and waning pattern ofdisease in most animals (Fig. 3A). Furthermore, similar to thereticular pattern of AA in humans, different hair patches experi-enced hair loss and regrowth independently (Fig. 3B); simulta-neous loss and regrowth in different regions was often apparent.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

developed complete hair loss or died, progressed to alopecia uni-versalis. A median of three cycles of hair loss and regrowth, as de-fined by a change in overall disease score $1, was seen (Table I).Therefore, 1MOG244.1 retrogenic mice develop a disease thatresembles reticular AA progressing to alopecia universalis.

Spontaneous T cell activation in 1MOG244.1 mice

We next examined T lymphocyte engraftment in 1MOG244.1mice.T cells were predominantly CD8+, with few CD4+ or coreceptornegative cells (Fig. 4A). Good splenic engraftment was seen inmice prior to the onset of disease, and T cell numbers were in-creased in age-matched (18.7 6 1.6 wk posttransplant, score 0–1;18.5 6 1.6 wk, score 3–5) alopecic mice with more advanceddisease (Fig. 4B; 3.8 6 1.5 3 106 cells, score 0–1; 6.9 6 2.2 3106 cells, score 3–5). An increase in T cell blasts, determined asthe percent of cells with increased forward and side scatter mea-surements, was also seen in 1MOG244.1 mice with more ad-vanced disease compared with those with no or early disease andwith simultaneously produced OT-1 mice (Fig. 4C, 16.5 6 5.9%,score 0–1; 50.1 6 9.7%, score 3–5; Supplemental Fig. 1A).1MOG244.1 AA was associated with manifest adenopathy andsplenomegaly compared with control OT-1 or C57BL/6 mice (Fig.

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.

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-

dinally. A, Kaplan–Meier analysis of disease-free survival for 1MOG244.1

(n = 63) and OT-1 (n = 10) TCR retrogenic mice. The 1MOG244.1 data are

pooled from several independently prepared cohorts of mice. A score $1

(.5% surface hair loss) was considered a positive event. B, Average

weight of groups of simultaneously produced 1MOG244.1 (n = 20) and

OT-1 (n = 10) TCR retrogenic mice is plotted. No significant difference is

seen.

FIGURE 3. Disease patterns in individual 1MOG244.1 mice. A, Plots

demonstrate disease scores of four representative individual 1MOG244.1

mice. Scores were calculated based on total body alopecia as described in

Materials and Methods and demonstrate the waxing and waning of disease

with ultimate progression to alopecia universalis. B, Regional hair loss

from four representative mice is plotted. The presence of alopecia within

the neck, left or right flanks, and back at an individual evaluation point are

indicated by the ♦, 4, d, and N symbols, respectively.

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4D), and moderately diminished TCR expression was frequentlyapparent on T cells from diseased animals (data not shown). Cumu-latively, these results indicate spontaneous activation of T cellsin 1MOG244.1 mice with the development of AA.The association of T cell activation with disease development was

confirmed by immunophenotyping. A dramatic increase of splenicT cells expressing the CD25 and CD69 activation markers was seenin concert with the progression of alopecia (Fig. 5). Disease waslikewise associated with markedly increased proportions of cellswith a memory/activated phenotype as defined by diminished lev-els of CD45Rb and CD62L and increased CD44 compared withC57BL/6 T cells. The percentage of memory, CD69+, and CD25+

T cells in 1MOG244.1 mice (disease scores of 1–2) was also in-creased compared with that in age-matched OT-1 retrogenics (Sup-plemental Fig. 1B, 1C, p , 0.01 for each). Notably, in individualmice, T cell activation measured by CD69 expression was elevatedin skin-draining LN compared with that in paired spleens, implyingthat T cell priming is more pronounced at these locations (Fig. 6,p , 0.05). Therefore, the 1MOG244.1 TCR guides T cells towardthe CD8 lineage. These T cells are spontaneously and specificallyactivated in retrogenic mice and differentiate into memory cells inassociation with the development of alopecia.To determine whether the AA was associated with elevated

