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Host antidonor effector T cells represent a major barrier to the successful engraftment of allogeneic donor hematopoietic progenitor and stem cells. Here, administration of a complex of IL-2 and anti-IL 2 antibodies (IAC) significantly enhanced donor chimerism early as well as long-term engraftment following reduced-intensity conditioning (RIC) and allogeneic major histocompatibility complex (MHC)-matched hematopoietic cell transplantion (HCT). Timing of administration of this complex was crucial: administration of IAC post-HCT more efficiently facilitated marrow engraftment than pre-HCT treatment. Donor chimerism persisted to >6 months post-HCT. Importantly, this approach clearly suppressed the emergence of host antidonor CD8 T cells 2 to 3 weeks post-HCT as assessed by tetramer staining. Following in vivo reactivation of IAC-treated and control recipients at >5 months post-HCT with donor antigen, only PBS-treated control marrow allograft recipients responded with tetramer-binding CD8 cells. In total, the present findings support the notion that the transient activation and expansion of host Tregs in situ post-HCT can be explored as a new approach to regulate host alloreactivity posttransplant. Interestingly, direct stimulation of recipient Treg cells in RIC recipients obviated a requirement for exogenous Treg cell transfusion in this model and may represent a viable alternative to, and/or complement the adaptive transfer of Treg populations in clinical HCT.

During the past decade, 2 major cytolytic pathays used by natural killer (NK) and T cells have been horoughly described. Both the perforin/granzyme nd death receptor (ie, fas/fas ligand [fasL]) pathways ltimately result in apoptosis of the targeted populaion [1]. Studies have been preformed using mutant nd knockout strains to investigate NK and T-cell mpairment in viral and tumor immune responses, as ell as in transplantation models, to investigate the ontribution of these pathways in resistance to allogeeic cell engraftment [2-9]. Several early studies ielded interesting results with regard to cytolytic deciencies and so-called allograft resistance. For examle, major histocompatibility complex (MHC)–hetrozygous recipients were observed to reject donor arental marrow despite granzyme B deficiency [4]. dditionally, lethally conditioned MHC-disparate reipients deficient in perforin or expressing defective asL were also able to reject allogeneic hematopoietic rafts after a standard dose of bone marrow (BM) was ransplanted [2]. By using a different approach in which diphtheria oxin was transgenically expressed under a granzyme B romoter to broadly eliminate cytolytic barrier cell opulations, results were obtained that were similar to hose described previously: no detection was reported f diminution in NK resistance to hematopoietic stem t

The lymphohematopoietic function of the spleen in mice varies dependent on age and hematopoietic requirements. A method was developed to study splenic repopulation of mature and progenitor cell populations by grafting neonatal or adult spleen tissue under the renal capsule of splenectomized mice. Two weeks following implant of irradiated syngeneic neonatal spleens into B6-Ly 5.1 or B6-gfp recipients, host lymphoid (B220(+), CD4/8(+)) and myeloid cells (CD11b(+)) had repopulated the splenic grafts and constituted the majority of cells contained in these heterotopic implants. Notably, the percentage of lymphoid and myeloid cells approximated adult levels in contrast to preimplant neonatal spleen levels. This observation indicated relatively rapid repopulation of the grafted tissue by adult host cells and suggests that the repopulation patterns were regulated by the host. Three months post-implantation, the cell composition in the graft remained comparable to adult levels. Microscopic examination demonstrated normal splenic architecture including follicles and red pulp. Lymphocytes within the graft were functional as indicated by their proliferation in response to lipopolysaccharide (LPS) and concanavalin A (ConA) stimulation. Progenitor cell activity determined by colony-forming unit interleukin-3 (CFU-IL-3) levels was also present in these grafts. Splenic implants were then assessed in transplant models following lethal irradiation and syngeneic or allogeneic bone marrow transplantation (BMT). Two weeks post-BMT, adult splenic tissue implants contained donor-derived B cells, T cells, and myeloid cell populations. As typically detected in the host spleen post-BMT, the grafted tissue also contained elevated levels of donor progenitor cells. By 3 months post-BMT, CFU-IL3 levels in the graft reflected the decreased levels characteristic of adult levels. The functional integrity of post-transplant splenocytes in the implants was also demonstrated by mitogenic responsiveness. In summary, this method should provide a useful model for the transfer of the splenic microenvironment to study the biology of the spleen in non-transplant and BMT settings.

