H60-specific CD8 Research Articles

PD-1 deficiency augments bone marrow failure in a minor-histocompatibility antigen mismatch lymphocyte infusion model

Although PD-1 blockade has revolutionized cancer immunotherapy, immune-related adverse events (irAEs) present life-threatening complications. Recent reports of aplastic anemia (AA) as irAEs implicate PD-1/PD-L1 as important in preventing immune-mediated destruction of the hematopoietic niche. Infusion of PD-1-deficient (PD-1 knockout [KO]) lymph node (LN) cells into minor-antigen mismatched mice resulted in early mortality, as well as more severe bone marrow (BM) hypoplasia, anemia, and BM microarchitecture disruption in PD-1 KO LN-infused mice relative to mice that received B6 LN cell infusion. Mice that received PD-1 KO LN cells had more CD8+ T-cell infiltration of the BM and greater expansion of H60-specific CD8+ T cells than did their B6 LN-infused counterparts. In the spleen, CD8+ T cells were skewed to an effector memory phenotype, suggesting accelerated differentiation of PD-1 KO T cells. Our data suggest that PD-1 dysregulation has a role in murine BM failure and vigilance in irAE monitoring may be desirable to treat early AA and related cytopenias.

Lack of central memory cell generation by PD-1-deficient T cells under helper-deficient conditions

Abstract An immune check point molecule PD-1 is associated with CD8 T cell exhaustion in tumor-infiltrating and chronic infection situations. Previously, we have demonstrated that CD8 T cells specific for a transplantation antigen, minor histocompatibility antigen H60, are exhausted when primed under CD4 helper-deficient conditions, showing PD-1hi phenotype, and the exhaustion is ascribed to inefficient antigen-clearance. We examined whether the helpless CD8 T cells could respond to re-challenge with H60 in absence of PD-1-mediated signaling using PD-1-deficient mice. Upon re-challenge with H60 of the PD-1-deficient mice, which had been primed against H60 under helper-deficient conditions, the H60-specific CD8 T cells expanded vigorously, exhibiting a memory-like response. Despite the restored proliferative potential to the antigen-re-encounter, the H60-specific PD-1-deficient CD8 T cells did not generate CD44hiCD6Lhicentral memory T cells under helper-deficient priming condition. They exhibited an exhausted status, with an increased CXCR3+KLRG1+ Tint cell fraction. When transferred to naïve hosts, PD-1-deficient helpless CD8 T cells showed reduced generation of central memory cells compared to their wild type counterparts. These results suggest that PD-1 signaling influences fate of effector CD8 T cells and provide information regarding CD8 T cell memory generation.

Subdominant H60 antigen-specific CD8 T-cell response precedes dominant H4 antigen-specific response during the initial phase of allogenic skin graft rejection.

In allogeneic transplantation, including the B6 anti-BALB.B settings, H60 and H4 are two representative dominant minor histocompatibility antigens that induce strong CD8 T-cell responses. With different distribution patterns, H60 expression is restricted to hematopoietic cells, whereas H4 is ubiquitously expressed. H60-specific CD8 T-cell response has been known to be dominant in most cases of B6 anti-BALB.B allo-responses, except in the case of skin transplantation. To understand the mechanism underlying the subdominance of H60 during allogeneic skin transplantation, we investigated the dynamics of the H60-specific CD8 T cells in B6 mice transplanted with allogeneic BALB.B tail skin. Unexpectedly, longitudinal bioluminescence imaging and flow cytometric analyses revealed that H60-specific CD8 T cells were not always subdominant to H4-specific cells but instead showed a brief dominance before the H4 response became predominant. H60-specific CD8 T cells could expand in the draining lymph node and migrate to the BALB.B allografts, indicating their active participation in the anti-BALB.B allo-response. Enhancing the frequencies of H60-reactive CD8 T cells prior to skin transplantation reversed the immune hierarchy between H60 and H4. Additionally, H60 became predominant when antigen presentation was limited to the direct pathway. However, when antigen presentation was restricted to the indirect pathway, the expansion of H60-specific CD8 T cells was limited, whereas H4-specific CD8 T cells expanded significantly, suggesting that the temporary immunodominance and eventual subdominance of H60 could be due to their reliance on the direct antigen presentation pathway. These results enhance our understanding of the immunodominance phenomenon following allogeneic tissue transplantation.