expression of skin-homing markers, we analyzed expression ofCD162 and CCR10. However, CD162 expression was not alteredrelative to that observed on B6 splenocytes, and CCR10 was similaror modestly diminished (data not shown). This suggests that thespontaneous activation of 1MOG244.1 T cells is not associatedwith 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 hadclassical features of AA. Characteristics of cicatricial alopecia thatcan normally be found in B6 mice were also seen (29), although themicroscopic lesions of AA predominated. Lymphocyte infiltrateswere observed in and around anagen and late-catagen stage hairfollicles, extending from the bulge downward to just above thehair bulb (Fig. 7A and not shown). Analysis of diseased skin,however, failed to correlate disease presence or magnitude withthe 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 fewneutrophils (Fig. 7 and data not shown). Small, fragmented, con-densed, and membrane-bound nuclear material consistent withapoptosis was a common finding in the affected follicular epithe-lium. Apoptosis was confirmed in these cells by positive labelingwith a cleaved caspase 3 Ab (data not shown). Special stains forbacteria and fungi were consistently negative in all sections ofskin 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 microscopicfindings. In early hair cycles in haired skin adjacent to alopecicareas, swarms of lymphocytes were present in and around intacthair follicles as described earlier. As the disease and folliculardestruction progressed over time, the density of hair follicles de-clined, and the absence of intact follicles became more prominent.In mice with alopecia universalis, the alopecic areas were devoid offollicles with only remnant clusters of hair bulbs in the deep dermisand few remaining lymphocytes in the dermal tissue.With lesion progression, superficial fibrous connective tissue or-

ganized parallel to the epidermis at the dermal–epidermal junc-

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

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

Retrogenic TCR Mice (n) Mean Median Range Mean Median Range Mean Median RangeMaximalScore

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 0OT-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, andthe 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.

FIGURE 4. T cell engraftment in 1MOG244.1 mice. A, Flow cytometric

analysis of splenocytes demonstrates a predominant CD8+ T cell engraft-

ment in 1MOG244.1 retrogenic mice compared with B6 controls. B and C,

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-

metrically determined forward and side scatter, are plotted. D, Gross photo-

graph of LN and spleens from a representative 1MOG244.1 mouse (score =

3), age-matched OT-1 mouse, and C57BL/6 mouse indicates adenop-

athy and splenomegaly in diseased 1MOG244.1 animals.

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tion, a sequela of the B6 alopecia and dermatitis complicating thismodel. In areas interpreted as containing long-standing injury,there were stretches of dermal tissue in which hair follicles wereabsent. Remnant follicular structures consisted of mild linear fi-brosis, dermal papilla, and small clusters of melanocytes. In sum-mary, histopathology was consistent with severe AA together withthe sequelae of concurrent cicatricial alopecia and dermatitispreviously described in B6 mice (29).

Class I MHC dependence of T cell response

To establish better the independent role of T cells in alopecia de-velopment, we determined whether they could transfer disease tounaffected mice. Splenic CD8+ T cells were isolated from mice withintermediate hair loss (disease score 3), and doses of 7.5 3 105 or1.53 106 T cells were transferred i.v. into otherwise unmanipulatedRag12/2 mice (Fig. 8A). All mice developed AA, and time to initialdisease was dose dependent (low dose, 636 6 d; high dose, 4362 d; p = 0.001). All mice also eventually progressed to alopeciauniversalis, and time to this was likewise dose dependent (lowdose, 133 6 17 d; high dose, 108 6 2 d; p = 0.03).