These studies investigate the involvement of the spleen in progenitor (PC) cell numbers and "cross-talk" with the marrow compartment following syngeneic or allogeneic bone marrow transplantation (BMT) in sham or fully splenectomized mice. Intact recipient B6 mice were lethally irradiated prior to transplant with T cell-depleted bone marrow (BM-TCD). The kinetics of PC reconstitution following i.v. transplant consistently revealed a dramatic increase in splenic colony-forming unit interleukin-3 (CFU IL-3) and CFU (high proliferative potential-(HPP) levels between days 5 and 12 post-BMT. Direct injection of TCD-BM into the recipient marrow cavity did not alter this pattern of reconstitution in the splenic compartment. In contrast to spleens from normal adult B6 mice containing 0.9% and 0.6% of the total combined splenic and marrow committed (CFU IL-3) and primitive (CFU-HPP) progenitors, respectively, spleens of syngeneic BMT recipients at day 12 contained a 10-fold increase (p < 0.001) over the progenitor levels in normal spleens. These splenic numbers decreased to normal, homeostatic levels by day 28 post-BMT. In contrast, the level of marrow CFU IL-3 progenitors continued to increase post-transplant, reaching near homeostatic levels by day 28 post-BMT. Interestingly, early seeding of 5- (and -6)carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled or green fluorescent protein (GFP) donor bone marrow cells (BMC) to the marrow compartment was not different in sham splenectomies or recipients splenectomized 14 days earlier. However, recipient splenectomy consistently resulted in significantly higher numbers of CFU IL-3 in the bone marrow during the first 2 weeks post-transplant compared to sham controls. These elevated levels exceeded the combined progenitor numbers of the splenic and marrow compartments of intact recipients. Notably, this increase in marrow progenitor activity in splenectomized recipients was observed after syngeneic as well as allogeneic BMT. Allogeneic transplants across major, or those limited to minor, histocompatibility antigen differences exhibited this increased marrow progenitor activity. Splenectomy performed 2 h post-transplant to assure "normal" marrow seeding also resulted in higher marrow progenitor activity. Thus, this "marrow response" to splenectomy is not induced by early "shunting" of infused BM cells to the marrow compartment. These results suggest that communication between the splenic and marrow compartments following syngeneic and allogeneic BMT exists during early hematopoietic reconstitution, one effect of which is to impact the compartmental distribution of donor progenitor cells. The role of the spleen on engraftment, chimerism, and tolerance in allogeneic BMT models are now under investigation.

Recognition of the ability of CD4(+)FoxP3(+) T cells (Treg) to influence the generation of peripheral immune responses has engendered enthusiasm for the development of strategies utilizing these cells to regulate immune responses in clinically important settings including transplantation, autoimmunity and cancer. A number of studies have reported effective regulation utilizing ex-vivo expansion approaches and subsequent transfer of Treg populations in experimental models. This commentary discusses recently emerging strategies to activate and expand Treg cells in situ which include antibodies, antigen presenting cells and the use of IL2 / anti-IL2 antibody complex. The development of reagents which can stimulate and / or remove Treg cells in situ would represent an important advance towards facilitating new opportunities to harness this compartment for the augmentation of 'wanted' or suppression of 'unwanted' immune responses. Simultaneous targeting of multiple molecules on Treg cells may ultimately enable more effective control of this regulatory sector.