PD-1-mediated deletion induces tolerance of CD8 T cell response specific for H60 under helpless condition (P2225)

Abstract H60 is a dominant minor H antigen, for which specific CD8 T cell response dominates over those for other minor H antigens in B6 anti-BALB.B response. The specific CD8 T cell response for H60 is CD4 help-dependent, as burst expansion of the specific cells during the primary and memory responses occurs only when CD4 T cell activation accompanies. When the CD4 help is not provided, primary expansion was limited and, then, recall expansion of the specific CD8 T cells is impaired even when the CD4 help is provided during the memory response. This tolerance phenomenon was ascribed to intrinsic property of H60-specific CD8 T cells activated under the helpless condition. The H60-specific CD8 T cells activated under helpless condition could develop into functionally active effector cells, migrating to inflamed sites. However, they were more prone to cell death with up-regulation of PD-1 on the H60-specific CD8 T cells activated under helpless condition. Recall expansion of H60-specific CD8 T was restored in the PD-1 deficient mice that had been primed under helpless condition, suggesting the significance of negative regulation by PD-1 in the tolerance induction of H60-specific CD8 T cell response after the helpless priming. These results will provide useful information for induction of tolerance after allogeneic transplantation.

A single-amino-acid variant of the H60 CD8 epitope generates specific immunity with diverse TCR recruitment.

TCR of CD8 T cells recognizes peptides of 8-9 amino acids in length (epitope) complexed with MHC class I. Peptide ligands differing from an epitope by one or two amino acids are thought to modulate the immune response specific to that epitope. H60 is a minor histocompatibility antigen for which the specific CD8 T-cell response dominates during alloresponse after MHC-matched allogeneic transplantation. In the present study, we developed a transgenic mouse (designated H60H Tg) expressing a variant of H60, designated H60H, in which the arginine residue at position 4 of the H60 epitope sequence (LTFNYRNL) is replaced by a histidine residue (LTFHYRNL). Immunization of female C57BL/6 mice with splenocytes from male H60H Tg induced a CD8 T cell primary response and memory response after re-challenge. The response was CD4 help-dependent, demonstrating the potency of H60H as a cellular antigen. The response induced by the H60H cellular antigen was comparable to that induced by H60 in its peak magnitude and overall immune kinetics. H60H challenge recruited broadly diverse TCRs to the specific response, shaping a TCR repertoire different from that of the natural H60 epitope. However, some of the TCRs did overlap between the H60H- and H60-specific CD8 T cells, suggesting that H60H might modulate the H60-specific response. These results may provide a basis for the modulation of the H60-specific CD8 T-cell response.

Requirement of CD4 Help for Induction of CD8 T Cell Response Specific for Virally Derived H60

CD40-CD40L-mediated help from CD4 T cells is essential to induce primary CD8 T cell responses specific to the non-inflammatory cell-based antigen H60. In this study, using H60 as a model antigen, we generated recombinant vaccinia viruses (rVVs) expressing the H60 CD8 epitope and investigated whether CD4 help was required to activate the CD8 T cell response specific to the virally expressed H60. The immune response after infection with rVVs expressing H60 was similar to that after immunization with H60 congenic splenocytes, with a peak frequency of H60-specific CD8 T cells detected in the blood on day 10 post-infection. A CD8 T cell response specific for virally derived H60 was not induced in CD4-depleted mice, but was in CD40-deficient mice. These results provide insights into the characterization of the CD8 T cell response specifically for antigens originating from cellular sources compared to viral sources.

Primary Vascularization of the Graft Determines the Immunodominance of Murine Minor H Antigens during Organ Transplantation

Grafts can be rejected even when matched for MHC because of differences in the minor histocompatibility Ags (mH-Ags). H4- and H60-derived epitopes are known as immunodominant mH-Ags in H2(b)-compatible BALB.B to C57BL/6 transplantation settings. Although multiple explanations have been provided to explain immunodominance of Ags, the role of vascularization of the graft is yet to be determined. In this study, we used heart (vascularized) and skin (nonvascularized) transplantations to determine the role of primary vascularization of the graft. A higher IFN-γ response toward H60 peptide occurs in heart recipients. In contrast, a higher IFN-γ response was generated against H4 peptide in skin transplant recipients. Peptide-loaded tetramer staining revealed a distinct antigenic hierarchy between heart and skin transplantation: H60-specific CD8(+) T cells were the most abundant after heart transplantation, whereas H4-specific CD8(+) T cells were more abundant after skin graft. Neither the tissue-specific distribution of mH-Ags nor the draining lymph node-derived dendritic cells correlated with the observed immunodominance. Interestingly, non-primarily vascularized cardiac allografts mimicked skin grafts in the observed immunodominance, and H60 immunodominance was observed in primarily vascularized skin grafts. However, T cell depletion from the BALB.B donor prior to cardiac allograft induces H4 immunodominance in vascularized cardiac allograft. Collectively, our data suggest that immediate transmigration of donor T cells via primary vascularization is responsible for the immunodominance of H60 mH-Ag in organ and tissue transplantation.