Lymphopenia may promote T cell survival and homeostaticexpansion. To examine whether the lymphopenia in the Rag12/2

recipients played a role in disease transfer, isolated CD8+ T cellswere transferred into either unirradiated (1 3 106 or 10 3 106

doses) or control sublethally irradiated (1 3 106 dose) C57BL/6recipients. Whereas the irradiated recipients showed a diseaseincidence similar to that observed after transfer into the Rag12/2

mice, the unirradiated recipients did not achieve a minimal scoreof 1. However, by 20–40 d after transfer, these mice manifestedsubstantial hair shedding with routine manipulation, somethingnot observed in control mice. The incidence and time course ofthis shedding was T-cell dose dependent (Supplemental Fig. 2).Similar to primary disease in 1MOG244.1 mice, shedding was re-gional. Therefore, CD8+ T cells can transfer AA into lymphore-plete recipients. However, disease penetrance and severity ismarkedly attenuated.Considering that the T cells in 1MOG244.1 mice were pre-

dominantly CD8+, we hypothesized that the alopecia would beclass I MHC dependent. To test this, we transferred ∼1.5 3 106

1MOG244.1 T cells into sublethally irradiated class I MHC-defi-cient b2m2/2 or class II MHC-deficient IAb2/2 mice. Whereas7 of 8 IAb2/2 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 aftertransfer; class II MHC is not required. Notably, in humans andC3H/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

To better delineate the effector potential of the spontaneously ac-tivated 1MOG244.1 T cells, we analyzed their proliferative re-sponse to stimulation with CD3- and CD28-specific Abs. Similaror only mildly diminished proliferative responses compared withthose of control B6 CD8+ T cells were seen regardless of disease

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.

FIGURE 6. CD69 expression on skin-draining LN and splenic T cells.

Paired splenic and skin-draining LN (axial, cervical, and inguinal) CD8+

T cells from 1MOG244.1 mice with varied disease scores (range 1–5) were

assessed for expression of the early T cell activation marker CD69. In-

creased expression was observed in the LN by paired t test (p , 0.05).

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score and in the presence or absence of exogenous IL-2 (Fig. 9).Therefore, disease progression in the 1MOG244.1 mice is notassociated with the development of overt anergy or proliferativedefects among T lymphocytes.In contrast to the proliferative responses, 1MOG244.1 cytokine

production differed substantially from that of control CD8+ B6T cells. IL-2 and IFNG generation by 1MOG244.1 T cells frommice without disease was modestly elevated compared with thatfrom control cells (Fig. 10A). Neither 1MOG244.1 nor controlcells produced detectable quantities of IL-4 or IL-10 (data notshown). However, moderate quantities of IL-17 were produced by1MOG244.1 T cells, a cytokine not detected from the B6 T cells.Therefore, even prior to clinical disease development, different Teffector responses are detected in 1MOG244.1 and control mice.In mice with intermediate disease (score = 2), dramatically

increased cytokine production was observed. An ∼3-fold increasein IL-2 production was accompanied by an ∼7-fold increase inIL-17 and.20-fold increase in IFNG production. Further, low butdetectable levels of IL-4 and IL-10 were seen. Progression tosevere disease (score = 5) was associated with additional dramaticenhancements in IL-17 (.10-fold), IL-4 (.100-fold), and IL-10(.10-fold) production compared with T cells from mice with

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 1MOG244.1and simultaneously produced OT-1 mice demonstrated markedlyincreased production of all cytokines by the 1MOG244.1 Tcells (Supplemental Fig. 3). Therefore, the cytokine profile of1MOG244.1 T cells varies both quantitatively and qualitativelywith disease state, a finding consistent with studies showing dy-namic changes in expression of immune response genes by arrayprofiling of C3H/HeJ mice during the course of skin graft-inducedAA (32). Early disease in 1MOG244.1 mice is primarily associ-ated with IFNG and lesser amounts of IL-17 production by pe-ripheral T cells, with progressive increases in IL-17 and Tc2 cyto-kines with advancing disease.To assess for cytokine production in target tissue, RNA was

isolated from skin specimens of 1MOG244.1 mice with alopecia orfrom control B6 mice. TNFA, IFNG, IL-17, and IL-10 were an-alyzed by quantitative PCR and normalized to levels present inConA-stimulated B6 splenocytes (Fig. 10B). Production of singlecytokines varied substantially between individual specimens, withoften several log differences in quantities.With the exception of positive testing for IFNG in 1 of 4 B6 skin