Abstract 2441 Poster Board II-418 Reducing and ultimately eliminating conditioning while enabling donor cell engraftment remains an elusive goal of allogeneic hematopoietic stem cell transplantation. The control of host resistance (HVG) to donor allogeneic transplantation antigens is pivotal for engraftment and the successful induction and maintenance of immune tolerance. Using a well characterized minor histocompatibility antigen (MiHA) matched allogeneic (HSCT) model, we have demonstrated a requirement for CD80/CD86 expression on donor APC in order to elicit resistance under reduced intensity conditioning (RIC) of 5.5Gy TBI. Based on this observation, we hypothesize that expansion of host Treg cells which may engage donor APC – the site of host anti-donor Tconv activation - is an advantageous strategy to effectively inhibit HVG responses. To address this hypothesis, we reduced TBI almost 30% to 4.0Gy, and found that even recipients transplanted with 4×106 CD80-/-CD86-/- T cell depleted donor BMC failed to engraft. Experiments were therefore initiated to target and rapidly expand host Tregs by administering IL-2 + anti-IL2 (IAC) complex to further strengthen suppression of HVG. C3H.SW (H2b) recipient mice were conditioned with 4.0Gy TBI (D-1) and B6-CD80-/-CD86-/- TCD-BM (H2b, 7×106) infused on the following day (D0). IAC was introduced on the day of conditioning (D-1) and then daily on days D 0, 1 and 2. We reported that anti-IL-2 + IL-2 complex can expand residual host Treg cells in RIC recipients (Blood, 113:733-743, 2009; Biol. Blood Marrow Transplantation. 15: 785-794, 2009). One month later, 1/3 of mice receiving complex – but not untreated controls demonstrated significant levels (15% B220+) of hematopoietic chimerism. These results, while encouraging, indicated that stimulation of the endogenous Treg compartment alone in 4.0 Gy recipients was not sufficient to regulate the HVG response. We then modified our approach by infusing 1×106 recipient Tregs obtained from normal C3H.SW mice one day following 4.0Gy RIC, i.e. at D0 and began expanding these cells with IAC injected early on D0 and again on D1 prior to the B6-CD80-/-CD86-/- TCD-BM graft – delayed one day and administered on D1. 100% of these recipients (n=3/gr) exhibited high donor chimerism (43.7+/-10.8 SD) 6 weeks post-HSCT, whereas all non-Treg infused control recipients rejected these marrow allografts. Results of 4 independent transplant experiments demonstrated 10/12 recipients contained similar levels of B220+ chimerism (mean donor: 53.9%) and significant T cell chimerism (mean donor: CD4, 19.8%; CD8, 35%). When both host Tregs and IAC were employed, strong chimerism was observed using 4 or 7×106 CD80-/-86-/- TCD-BM grafts. Notably, addition of 1×106 donor (i.e. B6-wt) Tregs and IAC treatment did not augment chimerism vs. IAC only in 4.0Gy conditioned C3H.SW recipients. These results suggest that the introduction of host Tregs 2 days prior to the BM graft allowed adequate time for IAC to mediate Treg expansion in the lymphopenic environment to generate sufficient Treg levels and together with the absence of donor APC B7 signaling to host Tconv cells overcome, i.e. suppressed HVG responses. Transplants were then performed using the identical Treg and IAC protocol with B6-wt TCD-BM which is readily rejected in 4.0 Gy TBI conditioned recipients. Despite the presence of B71/2 on B6-wt donor APC, one-third of the recipients receiving an infusion of host Tregs and IAC treatment exhibited chimerism, although lower (6.3%) than observed with B7.1/2 deficient donors. Transplants of wild type TCD-BM into MiHA mismatched recipients will determine how anti-B7.1/2 mab blockade, together with rapid in situ expansion of infused host Tregs post-TBI and pre-HSCT can be utilized to further diminish immunosuppressive TBI conditioning ( Disclosures: No relevant conflicts of interest to declare.

