major histocompatibility complex class ib antigens gamma/delta

1996 87: 827-837

BR Blazar, PA Taylor, A Panoskaltsis-Mortari, TA Barrett, JA Bluestone and DA Vallera major histocompatibility complex class Ib antigensgamma/delta expressing T cells with specificity for host nonclassical Lethal murine graft-versus-host disease induced by donor

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Lethal Murine Graft-Versus-Host Disease Induced by Donor ?/S Expressing T Cells With Specificity for Host Nonclassical Major Histocompatibility

Complex Class Ib Antigens By Bruce R. Blazar, Patricia A. Taylor, Angela Panoskaltsis-Mortar;, Terrence A. Barrett, Jeffrey A. Bluestone,

and Daniel A. Vallera

Although T-cell receptor (TCR) alp expressing cells have a well-known role in graft-versus-host disease (GVHD) generation, the role of TCR ylS expressing cells in this process has remained unclear. To elucidate the potential function of TCR y/S cells in GVHD, we have used transgenic (Tgl H-2d mice (termed G81 that express y/S heterodimers on a high proportion of peripheral T cells. In vitro, G8 Tg y/S T cells proliferate t o and kill C57BL/6 (B61 (H-2b) which express gene products (TIOb and T22b) from the nonclassical major histocompatibility complex (MHC) class Ib H-2T region. The infusion of G8 Tg (H-2Td) TCR y/S cells into lethally irradiated 1900 cGy total body irradiation (TBI)] B6 (H-2b) mice resulted in the generation of lethal GVHD characterized histologically by destruction of the spleen, liver, lung, and colon. Lethal GVHD was prevented by the injection of anti-TCR y/S monoclonal antibodies. Immunohistochemical analysis of B6 recipients post-bone marrow transplantation (BMT) confirmed that G8

N RODENTS AND HUMANS, donor T-cell receptor (TCR) alp’ cells have been shown to recognize host

major histocompatibility complex (MHC) and/or minor histocompatibility determinants and cause lethal graft-versus- host disease (GVHD).’.’ Host TCR alp cells are capable of rejecting allogeneic donor hematopoietic grafts by recognizing donor histocompatibility determinants.’ However, the in vitro removal of a high proportion of the TCR Q//? cells in bone marrow (BM) grafts is not uniform in preventing GVHD,’ which suggests that cells, which do not express TCR alp, may be involved in GVHD induction.

Several years ago, a second lineage of T cells that expressed TCR y and 6 chains, rather than TCR a and p chains, was We reasoned that the TCR a/p- cells that appear to participate in GVHD might be TCR y / 6 cells. TCR y/6 cells have been shown to localize to tissues including those potentially involved in GVHD (eg, skin, colon, lung and suggesting that these cells may preferentially migrate to GVHD target tissues. In contrast to the specificity of TCR alp cells for classical MHC class I and I1 antigens, some TCR $6 cells have been shown to have specificities for nonclassical MHC class Ib determinants in the H-2T r e g i ~ n , ’ ~ . ~ ‘ T region genes have sequence homol- ogy to classical MHC class I genes and the heavy chains of these genes are associated with fi2-microglobulin.22 Some of the T region genes can be ubiquitously expressed including organs such as the liver, intestine, skin, and lung, as well as on some normal and hematopoietic cells,16,22-24 thereby serv- ing as potential targets for donor antihost alloreactivity culminating in GVHD-induced destruction.

TO determine whether TCR y/6 cells could play a role in bone marrow transplantation (BMT) and GVHD, we used TCR y/6 expressing G8 transgenic (Tg) mice25 as GVHD donors. Because G8 Tg mice express a TCR y/6 transgene derived from an alloreactive cytolytic T lymphocyte (CTL) clone that lyses target cells derived from some strains of mice, we reasoned that the infusion of TCR y/6+ T cells

I

Blood, Vol 87, No 2 (January 15), 1996: pp 827-837

Tg TCR y/6 cells infiltrated GVHD target tissues (skin, liver, colon, and lung) and were absent in recipients treated with anti-TCR y/S monoclonal antibodies (MoAbsl but not anti- CD4 plus anti-CD8 MoAbs. In contrast, injection of TCR y/ Sf cells into irradiated (900 cGy TBI) B6.A-Tla’ BoyEg mice that do not express either TIOb or T22b did not induce lethal GVHD. Similarly, in a different GVHD system in which suble- thal irradiation without bone marrow (BM) rescue was used, B6 but not BG.A-Tla’/BoyEg mice were found to be susceptible to TCR yS’ cell mediated GVHD-induced lethality characterized by an aplasia syndrome. These results demonstrate that TCR y/S cells have the capacity to cause acute lethal GVHD in mice and suggest that nonclassical MHC class Ib gene products expressed on GVHD target organs are respon- sible for G8 Tg TCR y/S’ cell mediated lethality. 0 1996 by The American Society of Hematology.

into the appropriate recipient strain would be an ideal setting to test the hypothesis that cytolytic TCR $6 cells might contribute to GVHD. The availability of G8 mice allowed us to address a number of fundamental questions. Included among these are the following: ( 1 ) Can donor TCR y/6+ T cells specifically migrate to GVHD target organs in the absence of donor TCR @/p+ T-cell mediated tissue damage? (2) If TCR y/6+ T cells mediate tissue destruction, which GVHD target tissues are most susceptible? (3) Will the GVHD tissue pathology resemble GVHD mediated by TCR alp+ T cells? (4) Can TCR y/S+ T cells specifically recognize host alloantigens, rather than participate in GVHD in a nonspecific manner as would be the case for phagocytes and other inflammatory cell infiltrates? ( 5 ) If there is a specific allorecognition process mediated by TCR y/6+ T cells, what are the host alloantigenic determinants that serve to drive

From the Department of Pediatrics, Division of Bone Marrow Transplantation and the Department of Therapeutic Radiology, Uni- versity of Minnesota Hospital, Minneapolis, MN; the Department of Medicine, Section of Gastroenterology, Northwestern University Medical School, Chicago, IL; and Ben May Institute, University OJ

Chicago, Chicago IL. Submitted July 18, 1995; accepted September 5, 1995. Supported by the following grants: US Public Health Service

Grants No. ROI-A134495, R01 CA31618, and POI-AI35296 awarded by the National Cancer Institute and the National Institute of Allergy and Infectious Diseases, Department of Health and Hu- man Services, and a Scholar Award from the Edward Mallinckrodt Jr. Foundation (B.R.B.). This is no. 36 in a series on murine bone marrow transplantation across the MHC.