samples, none of the cytokines were detectable in the controlspecimens. Significant quantities of TNFA, IL-17, and IL-10 wereobserved in mice with disease scores of 1–3. IL-17 and IL-10production remained elevated in mice with alopecia universalis(score = 5), although TNFA production was diminished, with 2 of4 mice showing no detectable cytokine. This is consistent with thepresence of active inflammation and a regulatory response in theskin of mice with active disease.To determine the role of individual cytokines, a single large

cohort of 1MOG244.1 retrogenic mice was segregated into sub-groups that were treated with neutralizing Abs against TNFA (n =7), IL-17 (n = 5), or IFNG (n = 7), blocking IL-10R Ab (n = 7), oran Ab control (n = 8). Treatment began at 7 wk, at which timealopecia was first detected in the population, and continued for 5

FIGURE 7. Clinical appearance and histopathology of 1MOG244.1

mice. A, Skin sections (boxes) were isolated from mice with the indicated

alopecia scores and a clinically unaffected control B6 mouse with follic-

ular dystrophy unrelated to alopecia areata. H&E-stained paraffin sections

of representative fields at low magnification (320) and high magnification

(360; enlargement of boxed area) are shown. B, Immunohistochemistry

with T-specific CD3 Ab (brown) of skin sections from mice with the in-

dicated disease scores. Representative data from a total of 24 analyzed

1MOG244.1 skin sections is shown.

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).

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wk. The index diseased mice used to identify first symptoms in thepopulation were excluded from treatments and further analyses.Mice were monitored for disease incidence and severity in ablinded manner, and mice from the different treatment regimenswere co-housed in individual pens to minimize environmentaleffects. Kaplan–Meier and area-under-the-curve analyses failed tofind significant differences between any of the groups (data notshown). This suggests that either these cytokines primarily exerttheir effects prior to alopecia development or that they do not playstrong independent roles in alopecia progression and that redun-dant pathways are available.

DiscussionAA is a chronic, highly prevalent disfiguring condition, which anabundance of data now indicates is primarily mediated by auto-reactive T lymphocytes. Dissecting disease pathogenesis has beenhampered by the limitations in the animal model systems available.Both the C3H/HeJ mouse and DEBR rat model systems arecharacterized by low-incidence spontaneous disease first devel-oping late in life. Although adoptive transfer of lymphocytes or skingrafting can transfer disease, understanding the pathogenesis ofincipient spontaneous alopecia requires analysis of de novo diseasedevelopment. We characterize a new high-incidence retrogenicmodel for AA. It is to our knowledge the first described TCRtransgenic AA model system and the first in the B6 mouse strain.In both humans and rodents, AA is associated with the infil-

tration of both CD4+ and CD8+ T cells in the hair follicle, thoughCD8+ cells more specifically localize to the intrafollicular region(6–8). The relative roles of these T cell subsets are not fully

elucidated. McElwee and colleagues (33) found that s.c. injectedCD8+ T cells from alopecic mice promoted local disease at theinjection site at high incidence, whereas CD4+ T cells could pro-voke a more generalized alopecia although at lower incidence.This study differed from ours in mouse strain (C3H versus B6) and

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).

FIGURE 10. Cytokine production by 1MOG244.1 T cells and in

1MOG244.1 skin. A, CD8+ T lymphocytes from 1MOG244.1 or control

B6 mice were purified and stimulated as in Fig. 9. At 48 h, cell-free cy-

tokine was collected and concentrations of the indicated cytokines mea-

sured. B, RT-PCR was performed on skin specimens from 1MOG244.1

mice with alopecia (scores 1–3 or 5) or B6 controls. Cytokine levels were

compared with simultaneous quantitative PCR performed on B6 spleno-

cytes stimulated with ConA for 48 h, and relative quantity was calculated

using the comparative Ct approach. Circles indicate data from individual

mice, and bars indicate group averages. *p , 0.05 (by Kruskal–Wallis test

using Dunn’s multiple comparison test).