Abstract 66 We recently demonstrated that administration of IL-2 complexed to the anti IL-2 antibody JES6-A12 (IAC) induced stable chimerism and engraftment of donor HSCT (BBMT 15:785, 2009). Based on suppression of anti-donor MiHA-specific host T cells, it was concluded that IAC administration enhanced chimerism by suppression of HVG. We proposed that in situ manipulation of host Tregs was crucial to facilitating engraftment and establishing tolerance in this model and hypothesized that the enhanced chimerism induced by this strategy was a direct result of host Treg activation, expansion and function following engagement of the IL-2 receptor CD25. To directly test this hypothesis, B6 CD4−/− (H-2b, Ly9.1−) mice were infused with highly enriched CD4 cells from B6-WT or B6-IL-2Rβ−/− mice deficient in CD25 expression and 4 days later conditioned with 5.5 Gy TBI. One day later, these mice were transplanted with MHC-matched, MiHA-mismatched 4 × 106 BALB.B (H-2b, Ly9.1+) TCD-BM. At days +3 and +5, all recipients were administered IAC and subsequently assessed for peripheral donor chimerism. By 2 weeks post-HCT, untreated control mice had increased circulating levels of CD8TETRAMER+ T cells (representing specific host anti-donor H60 MiHA reactive T cells) vs. IAC-treated recipients. Three months post-HCT, CD4−/− recipients of WT but not IL-2Rβ−/− CD4 cells were chimeric as evidenced by high levels of circulating donor cells (60% vs. Disclosures: No relevant conflicts of interest to declare.

Engraftment and the induction of tolerance to donor antigens is a central aim of allogeneic hematopoietic cell transplantation (HCT). Currently, the administration of Treg cells is being examined as a new approach to support these processes. Based upon the reported ability of IL-2 anti-IL-2 mAb complex (IACX) to expand CD4+CD25+ FoxP3+ (Tregs) in vivo, we hypothesized that the administration of this complex to allogeneic HCT recipients may expand host Treg cells and inhibit host anti-donor responses, thereby effectively facilitating hematopoietic engraftment. To begin to address these questions, the effect of IACX treatment on the frequency of circulating Tregs was examined. Two days following the second injection of IACX in normal or reduced intensity conditioned (RIC) mice, the % of circulating Tregs in IACX treated mice was strikingly increased vs. PBS treated controls. Notably, the expression of Treg CD25 (MFI) was also significantly elevated (p

Surmounting the barrier mediated by host T cells against successful engraftment of MHC-matched allogeneic marrow is a crucial objective of hematopoietic stem cell transplant (HCT). We proposed that the ability of unmanipulated donor CD4+ CD25+ regulatory T cells (Tregs) to suppress activation of host effector CD8+ T cells would enhance donor HC engraftment between strains disparate for multiple minor HA (MiHA), and promote stable chimerism. C57BL/6 (H-2b, Ly9.1−) mice conditioned (5.5 Gy TBI) 24 hrs earlier, were transplanted with Tregs and TCD-BM from 129P3/J (H-2, Ly9.1+) mice. By four weeks post-HCT, the mean frequency of circulating donor-derived (total Ly9.1+) cells was 15.7 ± SE 5.9% in recipients of 4 × 106 TCD-BM + Tregs (4.5 × 105) compared to the absence of donor chimerism in recipients of allogeneic HCT only (0.3 ± 0.09%) - a finding also evident from the analysis of circulating donor B220+ cells (8.7 ± 3.0 vs. 0.2 ± 0.06%). Based on these findings, we hypothesized that pre-HCT infusion of Tregs into a lymphopenic, allogeneic host might enhance their facilitating function. Accordingly, Tregs (4 or 1.5 × 105) were transplanted 24hr. post-conditioning either 3 days prior to HCT or co-transplanted with (day 0) donor TCD-BM. The transplant was rejected in recipients not administered donor Tregs. The higher dose of Tregs administered pre-transplant or co-transplanted facilitated equivalent donor cell chimerism. In