Address reprint requests to Bruce R. Blazar, MD, Box 109 UMHC, 420 SE Delaware St, Minneapolis, M N 55455.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 I996 by The American Society of Hematology. 0006-4971/96/8702-0011$3.00/0

827




828 BLAZAR ET AL

the GVHD process and will the transgenic TCR y/6+ T cells from G8 mice retain the same functional properties as the CTL clone from which the transgenic strain was derived?

Our studies indicate that TCR y/6+ T cells can migrate to GVHD target tissues, cause tissue damage that has patho- logical similarities to GVHD caused by TCR a/P+ T cells, and mediate this destructive process via the recognition of nonclassical MHC class Ib antigens. Although naturally oc- cumng alloreactive TCR y/6+ T cells do exist at frequencies considerably lower than observed in G8 mice, the results we have obtained with these transgenic mice indicate that TCR y/6+ T cells have the capability of causing GVHD target destruction in mice. These data should provide the impetus for our laboratory and others to pursue studies to determine whether alloreactive TCR y/S+ T cells can contribute to GVHD destruction.

MATERIALS AND METHODS

Mice. C57BW6 (B6) (H-2h, TIOh), B6.A-TIaVBoyEg (H-2b, TI@), BIO.BR (H-2k, TIO”), and BALB/c (H-2d, TIOd) were purchased from The Jackson Laboratory (Bar Harbor, ME) or the Na- tional Institutes of Health (Bethesda, MD). Adult H - 2 ~ d TCR y/b expressing Tg mice (G8 Tg) were originally generated by the cross of a homozygote G8 Tgad male to BALB/c females and then back- crossed times five generations before establishing a breeding col- o n ~ . ~ ~ Mice were raised at the University of Minnesota in a specific pathogen-free, animal barrier facility. Donors were 4 to 6 weeks old, and recipients were 8 to 10 weeks old at the time of BMT.

Bone marrow transplantation. For all but one of the GVHD experiments, B6 recipients were conditioned with high-dose (9.0 Cy) total body irradiation (TBI) administered from a Philips RT 250 Orthovoltage Therapy Unit (Philips Medical Systems, Brookfield, WI) filtered through 0.35 mm Cu at a final absorbed dose rate of 0.41 Gyhinute at 225 kV and 17 mA.Z6 Donor BM was collected into RPMI 1640 medium by flushing it from the shafts of femurs and tibiae. Recipients (8 to I O mice per group per experiment) received 10 or 20 X 10‘ BM cells (as indicated) from BALB/c or G8 donors that had been T-cell depleted (TCD) with anti-Thyl.2 monoclonal antibody (MoAb) [hybridoma 30-H-12, rat IgG2b;’ provided by Dr David Sachs, Cambridge, MA (20 pg/mL)] + baby rabbit complement (C‘: Pelfreez, Rogers, AK) as previously described.z6 Single cell suspensions of lymph node (LN) cells or splenocytes were obtained (as a source of GVHD-causing effector cells) by passing minced LN or spleen cells through a wire mesh and collecting them into RPMI 1640.** Some groups received a total of 25 X 10‘ splenocytes or 1 to 3 X IO‘ TCR y/6 expressing Tg or I x IO6 BALBlc non-Tg LN T cells with or without CD4 depletion (as indicated), which removed the vast majority of TCR U / @ expressing, non-Tg T cells. The final composition of T cells in the donor graft was determined by flow cytometry. When indicated, mice were given anti-CD4 MoAb (hybridoma GKI .S, rat IgG2b, obtained from Dr Frank Fitch, University of Chicago, Chicago, IL, 400 pg weekly),29 anti-CD8 (hybridoma 2.43, rat IgG2b, obtained from Dr Frank Fitch),’” or anti-TCR y/6 MoAbs [hamster IgG MoAbs: pan- TCR y/6 hybridoma UC7-13DS”: 400 pg + anti-Vy 2 hybridoma UC3-10A6”: 400 pg, weekly)] intraperitoneally (IP) from days - 1 through +41 post-BMT or until time of elective sacrifice.

To induce GVHD by TCR y/6+ T cells in the absence of BM rescue, B6 or B6.Tlaa recipients were sublethally irradiated (7.0 Gy TBI) and injected with highly purified TCR y/6’ cells. This procedure was adapted from a procedure developed by Sprent et a132 who recently showed that highly purified donor TCR a/D+ T cells could mediate lethal GVHD when infused into sublethally irradiated recipi-

ents. TCR y/6+ CD4- CD8- T cells were highly enriched by treating the LN cells with anti-CD4 (GK1.5) + anti-CD8 (2.43) (20 p&nL each) MoAbs + C’ as described above for anti-Thy 1.2 + C’ depletion. Cells were washed, resuspended, and passed through an anti- Ig column (Biotex, Edmonton, Canada). The eluates were washed. resuspended, and given at a final TCR y/6+ cell dose of 2 X I O ” per mouse. Mice were given anti-CD8 + anti-NK1.1 (hybridoma PK-136, rat IgG2a, provided by Dr Gloria Koo, Merck-Sharpe- Dohme, Rahway, NJ)33 MoAbs (400 pg weekly from days - 1 through + day 21 post-BMT) to prevent host cells from rejecting the T-cell inocula. Hematocrit values were obtained as previously describedzx at periodic intervals post-BMT as an indicator of the possible BM destructive effects of infused LN cells, as has been reported for TCR a/@’ T-cell mediated GVHD in sublethally irradiated recipients.”