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route of cell administration (s.c. versus i.v.), potentially explainingdifferences in outcome. The localized reaction in that study mayhave resulted from immune exhaustion in the transferred cell pop-ulation or a failure to migrate from the skin, effects obviated byour transfers into lymphopenic mice, which will promote cell sur-vival and homeostatic expansion, and i.v. transfer, which will pro-mote dissemination of the transferred cells. In contrast, in studiesof human scalp explants, cooperation between CD4+ and CD8+

T cells was required for reproducible hair loss (34). Likewise,depletion of CD4+ or CD8+ T cells with mAb proved transientlytherapeutic in DEBR rats (35, 36). With development of alopecia,MHC class I and II is upregulated on follicular epithelium, po-tentially supporting both CD4+ and CD8+ T cell responses (3).One role for CD4+ T cells is to provide help for the formation of

pathologic CD8+ T cells (37). A key finding of our analysis is thatclass I MHC-restricted T cells are capable of independently me-diating alopecia development and progression. Few CD4+ T cellsare present in 1MOG244.1 retrogenic mice (Fig. 4A). Further,purified adoptively transferred CD8+ T cells mediated diseaseafter transfer into Rag12/2 mice but failed to induce alopeciain class I MHC-deficient b2m2/2 recipients. In contrast, class IIMHC was not required for disease development after adoptivetransfer. This implies that class I MHC-restricted 1MOG244.1CD8+ T cells are capable of independently producing effectorresponses necessary for AA development and full progression. Analternative possibility is that the CD4+ T cells provided help to theCD8+ cells prior to transfer. We consider this improbable, as itwould seem unlikely that the very small number of these cellspresent would be effectively stimulated by a class I-restrictedautoantigen in the absence of a CD8 coreceptor. More likely,the relatively high numbers of monoclonal 1MOG244.1 CD8+

T cells bypasses requirements for CD4+ T cell help, as has beenobserved elsewhere (38). Indeed, alopecia was only weakly in-duced after transfer into wild-type recipients, contrasting with themore severe disease after transfers into Rag12/2 or sublethallyirradiated mice. Disease in these wild-type recipients was regionaland associated with infiltrates of CD3+ cells. Potentially, the ho-meostatic stimulus of the lymphodeplete microenvironment helpssustain the 1MOG244.1 T cells and compensates for the absenceof CD4+ T cell help. The development of model systems assessingmonospecific CD4+ T cell responses, similar to that in this studywith CD8+ cells, will assist in fully evaluating the role of Th cells.Notably, unlike CD8+ T cells, both regulatory and effector func-tions have been ascribed to CD4+ T cells in AA (39, 40).The effector pathways used by the 1MOG244.1 T cells remain

to be defined. Several cytokines have been implicated in AA patho-genesis. TNFA affects skin in multiple ways, and polymorphismsin it have been associated with AA (41). Notably, despite beinggenerally proinflammatory, blockade of TNFA is not only ineffec-tive in AA but also has been associated in case reports with AAdevelopment (42–45). Biologically, TNFA has been shown toblock the IFN-mediated MHC upregulation in hair follicles thatpromotes AA (3, 46). A pathologic role for IFNG is more estab-lished in model systems, and the effector T cell response in AAappears primarily Th1/Tc1 (32, 47). Ifng2/2 C3H/HeJ mice werefound to be resistant to AA (48). IFNG treatment was also asso-ciated with enhanced disease in one study (49) but not a second(50). It was therefore curious that relatively small amounts ofIFNG were observed in skin sections in 1MOG244.1 mice (Fig.10B). However, this may reflect the limited number of T cellspresent within the skin mass, which may serve as the primarysource of the cytokine. Indeed, similar findings were present ina cross-sectional transcriptome study done at 2-wk intervals ofthe C3H/HeJ skin graft model (32) and confirmed in a more recent

transcriptome study at 5-wk intervals (J.P. Sundberg, unpublisheddata). IL-17 is prominently induced in the 1MOG 244.1 T cellsduring alopecia development, and though not well studied in thecontext of AA, its significance in other inflammatory autoimmuneconditions is well recognized. IL-10 is widely considered to playa regulatory role in AA and was found to be upregulated in C3H/HeJ mice that were resistant to AA through grafting of alopecicskin (40). However, C3H AA lesional skin grafts placed on IL102/2

mice paradoxically also showed decreased transfer of diseasecompared with wild-type controls (51). Therefore, these cytokineshave complex roles in AA, with seemingly mixed pro- and anti-inflammatory activities.mAb inhibition of TNFA, IFNG, IL-17, and IL-10 in