contrast, administration of 1.5 × 105 Tregs resulted in chimerism only when this donor population was infused 3 days prior to HCT (1.6±0.9%). To test the hypothesis that donor Tregs suppressed host anti-donor specific CD8 T cells, we monitored host CD8+ T cell responses to the immunodominant donor epitope H60 by tetramer staining (LTFNYRNL/Kb). One month post-HCT of TCD-BM alone, the frequency of circulating tetramer+ CD8+ cells in these recipients rejecting the marrow graft was clearly greater compared to the frequency in recipients of TCD-BM + Tregs which engrafted (p0.05). Notably, transplants using unmanipulated host Tregs failed to support engraftment. These findings illustrate that donor Tregs support chimerism via suppression of host anti-donor antigen-specific T cells. Finally, the persistence of donor cells in circulation as well as in lymphoid tissues 3 months post-HCT indicated that long-term, stable chimerism was established by this Treg administration regimen. In total, our results demonstrate that donor Tregs promote MHC-matched allogeneic marrow engraftment under conditions of reduced intensity conditioning and that the strategy of pre-HCT Treg infusion supports the notion that antigen driven expansion of donor anti-host reactive Treg cells prior to stem cell transplants can increase their efficacy to facilitate hematopoietic engraftment. Experiments to study transient vs. long-term engraftment and expansion of donor Treg cells are being examined.

T cell populations specific for transplantation antigens have been detected in sensitized individuals following multiple blood transfusions, marrow transplants as well as in multiparous females. Resistance to allogeneic hematopoietic cell transplant (HCT) in such sensitized individuals is consistent with the presence of a host memory T cell (TM) population specific for donor cell antigens. We hypothesized that a single donor minor histocompatibility (MiHA) epitope could elicit antigen-specific CD8 TM capable of resisting MHC-matched allogeneic hematopoietic cell engraftment. To address this question, CD8 TM were generated against a single MiHA epitope to determine if such cells could mediate resistance after ablative TBI conditioning. B6 mice were sensitized 2X to the H60 immunodominant MiHA epitope utilizing bone marrow-derived dendritic cells pulsed with the H60 (LTFNYRNL) peptide. Three weeks following booster sensitization, CD8 T cells were detected by tetramer staining in peripheral blood samples. These T cells exhibited a phenotype characteristic of memory cells (CD44hi, Ly 6C+). B6 (H2b) mice containing CD8+ H60+ T cells were subsequently conditioned with 9.0 Gy TBI and transplanted with 5 × 106 BALB.B (H2b) BM-TCD. One week post-transplant, naive recipients of BALB.B (H60+) or B6-H60 congenic TCD-BM contained >10-fold higher levels of circulating donor cells than the B6 dendritic cell/peptide sensitized recipients. Donor progenitor cells were also found to be significantly reduced in sensitized recipients of allogeneic TCD-BM at this time. Two weeks post-HCT, recipients of syngeneic marrow exhibited >10-fold greater frequency of circulating donor cells compared to recipients of MHC-matched allogeneic marrow (< 5% donor chimerism was detected). These findings demonstrate that host T cells with specificity against a single donor MiHA determinant are sufficient to induce resistance to MHC-matched allogeneic marrow engraftment. Such observations regarding the effector response of HVG contrast those by donor T cell responses post-transplant in which single MiHA differences fail to induce GVHD. Finally, heterologous immunity to virus has been reported to generate allo-reactive TM cells. Since such TM repertoires could include specificity for MiHA immunodominant epitopes, the presence of TM populations that can mediate resistance in “naive” recipients may be more prevalent than hitherto appreciated.