Pathologic examination oftissues. Mice were killed, autopsied, and tissues were taken for histopathologic analysis. All samples were embedded in optimal cutting temperature (OCT) compound (Miles, Inc, Elkhart, IN). snap frozen in liquid nitrogen and stored at -80°C. Serial 4 pm sections were cut (IEC Minotome), thaw mounted onto glass slides and fixed for 5 minutes in acetone. Slides were stained with hematoxylin and eosin for histopathological assessment.’6 Or- gans were scored positive for GVHD if there was single cell necrosis (skin, colon), crypt dropout (colon), peri-ponal infiltrate with acute necrosis (liver), or endothelialitis with a lymphocytic infiltrate (lung).’” In previous studies, these features were present only in mice with active GVHD and not in irradiated recipients of either syngeneic or anti-Thy1.2 + C‘ treated fully allogeneic BM.

Immunohisrochemisty. Frozen tissues (spleen, liver, and colon) were also stained for cell surface antigenic determinants. After blocking with normal horse serum, sections were incubated with biotinylated MoAbs (purchased from PharMingen, San Diego, CA) specific for the following molecules: TCR y/6 (GL3),” TCR a/ D (hybridoma H57-597, hamster IgG),’4 CD4 (GK1.5). or CD8g (hybridoma 53-5.8, rat IgG1).27 Detection with peroxidase-conjugated avidin-biotin complex and 3,3’-diaminobenzidine (DAB) as chromogen was performed essentially as describedx5 with reagents purchased from Vector Laboratories, Inc (Burlingame, CA). The frequency of immunoperoxidase positive cells in tissue sections was quantitated using standard point counting technique as previously described36 and expressed as the number of positive staining cells per mm2 averaged in at least seven representative fields or as a percentage of nucleated cells (lung).

In v i m prolijieration of G8 cells. G8 LN cells were depleted of CD4’ and CD8’ cells using anti-CD4 (hybridoma GK1.5) + anti- CD8 (hybridoma 2.43) MoAbs (20 p g h L each) + C‘. LN cells were then passaged through an anti-Ig column to remove B cells and macrophages. Washed and resuspended responders were mixed with irradiated (30 Cy) anti-Thy1.2 + C‘ treated splenocyte stimulators from B6, B6.Tlaa, or B 1O.BR mice and suspended in Dulbecco’s minimal essential medium (Bio Whittaker, Walkersville, MD), 10% fetal calf serum (Hyclone, Logan, UT), 2-mercaptoethanol (5 X mol/L) (Sigma, St Louis, MO), 10 mmolL hepes buffer, I mmol/ L sodium pyruvate (GIBCO BRL, Grand Island, NY), and amino acid supplements ( 1 .S mmol/L L-glutamine, L-arginine, and L-aspar- agine) (Sigma), antibiotics (penicillin: 1 0 0 units/mL, streptomycin: 100 pg/mL) (Sigma). A total of lo5 cell responders and 5 X I O 5 irradiated stimulators were plated into 96-well round bottom (Costar, Cambridge, MA) plates and placed at 37°C and 10% CO, for 2 to 6 days. Tritiated thymidine ( 1 pCi) was added 16 hours before harvesting and counting, in the presence of scintillation fluid, on a beta counter.

Flow cytometty analysis. Single cell preparations were suspended in buffer (phosphate-buffered saline [PBS] + 5% colostrum- free bovine serum + 0.015% sodium azide). Pelleted cells were




y/6 T CELLS IN MURINE GVHD

incubated for 15 minutes at 4°C with 0.4 pg of an anti-Fc receptor MoAb (clone 2.4G2, provided by Dr Jay Unkless, Rockefeller Uni- versity, New York, NY) to prevent Fc binding.37 Optimal concentra- tions of directly conjugated (biotin, PE, and fluorescein isothiocya- nate [FITC] labeled) MoAbs were added to a total volume of 100 to 130 pL and incubated 1 hour at 4°C. For the biotin-labeled MoAb, fluorescence was indirectly measured by adding streptavidin-labeled Red 613 (GIBCO) for an additional 1 hour at 4°C. After final wash- ing, cells were fixed in 1% formaldehyde. For analysis of LN cells or splenocytes, anti-TCR y/6 (UC7-13D5), anti-TCR alp (H57-597), anti-CD4 (GK1.5), anti-CD8 (53.6.7, rat IgG2a), B220 (CD45R; hybridoma Ra3-6B2, rat IgG2a),18 CDllb (Mac-l; hybridoma MI/ 70, rat IgG2b)39 MoAbs were either purchased from PharMingen or were conjugated in our laboratory as previously described.26

For analysis of donor or host origin of repopulating cells post- BMT, two-color flow cytometry was performed as described previo~sly.~~ Peripheral blood cells were costained with anti-H-2Kb- biotin streptavidin-phyocoerythrin (clone EH-144, mouse IgG, provided by Dr T.V. Rajan, University of Connecticut, Famington, CT) and anti-H-2Dd-FITC [hybridoma 34-5-83, mouse IgG2a,& provided by Dr David Sachs] after resuspending in buffer. An irrelevant anti- human MoAb [3AIE, rat IgG24' provided by Dr Barton Haynes, Duke University, Chapel Hill, NC] was used for background subtrac- tion in each sample. All samples were analyzed on a FACscan (Bec- ton-Dickinson, Mountain View, CA) using consort-30 software. A minimum of 20,000 events was examined.