Lack of CD4 help induces tolerance of CD8 T cell response for H60, a dominant minor histocompatibility antigen. (169.46)

Abstract Mismatch on minor histocompatibility (H) antigens is a major obstacle for the success of transplantation between MHC-matched individuals, generating CD4 and CD8 T cell responses specific for the disparate allo-minor H antigens. H60 is a dominant minor H antigen, for which specific CD8 T cell response dominates over those for other minor H antigens in B6 anti-BALB.B response. In our previous study, the H60-specific CD8 T cell response was found to be Th dependent, requiring CD4 T cell activation for both the primary and memory expansion. Here, we show that CD4 help deficiency during the induction of the H60-specific CD8 T cell response not only forbids the primary blast expansion but also prevents the specific memory response development, implying the tolerance induction of the H60 specific CD8 T cells under helpless condition. H60-specific CD8 T cells activated under the helpless condition were more prone to cell death than those under helped condition. Tolerance induction on the H60-specific CD8 T cells rendered the total CD8 T cell immunity of B6 mice to BALB.B to be diminished, while it remained unaffected by matching on H60. The results demonstrate that CD4 help provision influences CD8 immunity against minor H antigens and suggest that tolerance of CD8 T cell response to dominant antigen would be applicable for controlling transplantation rejection or GVHD.

CD8 T cell response for H60 requires CD4 T cell help when the antigen is expressed as a viral antigen (132.36)

Abstract CD4 helper T cell is critical for the generation of primary and memory CD8 T cells specific for non-inflammatory antigens. Minor histocompatibility antigens are representative non-inflammatory cellular antigens that require CD4 help for the response induction, and we have shown that CD8 T cell response for minor histocompatibility antigen H60 is dependent on CD4 help not only for the primary response but also for the secondary response inductions. In this study, we addressed a question whether H60 epitope as a viral antigen would require CD4 help for specific response induction. A recombinant vaccinia virus expressing H60-CD8 epitope and HY-CD4 epitope (rVV-H60HY) was used for immunization of B6 female mice. Immunization with the recombinant virus induced a significant amount of H60-specific CD8 T cell proliferation with a peak frequency around 10~20% of total blood CD8 T cells. The response was CD4 help dependent both after the primary and secondary viral challenges. The CD4 help could be substituted by combined treatment of TLR agonists and anti-CD40 antibody. Our results demonstrate that CD4 help is required for appropriate CD8 T cell responses specific for H60 not only as cellular antigen but also as viral antigen, and the signaling through TLR and CD40 would license antigen presenting cells to appropriately activate antigen-specific CD8 T cells.

Influence of partial agonist expression on CD8 T cell responses for non-self antigens (132.35)

Abstract Differences in affinity of TCR for peptide/MHC complex induced by single amino acid substitutions in the peptide dramatically alter recognition by T cell in the periphery. With H60 as an antigenic model, we investigated the influence of transgenic expression of natural H60 antigen (H60N Tg) or of H60 antigen with single amino acid substitution at position 4 to histidine (H60H Tg) on H60H- or H60N-specific CD8 T cell responses, respectively. Immunization of B6 female mice with splenocytes from male H60NTg or H60H Tg mice induced strong CD8 T cell responses against each epitope, H60N or H60H, in CD4 T cell help dependent ways. While the immunization with splenocytes from male H60H Tg induced proliferation of H60-specific CD8 T cell responses in the female H60N Tg mice, CD8 T cell response for H60H induced in female H60N Tg showed significant reduction in the magnitude of H60H-tetramer-binding CD8 T cells at the peak time. And differences in TCR V beta usages of H60H-reactive CD8 T cells were also found between in the female B6 female and female H60N Tg hosts. Our data suggest that expression of proteins with amino acid changes influences the antigen-specific CD8 T cell responses and repertoire of TCRS involved in the responses.

Memory CD8. + T Cells Specific for Minor Histocompatibility Antigen H60 Can Mediate the Graft-Versus-Leukemia Effect.