We are interested in investigating the survival and location of host CD8 memory (TM) cells following allogeneic hematopoietic cell transplants (HCT). The H60 antigen dominates the immune response in B6 mice primed with BALB.B antigens (B6BALB.B). In such primed recipients, transplant of BALB.B MiHA-disparate marrow BM induces CD8 TM responses that mediate resistance to bone marrow engraftment. Therefore an H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to detect host H60-specific CD8 TM in the spleen and marrow compartments. In the marrow compartment, the frequency of H60+ cells amongst the CD8+ T cell population was significantly (p = < 0.05) higher compared to spleen levels. In both BM and spleen, >90% of CD8+ H60+ cells expressed the central memory phenotype (CD44+, Ly6C+ CD25−, CD69−). To mediate resistance to progenitor cell engraftment, H60-specific effector CD8+ TM must first survive the immediate post-HCT milieu in the hemopoietic compartments. We observed a dose-dependent increase in percent of CD8+ T cells expressing the H60 TCR in the spleen as well as bone marrow in B6BALB.B mice irradiated at 3.0, 6.0 (non-ablative) and 9.0 Gy (ablative) 24 hrs post-HCT. Five days post-HCT, CD8+ H60+ cells were also readily detectable. At this time, resistance to engraftment assessed by IL-3 progenitor assay was present in sensitized, ablatively conditioned recipient mice transplanted with 5 X 106 BALB.B or congenic H60 TCD-BM. We then utilized a “double transplant” model to determine the compartmental distribution and function of MiHA-specific TM at later intervals (14 days) post-HCT. B6 mice containing CD8 TM were ablatively conditioned and 24 hrs later received syngeneic B6 (Ly 5.2) BMT. Twelve days following this syngeneic HCT, the mice were irradiated at 4.5 Gy and administered a second HCT consisting of either syngeneic (Ly 5.1) or allogeneic HCT (5 X 106 TCD-BM). These recipients were assessed for donor progenitors 10 days later. At this time point, H60+ CD8 TM were again readily detectable in both compartments, indicating that these TM effectors survived ablative (and subsequent non-ablative) conditioning and were present at this time post-transplant. Resistance to allogeneic marrow engraftment as assessed by IL-3 progenitor assay was detected after this subsequent transplant with BALB.B BM. These findings indicate that host TM survive and function following ablative conditioning and HCT. To evaluate if the viability of host CD8 TM differed in the host compartments following HCT, Annexin V and 7-AAD staining was performed. A higher frequency of non-viable CD8+ H60+ T cells in the spleen (> 50%) compared to the BM (

Lymphopenia following bone marrow transplant (BMT) can induce expansion of donor T cells which help contribute to reconstitution in recipients. We are interested in elucidating the responses of transplanted CD8 T memory (TM) to lymphopenic signals in the recipient splenic and marrow environments. To compare proliferative responses of marrow and spleen populating antigen-specific CD8 TM to lymphopenic signals, purified (95% pure) CFSE labeled B6 CD8 TM were co-transplanted with syngeneic or allogeneic TCD-BM to ablatively conditioned (9.0 Gy) syngeneic B6 recipients. Antigen-specific CD8 TM were obtianed following priming B6 mice against BALB.B antigens. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to identify immunodominant H60-specific CD8+ cells which expressed a central memory (TM) phenotype (CD44+, Ly6C+). Three days post-transplant of these CD8 TM, recipient spleen and marrow cells were analyzed for CFSE dilution in gated H60+CD8+ T cells. Transferred H60+ CD8+ cells in the marrow compartment consistently showed greater (5 – 6 cell divisions) proliferative activity compared to those populating the spleen (1 – 2 divisions) in recipients of syngeneic (i.e. homeostatic) as well as allogeneic TCD-BM. Thus, the marrow compartment apparently provided greater support for the early homeostatic proliferation of these MiHA-specific CD8 TM. Furthermore, the presence of antigen in B6 recipients of allogeneic BM did not significantly enhance the proliferation of transferred CD8+ TM, indicating that homeostatic signals induced greater overall proliferation than antigen