Statistical analyses. In vitro proliferative data and flow cytometry values are listed as mean 2 1 standard error of the mean. Group comparisons of continuous data were made by Student's t-test. Sur- vival data were analyzed by lifetable methods using the Mantel- Peto-Cox summary of chi-square?* Actuarial survival rates (the proportion of mice surviving on each day post-BMT) are shown. Probability ( P ) values < .05 were considered significant and P values <.l but >.05 were considered to be statistical trends.

RESULTS

Irradiated B6 recipients of Tg TCR y/6 enriched LN cells develop lethal acute GVHD. To select a recipient for which the donor y/6 T cells have the appropriate TCR for allorecognition, we screened three fully allogeneic B6/B10 strains differing at MHC and T region loci for in vitro proliferative responsiveness. G8 LN cells (Fig 1) enriched for TCR y/6 cells and depleted of CD4+ and CD8+ TCR a/p cells re- sponded vigorously to irradiated, TCD stimulators from B6 mice. A lower level of response was noted when B1O.BR splenocytes were used as stimulators while the lowest response was seen when B6.Tla" stimulators were used. The potency of CTL killing of the G8 clone has been reported to be highest against B6 blasts followed by B1O.BR and B6.Tla" blasts, which were roughly eq~iva1ent.l~

Based on these studies, B6 mice were irradiated and used as recipients for GVHD experiments. Control BALB/c nontransgenic LN cells (no detectable TCR y/6+ cells, 78% TCR alp' cells: 58% CD4+, 17% CD8', G8 Tg LN cells (48% TCR y/6+, 6% TCR alp' cells: 12% CD4'; 2% CD8+), or BALB/c splenocytes (5 1% TCR y/6+ cells, 32% TCR a/ p' cells: 22% CD4', 8% CD8') were harvested as a source of mature T cells. Two-color flow cytometry has shown that the vast majority of the TCR y6+ cells do not express the CD4, while a small proportion do express the CD8 receptor (data not shown). Therefore, in initial experiments, we sought to reduce the number of TCR alp cells while preserv-

829

1

h

r

X

"1

DAYS IN CULTURE

Fig 1. In vitro proliferative response of LN TCR y/6+ cells to allogeneic splenic stimulators. G8 lymph node cells were depleted of CD4+ and CD8+ cells using MoAbs + C' and then passaged through an anti- Ig column before mixing with irradiated anti-Thy 1.2 + C' treated splenocyte stimulators from B6 (O), B6.Tla" (A), or B1O.BR (0) in a 1:5 ratio. A total of lo5 cells responders were plated in microtiter wells and pulsed after 2, 3, or 4 days as shown on the x-axis. Cpm x lo-' are listed on the y-axis on a log,, scale. Cpm x 10-3 for autologous responses were 0.16 (day 21, 1.2 (day 3). and 0.89 (day 4).

ing TCR $6 cells co-expressing CD8 molecules (which may be needed for an optimal GVHD effect). G8 LN cells were treated with anti-CD4 + C', which removed all detectable CD4+ cells. One million BALB/c TCR alp+ cells or an identical number of G8 TCR $6 cells was infused in vivo along with TCD BALB/c donor BM (20 X lo6 cells). Addi- tional groups received TCD BALB/c splenocytes (GVHD negative control) or non-TCD splenocytes (25 X lo6) (GVHD positive control).

By day 47 post-BMT, recipients of G8 LN cells had a 50% actuarial survival rate with all deaths associated with clinical signs of severe GVHD and occurring between days 42 to 47 post-BMT. Because the remaining murine recipients of G8 LN cells that were alive on day 47 post-BMT were moribund with a mean weight loss of 40% of their pre-BMT body weight, these recipients were electively killed, and the experiment was terminated. Recipients of an equal number of TCR alp+ LN cells did not develop lethal GVHD and had a 75% actuarial survival rate and mean weight that exceeded pre-BMT body weight at the time of experiment termination. Recipients of TCD splenocytes had mean body weights that exceeded pre-BMT body weights and had an 88% actuarial survival rate that was substantially ( P = ,079) higher than recipients of G8 LN cells but comparable ( P = .28) to recipients of BALB/c LN cells. Recipients of 25 X lo6 BALBfc splenocytes containing 8 X lo6 TCR alp+ cells had similar (P = .45) actuarial survival rate (63%) and post- BMT body weight losses (33% of pre-BMT body weights) as those given only l X lo6 TCR $6' cells. These data suggest that G8 Tg TCR y6 cells are potent inducers of lethal GVHD and TCR (YIP+ cells must be given in doses in excess of 1 X lo6 to cause lethal GVHD in B6 mice.

As further proof that G8 TCR y/6+ cells were required for GVHD generation, nondepleted G8 LN cells were used




BLAZAR ET AL 830

A

0.6 1 0.4 -

0.2 -

L f f

'L: n = Wgroup

0.0 ' . I ' I " , : . I ' I . I . 0 2 0 4 0 6 y 8 0 1 0 0 1 2 0 1 4 0

G8 TCD BM + G8 LN cells (3 x lose TCA 716 Cells) DAYS POST-BMT + no mAb

2 4 1 ,.o'o + anti-TCR mAbs

G0 TCD BM + G8 LN cells

P' 22

G8 TCD BM + no LN cells 20

18

16 G8 TCD BM + G8 LN cells + anti-CD4 mAb

14

12 - 2 1 8 3 8 5 8 7 8 9 8 118 138

DAYS POST-BMT

to determine if the in vivo depletion of CD4' T cells from G8 LN cells altered GVHD generation by removing a small population of CD4' TCR y/6+ T cells or by precluding a GVHD enhancing effect by CD4' TCR a/@+ cells. The final composition of the infused LN inocula contained 55% TCR y/S', 10% TCR alp' cells: 17% CD4+, 4% CD8+ cells. A total of 3 X lo6 TCR y/S+ cells was administered along with G8 TCD BM (20 X lo6 cells). Recipients receiving G8 LN cells were given no MoAb, anti-CD4 MoAb (400 pg weekly) or anti-TCR ylS MoAbs (UC7-13D5: 100 Mg + UC3-10A6: 400 pg weekly) from days -1 through +41 post-BMT. Re- cipients of supplemental G8 LN cells + no MoAb all succumbed to lethal GVHD by day 75 post-BMT (Fig 2A).