1MOG244.1 mice did not lead to significant differences in diseaseincidence or severity compared with controls, suggesting that theydo not play essential roles in the CD8+ T cell AA response. Thisinterpretation must be tempered, however, by the fact that mAbinhibition was performed for a period of just 5 wk beginning at thetime of anticipated clinical disease development, and it is possiblethat critical cytokine actions occur earlier than this. It is alsopossible that functional redundancy among cytokines in this modelpermits normal disease development despite blockade of indi-vidual cytokines. The generation of 1MOG244.1 retrogenicsin Rag2/2 mice bred for select cytokine deficiencies will be im-portant to evaluate further the role of individual cytokines.Importantly, no Ag has been identified that is recognized by AA-

associated T cells. The 1MOG244.1 T cell clone independentlymediates AA and may therefore serve as a tool for Ag screening.The loss of immune privilege in the hair follicle in associationwith AA suggests that follicle-associated Ags stimulate autoim-munity. Melanocytes have also been hypothesized to be a target,and decreased numbers of follicle-associated melanocytes are ob-served in cases of AA (52). Likewise, stimulation of T cells withmelanocyte-derived peptides enhanced AA in an experimentalmodel, and immunotherapy of a mouse model of melanoma led tothe development of alopecia (9, 53). However, AA is not intrin-sically associated with melanocyte loss, and though graying wasseen in hairs in the 1MOG244.1 model (Fig. 7), this was typicallyobserved only after several rounds of hair loss and regrowth andnot uniformly seen, particularly in early disease states. We there-fore consider it more probable that the 1MOG244.1 T cells arespecific for Ags that are not melanocyte associated, and that by-stander loss of follicular melanocytes with progressive cycles ofalopecia leads to amelanosis. Keratins have also been implicatedas immunologic targets in AA (54) and may serve as good can-didates for screening with the 1MOG244.1 TCR.Although alopecia universalis ultimately developed in all mice,

disease was cyclical and highly regional. Alopecic regions were notdefined by dermatomes or other obvious borders and were oftenirregular in shape and with distinct borders. Histologic studies wereunable to associate the follicular loss with specific hair folliclephases. Further, cyclical disease was regional, and at a single timepoint distinct patches showed hair loss or regrowth. Patchy baldnessis characteristic of the most common forms of alopecia areata inpeople, too, and the simultaneous growth and loss of hair is ap-parent in the reticular variant of the disease. This seemingly ar-bitrary regionalism may indicate that disease is unassociated witheither global cycles of T cell functional potential or with hair cyclechanges. Alternatively, studies of hair cycle in mice, including B6mice, have demonstrated complex hair cycle domains in individualanimals (55). Indeed, some mutant mice possess a cyclical alo-pecia where hair shafts dissociate from the follicles during speci-fic hair cycle stages, and patchiness is observed in these circum-stances.

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In summary, we describe a novel mouse model for AA on theB6 background with similarities to the reticular variant thoughprogressing to alopecia universalis. Our findings indicate thatmonoclonal CD8+ T cells can independently mediate spontaneousAA in a class I MHC-dependent manner and that progressivedisease is associated with systemic T cell expansion and degen-erate cytokine production. The 1MOG244.1 T cells further pro-vide an opportunity to better define Ags responsible for AA anddisease pathophysiology.

AcknowledgmentsWe thank Richard Cross, Greig Lennon, Stephanie Morgan, and the Immu-

nology Core Flow Cytometry facility for assistance with flow cytometric

sorting.

DisclosuresThe authors have no financial conflicts of interest.

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