activation in the simultaneous presence of both signals early post-transplant. To examine for non-proliferative functional activity in transplanted antigen-specific CD8 TM, degranulation was assessed by surface expression of LAMP-1 (CD8 107a) which was analyzed 3 days after the transfer of purified splenic CD8 T cells from B6BALB.B into lethally irradiated (9.0 Gy) syngeneic B6 mice. Analysis of recipient spleeen and marrow showed that transferred antigen-specific in these tissues markedly upregulated surface LAMP-1 expression. Interestingly, there was greater frequency (~90%) of CD8+ TM in the spleen expressing LAMP-1 than in the bone marrow (56%). To determine if this degranulation response was limited to transplanted TM, a homogeneous population of naive T cells (TN) from OT-1 RAG−/− TCR tg mice was prepared and transplanted in an identical manner. LAMP-1 expression was marginally detectable in these cells. In total, these results suggest that transplanted CD8 TM and TN respond differently in the homeostatic environment post-transplant. Moreover the observations may suggest that CD8 TM can exhibit effector-like activity in the lymphopenic milieu immediately post-transplant. These findings have implications for the relative contribution of hematopoietic compartments during immune reconstitution after HCT.

Minor histocompatibility (MiHA) antigens induce CD8+ T-cell responses that mediate resistance to bone marrow engraftment in an MHC-identical, MiHA-disparate marrow transplant model, in which recipients are sensitized to donor antigens (BALB.B à B6BALB.B) prior to BMT. The H60 H antigen has been shown to dominate the immune response in B6 mice primed with BALB.B antigens (B6BALB.B). We initially sought to determine the distribution and partially characterize the phenotype of H60-specific CD8+ cells in the blood, spleen and marrow compartments of B6 mice primed with 3 x 107 BALB.B lymphoid cells and ≥3 weeks later boosted with 2 x 107 cells. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to detect H60-specific CD8+ cells in these compartments. Eight days following the second immunization, the mean frequency of circulating H60-specific cells was 12.2% ± SE 0.88 of CD8+ cells (range: 5.6 – 20.5%). The frequency of splenic H60-specific CD8+ cells was equivalent to that of circulating antigen-specific cells, thus peripheral blood levels of H60-specific CD8+ cells appear to be representative of those resident in the spleen. Interestingly, in the marrow compartment, the frequency of H-60+ cells amongst the CD8 T cell population was higher compared to peripheral blood and spleen levels, suggesting that H60-specific CD8+ cells in this compartment may comprise both migrant cells from the periphery and resident cells in the marrow elicited during priming. In both BM and spleen, >90% of CD8+ H60+ cells expressed the memory phenotype(CD44+, Ly6C+) and as expected, did not express early activation markers (CD25, CD69). To mediate resistance to progenitor cell engraftment, H60-specific effector CD8+ cell must first survive the immediate post-BMT milieu in the hemopoietic compartments. To examine this question, B6BALB.B mice irradiated at 3.0, 6.0 and 9.0 Gy were analyzed 24 hours later for the presence of H60-specific CD8+ cells in the spleen and marrow compartments. Although there was an expected irradiation dose-dependent decrease in absolute numbers of CD8+ H60+ cells in the two compartments, there was a dose-dependent increase in percent of CD8+ T cells expressing the H60 TCR in both compartments. This observation indicates enhanced survival of these antigen-specific CD8+ memory T cells post-conditioning. Preliminary results indicate that 24h post-BMT into 9.0 Gy TBI recipients, there was BrDU uptake in marrow and splenic CD8+H60+ T cells in B6BALB.B transplanted with 1 x 107 BALB.B BM-TCD. Approximately 80% of CD8+H60+ T cells in the marrow compartment of primed recipients of BALB.B cells exhibited proliferation by BrDU uptake. Thus, donor MiHA-disparate marrow grafts elicit antigen-driven proliferation early post-BMT by CD8+ memory T cells in both compartments consistent with the potential importance of these cells in mediating resistance against progenitor engraftment across these MiHA differences.