Fig 2. Actuarial survival rates and post-BMT mean weight curves in B6 recipients of pan- TCD G8 BM + G8 LN cells or no supplemental G8 LN. Pan-TCD BM (20 x lo6 cells) from G8 donors was coinfused along with 3 x lo6 TCR ylS' cells obtained from G8 LN or no supplemental G8 LN cells into lethally irradiated 19.0 Gy TB1 by x-ray1 B6 recipients In = Blgroup). Some groups as indicated received anti-CD4 or anti-TCR ylS MoAbs beginning on day -1 and contin- uing weekly through day 41 post-BMT. The actuarial survival (A) and post-BMT mean weight curves (B) are plotted.

Recipients of the same inocula with anti-CD4 MoAb uni- formly succumbed to lethal GVHD by day 88 post-BMT, which was marginally (P = ,058) superior to recipients of no MoAb. Both of these groups of mice had a significantly lower ( P 5 .0075) actuarial survival rate as compared with recipients of either no supplemental G8 LN cells (75%) or supplemental G8 LN cells + anti-TCR y lb MoAbs (84%). Weight curves (Fig 2B) were consistent with the clinical evidence of GVHD in recipients of G8 LN cells + no MoAb or anti-CD4 MoAb and the absence of GVHD in recipients of no G8 LN cells or G8 LN cells + anti-TCR y /b MoAbs. Thus, TCR ylS' cells can mediate GVHD in the absence of CD4' T-cell help, although CD4+ T cells may either speed




y/6 T CELLS IN MURINE GVHD 831

the onset of or contribute to the tissue destructive properties of TCR y/6+ T-cell mediated lethal GVHD.

At 5 months post-BMT, splenocytes were obtained in three representative mice per group that received G8 TCD BM + no LN cells + no MoAb or G8 TCD BM + G8 LN cells + anti-TCR $6 MoAbs. There were comparable mean percentages of splenic TCR (YIP' cells (12% to 13%) in these groups of mice. However, recipients of G8 TCD BM + G8 LN cells + anti-TCR y/6 MoAbs had a modest in- crease ( P = .047) in TCR y/6 cells as compared with recipients of G8 TCD BM + no LN cells + no MoAb (5% ? 0% v 3% 2 0%, respectively), although it is unclear if these differences are biologically significant. Mean numbers of splenocytes and percentages of CD4+, CDV, B220+ or Mac- l + cells did not significantly differ in these two groups. Recipients in both groups had high levels of alloengraftment with mean donor cell percentages greater than 89% and mean host cell percentages less than 5%. These data demonstrate that donor G8 LN-derived TCR y/S+ cells contribute to splenic TCR y/6+ T-cell repopulation.

To see if higher numbers of TCR y/6+ cells could be detected early post-BMT than were found late post-BMT, in a separate experiment, spleens taken from three representative B6 recipients of G8 TCD BM (20 X lo6 cells) + 3 X lo6 G8 TCR y/6+ cells were individually phenotyped early (day 9) post-BMT using two-color flow cytometry. At this time, there was a mean value 2 1 standard error of the mean of 24.6 ? 10.0 X lo6 splenocytes, 12% ? 1% donor TCR y/6+ cells, and 0% 2 0% donor or host CD4+ or CD8+ cells. Because only 3 X lo6 total G8 TCR y/6+ cells were infused, most of which probably localized to areas other than the spleen and approximately 2.8 X lo6 TCR y/6+ cells are detectable in the spleen on day 9 post-BMT, there is, apparently, a considerable expansion of these cells in vivo.

TCR y6+ cells injiltrate GVHD target tissues. To con- firm that TCR y/6+ cells were involved at the tissue level in the GVHD destructive process, tissue histology and immunohistochemistry were performed on day 7 post-BMT in irradiated B6 mice that received G8 TCD BM (20 X lo6 cells) + no LN or 3 X lo6 G8 TCR y/6+ LN cells with or without either anti-TCR yl6 or anti-CD4 + anti-CD8 MoAbs. As early as day 7 post-BMT (Fig 3), there was a massive infiltration of the liver and the colon, a destructive process present in the spleen that was evident in recipients of G8 LN cells, but absent in recipients of G8 LN + anti- TCR y/6 MoAbs. Mice receiving supplemental G8 LN cells + anti-CD4 and CD8 MoAbs had the splenic destructive process and moderate involvement in the liver and colon. In situ immunohistochemistry demonstrated that TCR y6 cells predominated in the skin, liver, colon, and lung in recipients receiving G8 LN cells and no MoAb or anti-CD4 + anti- CD8 MoAbs (Fig 4, Table 1). Recipients of supplemental G8 LN cells + anti-TCR y6 MoAbs had few TCR y6+ or TCR cup+ cells infiltrating these tissues. In a separate experiment in which tissue immunohistochemistry was performed in moribund B6 mice dying of GVHD, TCR y6+ cells were present in GVHD target tissues at the time of elective killing 74 days after infusion of G8 LN cells (3 x IO6 TCR y/6+ cells) into mice that received G8 TCD BM

(20 X lo6 cells) and no MoAb or anti-CD4 MoAb but were virtually absent in recipients of supplemental G8 LN cells with anti-TCR y6 MoAbs (data not shown). These data, along with the survival and weight results shown in Fig 2 indicate that TCR y6' cells are required for the GVHD- induced mortality and implicate TCR y/6' cell infiltration in the tissue destructive process of GVHD.

Preferential GVHD causing response of donor TCR y6 cells to p rather than P antigens expressed in the host. Because G8 TCR y/S+ cells respond strongly to Tb+ but weakly to T+ targets, we performed our GVHD studies in B6 congenic mice differing in the TL region [B6: Tb+ v B6: Tla"+ (B6.Tlaa)]. Irradiated B6 or B6.Tla" recipients were given G8 TCD BM (10 X lo6 cells) + 3 X lo6 G8 TCR y / 6' LN cells (containing 46% TCR y/6+, 8% TCR (YIP+ cells: 10% CD4+, 8% CD8+ cells). Recipients expressing T" region determinants had a significantly (P = .0062) higher actuarial survival rate as compared with congenic recipients expressing Tb region determinants (Fig SA). Weight data (Fig 5B) and clinical findings were consistent with an acute GVHD- induced lethality. These results suggest that the TCR y/6+ LN cells are recognizing T region encoded antigens on GVHD target tissues.

In a second approach, B6 were sublethally irradiated and infused with highly purified G8 y/6 TCR LN cells. One advantage of this model system is that the effect of T cells can be measured without being affected by donor BM cells that could either counterregulate or facilitate the GVHD process. To determine if Tla" expression was sufficient for GVHD generation in sublethally irradiated mice, B6 or B6.Tl" mice were sublethally irradiated and infused with 3 X lo6 G8 TCR y/6 LN cells (TCR y/6+: 87%; TCR a@+: 6%; CD4+: 0%; CD8+: 0%; B220': 2%; Mac-l+: 2%) without donor BM. By day 14 post-BMT, B6 mice receiving enriched G8 TCR y/6+ LN cells had evidence of hematopoietic failure. Hematocrit values in these mice were 10.8% 2 2.0%, which were significantly (P < .001) lower than the values of 24.3% 2 1.7% observed in B6.Tla recipients of the same inocula. By day 21, all but three of the B6 recipients had died and the hematocrit values in each mouse were 59.0% versus mean values of 27.4% 2 3.6% in the B6.Tl" mice. B6 recipients had a profound and rapid early post- BMT weight loss with mean body weights falling more than 26% by 3 weeks post-BMT, in contrast to less than 2% weight loss in the B6.Tla mice. By 1 month post-BMT, actuarial survival rates were 0% for B6 recipients, which was significantly ( P = .0015) lower than the 100% actuarial survival rates in the B6.Tla" recipients (Fig 6A). Weight data were consistent with a rapidly lethal acute GVHD process in B6 but not B6.Tla" recipients (Fig 6B). These data suggest that host T region determinants are recognition molecules for donor TCR yl6+ T cells in the GVHD destructive process.

DISCUSSION

In this report, we show that G8 Tg TCR y/6+ cells can mediate acute lethal GVHD when transferred into irradiated B6 recipients expressing Tb determinants. In our studies, antigenic determinant@) encoded by Tb region genes appear to serve as recognition molecules for donor TCR y/S+ cells




832 BLAZAR ET AL

Fig 3. Histologic assessment of GVHD in recipients of TCR y/S' LN cells. Haematoxylin and eosin stained cryostat sections of liver, colon, and spleen were taken 7 days post-BMT in irradiated B6 mice receiving G8 TCD BM (20 x 10' cells) + no LN, G8 TCD BM + LN (containing 3 x 10' TCR ylS' cells), alone, or with anti-TCR y8 MoAbs or anti-CD4 + anti-CD8 MoAbs. Mice receiving G8 TCD BM + LN alone or with anti- CD4 plus anti-CD8 MoAbs had evidence of perivascular and peri-bile duct mononuclear cell infiltrates in the liver. Mononuclear cell infiltrates were also seen invading the colonic mucosa of these same groups of mice along with a splenic destructive process exhibiting many apoptotic- looking cells (arrows) and numerous large syncytial aggregates. Mice treated with anti-TCR yS MoAbs had no evidence of infiltrates in GVHD target organs. (Original magnification x 50.)

Fig 4. lmmunoperoxidase staining of liver, colon, and skin taken 7 days post-BMT in B6 mice receiving TCR y/S' cells. Irradiated recipients received G8 TCD BM (20 x 10' cells) + LN (containing 3 x 10' TCR ylS' cells) with either anti-TCR y~5 MoAbs or anti-CD4 plus anti-CD8 MoAbs. Cryostat sections were stained for either yS TCR using GL3 biotinylated MoAb or ap TCR using H57-597 biotinylated MoAb. Anti-yS TCR treated mice, which had no evidence of GVHD, had no y6+ TCR cells and scant crp' TCR cells in GVHD target organs. In contrast, the GVHD target organs of mice receiving anti-CD4 + anti-CD8 MoAbs had invasive mononuclear cell infiltrates consisting predominantly of y8' TCR cells and rare ap' TCR cells. (DAB chromogen, methyl green counterstain, original magnification x 100).




yl6 T CELLS IN MURINE GVHD 833

Table 1. Frequency of TCR y/S+ and TCR alp' Cells Infiltrating GVHD Target Tissues Obtained From Lethally Irradiated B6 Recipients of pan-TCD G8 BM With or Without Supplemental G8 LN Cells

Skin Liver Colon Lung

G8 LN MoAb In Vivo Day Post-BMT TCRaS TCR y6 TCRa/3 TCRy6 TCRaS TCR y6 TCR TCR 7 6

None None 7 0.8 8.5 0.2 0.1 1 .o 0.5 0.0% 0.0% 3 x 10' None 7 0.4 10.4 0.3 11.1 1.6 3.8 0.2% 6.8% 3 x IO' Anti-CD4 + 8 7 0.0 22.0 0.0 13.0 0.3 89.0 0.0% 1 .O% 3 x IO' Anti-TCR yb 7 0.1 0.0 1 .o 0.0 3.6 0.4 0.6% 0.0%