Memory T cell (TM) populations specific for transplantation Ags may arise from sensitization due to blood transfusions, tissue transplants or in multiparous females. In each of these scenarios, memory T cells (TM) are likely generated and have been shown to persist in such individuals for extended time periods. Heightened resistance to allogeneic marrow engraftment in certain individuals is therefore consistent with the presence of anti-donor TM. CD8 TM were generated against a single minor H Ag (MiHA) epitope to determine if such cells could inhibit allogeneic marrow engraftment. The present results demonstrate that B6 mice sensitized to a single immunodominant (H60) epitope efficiently reject donor marrow allografts expressing this MiHA alone or together with multiple minor transplantation antigens, even following ablative TBI conditioning. To further address the survival and function of these CD8 TM, sensitized mice were ablatively conditioned and administered a syngeneic HCT. CD8+H60TCR+ TM were clearly detected up to two weeks later in such recipients. Additionally, the memory cells present were capable of mediating effector responses as evidenced by their ability to resist a second, allogeneic HCT. In summary, these observations highlight the increased risk of resistance in the presence of anti-donor antigen specific CD8 TM due to their ability to survive and function even following rigorous conditioning and HCT.

Reduced-intensity conditioning regimens for transplant recipients have heightened awareness of immunologic resistance to allogeneic bone marrow transplants (BMT). Although T cell-mediated cytotoxicity has been assumed to play a role in the resistance against donor allogeneic hematopoietic stem and progenitor cell grafts, several studies have reported relatively unimpaired resistance by recipients who lack perforin, Fas ligand (FasL), and other cytotoxic mediators. This study compared the early kinetics of T cell-mediated resistance in B6 (H2b) cytotoxically normal versus deficient recipients after transplantation with major histocompatibility complex-matched, minor histocompatibility antigen (MiHA)-mismatched allogeneic marrow grafts. Wild-type B6 or cytotoxic double-deficient perforin−/−/gld+/+ (B6-cdd) mice were sensitized against major histocompatibility complex-matched BALB.B or C3H.SW (H2b) MiHA and transplanted with a high dose (1 × 107) of T cell-depleted bone marrow. CD8 T memory cells were shown to be present in recipients before BMT, and anti-CD8 monoclonal antibody infusion abolished resistance, thus demonstrating that CD8 T cells are the host effector population. Donor-committed and high proliferative potential progenitor numbers were markedly diminished by 48 hours after transplantation in both wild-type B6 and B6-cdd anti-donor MiHA-sensitized recipients. These observations indicate that the resistance pathway used in the cytotoxic deficient mice was both potent and rapidly induced—consistent with a CD8 memory T-cell response. To examine the role of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)- and TL1A-mediated cytotoxicity in this strong resistance, newly generated monoclonal antibodies specific for these ligands were administered to B6-cdd recipients sensitized to donor antigens. Recipients of syngeneic B6-gfp bone marrow exhibited significant donor colony-forming unit numbers after BMT. In contrast, low or absent colony-forming unit levels were detected in allogeneic recipients, including those that lacked perforin and FasL and that received anti-TWEAK, anti-tumor necrosis factor-related apoptosis-inducing ligand, and anti-TL1A monoclonal antibodies. These findings extend previous observations by demonstrating the existence of a rapidly effected resistance pathway mediated by memory CD8 effector T cells independent of the 2 major pathways of cytotoxicity. Together with previous findings, these results support the notion that effector cells derived from memory CD8 T-cell populations can mediate strong resistance against donor allogeneic MiHA-disparate hematopoietic engraftment by using a mechanism that is independent of the contribution of perforin, FasL, and the known death ligand receptor pathways.