B6 mice were lethally irradiated (900 cGy TBI) and infused with pan-TCD G8 BM + 3 x 10' G8 LN cells. Some groups were also given anti- CD4 + anti-CDIMoAbs or anti-TCR yl6MoAbs on day -1 of BMT. Frozen tissue sections were obtained 7 days post-BMT and stained for the expression of TCR a/O+ or TCR y/6' cells. Data are expressed as the frequency of positive cells/mm* for all but the lung tissue, which is expressed as percentage of nucleated cells. Seven separate fields per tissue were analyzed. The data shown are representative of two mice studied. An illustration of these data from the MoAb treated groups is shown in Fig 4.

as congenic B6.A-Tla"/BoyEg (T") recipients do not experi- ence an acute lethal GVHD as do B6-Tb recipients. T-region products, in contrast to classical MHC class I gene products, are relatively nonpolymorphic, do not always require peptide binding to confer specificity to T-cell clone^,'^*^' and are poorly immunogenic to TCR a//?+ ~ e l l s . ~ ~ * ~ ~ As such, T region determinants, such as TL antigens, do not elicit a CTL response by TCR a/@+ cells."*45 However, when expressed in the skin at high levels in transgenic mice, grafting of skin from transgenic B6 mice encoding the TL gene product, T3b, onto TI- recipients results in the generation of CD8+ TCR alp+ T3b specific CTLs and rejection of these skin Thus, under certain conditions, T region determinants can serve as transplantation antigens for TCR alp+ T cells in vivo. In this study, we now report that T region determinants, even expressed at normal levels in nontransgenic mice, can serve as transplantation antigens for TCR y/6+ cells.

The in vitro reactivity of TCR y/S+ cells to T region antigenic determinants has been demon~trated.'~"~ Two T region gene products, TIOb and T22b, are known ligands for TCR y/6+ cells as measured by in vitro proliferative and CTL responses.'8-2' Evidence also exists to show that Tb gene products expressed in vivo cause G8 TCR y/S+ cells to be deleted in the thymus and peripheral lymphoid organs.25 G8 TCR y/6+ cells that localize to the intestinal epithelium be- come activated in response to host alloantigen with a proportion of intestinal intraepithelial lymphocytes undergoing clonal deletion in the ~ e r i p h e r y . ~ , ~ ~ The remaining nonde- leted G8 intestinal intraepithelial TCR y/6+ cells are specifically nonresponsive to Tb alloantigens,M347 which was determined by transferring G8 TCD BM cells into irradiated (8.0 Gy TBI) Tb bearing host (BALB/c X C57BU6) F1 mice.

The data reported here clearly demonstrate that highly enriched G8 Tg TCR y/6+ cells can mediate acute lethal GVHD when transferred into irradiated B6 hosts. The tissue pathology of TCR y/S+ cell mediated GVHD closely resem- bles that of TCR cell mediated GVHD.26 TCR y/6+ cells localized to the same sites with preferential migration into epithelial surfaces in the lung, liver, and colon. While TCR y/S+ cells are typically situated in each of these loca- tion~,". '~ the endothelial linings of blood and lymphatic ves- sels are not typically occupied by these cells. One exception to the similarity of TCR y/6+ to TCR alp+ GVHD is the skin where TCR y/6+ cells were predominantly present in

the epidermis and to a lesser extent throughout the dermis and juxtaposed to hair follicles as seen in TCR alp+ cell mediated GVHD. Thus, the GVHD destruction does not appear to simply be the result of normal homing of TCR yl6' cells to these organs, but rather represents the result of a tissue-specific process culminating in tissue damage.

Our studies show that lethal GVHD can be induced in irradiated B6 recipients of highly purified G8 transgenic TCR y/6+ cells. We have also shown that there is a preferential and early post-BMT infiltration of GVHD target tissues with TCR y/S+ cells in recipients dying of rapidly progres- sive GVHD. In the initial experiments, the small number of residual TCR a/@' cells present in these LN preparations is unlikely to contribute significantly to the GVHD-induced mortality because as many as 1 X lo6 TCR alp' cells from BALB/c (H-2d') donors was not sufficient to mediate GVHD in heavily irradiated B6 recipients in contrast to the GVHD- induced lethality observed when 1 to 3 X lo6 TCR y/6+ cells were infused in the same experiment. Moreover, the infusion of anti-CD4 or anti-CD4 + anti-CD8 MoAbs in B6 recipients of TCR y/6+ cells did not protect these mice from the lethality or tissue pathology seen in nontreated mice in striking contrast to recipients of anti-TCR y/6 MoAbs. However, because the infusion of anti-CD4 MoAb in recipients of highly purified TCR y/6+ cells did provide a transient, albeit modest protective effect, it is possible that the small numbers of CD4+ T cells produced cytokines (eg, interleu- kin-2 [IL-2]), which helped to drive TCR y/6+ proliferation or were directly involved in the tissue destructive effects of TCR y/6+ T cells. Nonetheless, highly purified TCR y/6+ cells in the absence of detectable CD4+ or CD8+ T cells, caused lethal GVHD when infused into sublethally irradiated B6 recipients. These data, along with the demonstration that TCR y/6+ cells persist in the tissues of moribund mice dying of GVHD later post-BMT support our contention that TCR a/@+ cells are not required for GVHD-induced lethality.

An advantage in using G8 mice as donors for these experiments was the fact that the transgenic TCR y/6+ cells express a TCR with specificity for T region determinants expressed on the B6 host strain and thus are present in high frequency in the donor LN inoculum. We speculated that biasing the GVHD system toward the infusion of high numbers of transgenic TCR y/6+ T cells, most of which were expressing a TCR that would enable these cells to be host alloreactive,




BLAZAR ET AL

0.8 -

0.6 -

0.4 -

0.2/

1 n = 1Dlgroup

A..? B6.T recipient of G8 TCD BM + G8 TCR 716 LN cells (3 x lo&)

f b."" p

B6 (T ) recipient of G8 TCD BM + G8 TCR V 6 LN cells (3 x 1Oe6)

b

u . u , . , . * . , . . . . . , - , 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0

DAYS POST-BMT

22 - B6.T' recipient of Q8 TCD BM + G8 TCR V 6 LN cells (3 x lOe6)

Fig 5. Actuarial survival rates and pod-BMT mean weight curves in B6 or B6.Tla' recipients of pan-TCD G8 BM + G8 LN cells or no supplemental Q8 LN.

B6 (Tb ) reclpionts of Q8 TCD BM + Pan-TCD G8 BM (10 x l@ cells) G8 TCR 716 LN cells (3 X 1006) was coinfused along with 3 x 10'

G8 TCR y/6* LN cells into heavily irradiated (900 Coy TBi) B6 or B6.Tlao recipients (n = 8/groupl.

14 -

12 - 2 8 1 8 2 8 3 8 4 8 The actuarial survival (A) and

DAYS POST-BMT

would optimize the likelihood of determining whether TCR y/6+ T cells could have the capacity to migrate to and cause destruction in GVHD target tissues. However, even transgenic T cells with alloreactive capabilities are not always capable of mediating GVHD in vivo:' Therefore, it was particularly interesting that transgenic TCR y/6+ T cells were able to do so. In support of the hypothesis that TCR y/6+ T cells may play a role in GVHD tissue destruction, investigators have recently reported that the infusion of an anti-TCR y/6 MoAb (UC7-13D5) into non-irradiated (C57BU6 X DBN2)Fl (H-2") recipients of 2 X 10' B6 parental splenocytes reduced host TCR y/6+ intraepithelial

post-BMT mean weight curves (B) are plotted.

intestinal lymphocytes, reduced apoptosis and mitosis of crypt cells in the intestinal mucosa, and reduced the infiltration of donor-derived T cells into the epitheli~m.4~ No effects on splenomegaly, an in vivo measurement of the graft-versus-host response, was found. These previously reported results suggested that TCR y/6+ cells may have the capacity to participate in the enteropathy of acute GVHD in recipients of donor cell inocula with normal numbers of nontransgenic T-cell populations. Additional studies to determine how im- portant TCR y/S+ T cells may be in mediating GVHD lethality and tissue destruction in other organs both in the presence or absence of TCR alp+ T cells are clearly warranted. The




y/6 T CELLS IN MURINE GVHD

A 1.0 r

0.a -

0.6 -

0.4 -

0.2 -

&.. . .. . . . . 4

n = Elgroup

"." . 0 1 0 2 0 S O

- . DAYS POST-BUT

.S . - 1 S 1 0

DAYS POST-BMT

Fig 6. Actuarial survival rates and post-BMT mean weight data in sublethally irradiated B6 or B6.Tla' recipients of highly enriched TCR y/6* G8 LN T cells. TCR y/6+ G8 LN cells (3 x l@) ware infused into sublethally irradiated (700 cGy TBll B6 or B6.Tla' recipients (n = 8/ group). The actuarial survival (A) and post-BMT mean weight curves (B) ara plotted.

availability of TCR cy- or TCR p- chain deletional mice5' will aid in these studies, as the in vivo infusion of an anti- TCR alp' MoAb," an alternative approach to the use of deletional mice, could activate TCR alp' T cells by TCR cross-linking. Attempts to infuse large numbers of highly purified TCR y/6+ T cells from nontransgenic mice will be hampered by their relatively low frequency as compared with TCR a/p' T cells.

An interesting aspect of the current study is the generation of acute lethal GVHD associated with hematopoietic failure that was observed when highly purified G8 Tg TCR y/6+ cells were infused into sublethally irradiated B6 recipients. T region determinants appear to be critical target antigens for GVHD-induced lethality in this sublethally irradiated system. Even though T region antigens have been shown to be expressed on cells of hematopoietic direct evidence that T region determinants are expressed on hema-

a35

topoietic cells necessary to sustain the recipient, has to our knowledge, been lacking. Because the infusion of TCR y / 6+ cells into sublethally irradiated B6 recipients caused a rapidly lethal aplasia syndrome, these data would suggest that hematopoietic cells and/or stromal cells express T region determinants in sufficient density to serve as target antigens for TCR y/6+ cells. Given that GVHD-induced aplasia results from the recognition of TlO' or T22' gene products, it is interesting to note that sublethally irradiated BlO.BR, a strain that expresses T" determinants, also succumbed to TCR y/6+ cell mediated GVHD between 2 to 3 weeks post- BMT, which was associated with profound anemia (data not shown). Because B1O.BR mice have a number of polymor- phisms in the Qa-T intervalI5 and are superior targets for G8 TCR y/6+ cell proliferative responses as compared with B6.Tla" stimulators, it is possible that the protein products of one or more genes in the Qa-T region interval either share a common epitope with TlO' or T22b gene products or have cross-reactive epitopes that are sufficiently antigenic for G8 TCR y/6+ cells to mediate a GVHD response in vivo.

What is known about TCR yl6' T cells that may be rele- vant to GVHD? Human alloreactive TCR y/6' cytotoxic T- cell clones with HLA class I restriction elements have been generated by Ciccone et al:' raising the possibility that such alloreactivity could be generated in vivo following the in vivo infusion of mature T lymphocytes. Other investigators have shown that TCR y/6+ T cells with alloreactivity can be generated from bulk cultures, although these TCR y/6+ T cells were nonspecific in their lytic proper tie^.^' Viale et als3 have shown that patients with acute GVHD have higher numbers of circulating TCR y/6+ cells than those without acute GVHD.

While it is unclear to what extent TCR y/6+ T cells contribute to GVHD target tissue damage and lethality in nontransgenic murine BMT model systems or in humans, consideration of the potential role of TCR y/6+ cells is warranted. Such studies may lead to a better understanding of the biological properties of TCR y/6+ T cells in the BMT setting which might ultimately translate into improved GVHD prophylactic and/or treatment strategies.

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major histocompatibility complex class ib antigens gamma/delta

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