Abstract 1332Poster Board I-354In allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells mediate graft-vs-leukemia (GVL) but also cause graft-vs.-host disease (GVHD). Our previous studies indicate that memory T cells (TM) induce less GVHD than do naïve T cells (TN). Therefore the selective infusion of memory T cells could improve immune reconstitution with less GVHD. However, human memory T cells have a more restricted TCR repertoire than do TN and therefore may be less effective in mediating GVL. One approach for addressing this concern would be to vaccinate donors against a single miHA expressed by host leukemic cells. Then the selective transfer of memory T cells could improve both GVL and immune reconstitution with less GVHD. Questions remain about how best to apply this strategy. For example, differences in target antigen distribution could affect the re-expansion of transferred memory cells and determine whether vaccination augments GVHD. Also, different types of memory cells could behave differently. To begin to optimize this approach in mouse models we chose as our target antigen the H-2Kb-restricted minor histocompatibility antigen (miHA) H60. We cloned the H60 peptide sequence (LTFNYRNL) into a genetic construct encoding the heavy chain of a monoclonal antibody against DEC205. Donor C3H.SW (H-2b, H60-) mice were vaccinated with a single injection of anti-DEC205-H60 plus an agonist antibody against CD40 (FGK45). By two months post vaccination, H60-reactive memory cells were a stable population that comprised approximately 4-8% of splenic and peripheral blood CD8 cells. Approximately 50% of H60-tetramer+ cells had central memory phenotype, which could be ideal as central memory T cells mount strong anti-tumor responses. Similar results were obtained by using a prime/boost approach with H60-pulsed DCs. To test their function, CD44+ memory CD8 cells from H60-vaccinated mice were sort-purified and transferred into recipient B6 mice congenic for H60 (B6.H60; expression hematopoietically restricted ). By day 7 post transplant, H60-specific CD8+ TM from vaccinated mice comprised 70-90% of total splenic and blood CD8 cells, as compared to 1-5% in recipients of TM from unvaccinated mice. In contrast to anti-H60 responses by naïve CD8 cells, expansion of H60-tetramer+ cells from H60-vaccinated mice did not require CD4 help. To test whether target antigen distribution affects the re-expansion of H60-reactive cells from H60-vaccinated mice, expansion was compared in B6.H60 and B6.H60<right arrow>B6.actH60 (ubiquitous expression of H60 driven by an actin promoter) bone marrow chimeras. Approximately 60% of splenic and lymph node CD8 cells in both recipient groups were H60-tetramer+, though there was a trend towards increased overall numbers of tetramer+ cells in B6.H60 recipients. We are currently testing the GVL potency of TM from H60-vaccinated against mouse models of chronic phase (CP-CML) and blast crisis chronic myelogenous leukemia (BC-CML). In an ongoing experiment, very low numbers of CD8+ TM from H60-vaccinated mice mediate potent GVL against CP-CML relative to CD8+ TM from unvaccinated donors. Small numbers of CD8+ TM cells from vaccinated mice also mediated GVL against BC-CML, which is typically GVL resistant. Future studies will better define the potency of TM from H60-vaccinated mice and test these cells against CP-CML and BC-CML that do or do not express H60 but are otherwise identical. GVHD effect will also be tested. DisclosuresNo relevant conflicts of interest to declare.

Alloreactive CD8+ Effector T Cells Proliferate and Persist Upon Chronic Exposure to Alloantigens through Reactivation of Stem Cell Transcriptional Programs.

Abstract 2444Poster Board II-421Alloreactive effector T cells are the central to graft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). In GVHD host antigens are never cleared and alloreactive effector T cells are continuously generated over a period of several months or longer, but their suppression and control have proven to be difficult in practice. Using mouse models of GVHD directed against minor histocompatibility antigens (miHAs), we demonstrate that alloreactive effector T cells proliferate and persist upon chronic exposure to alloantigens via reactivation of stem cell transcriptional programs normally expressed in embryonic stem cells and neural stem cells. Many activated stem cell genes in effector T cells were distinct from those in memory T cells and were maintained at high levels upon T cell receptor activation, suggesting a specific role in chronically activated effector T cells. One of these genes, Ezh2, encodes a chromatin modifying enzyme essential to the proliferation, survival and differentiation of stem cells, was upregulated in CD8+ effector T cells upon antigenic stimulation and downregulated when the antigen was withdrawn. Pharmacologically inactivation of EZH2 with 3-Deazaneplanocin A inhibited effector T cell proliferation and survival. Silencing Ezh2 independently validated that Ezh2 was important for regulating effector T cell proliferation and expression of many stem cell genes. To further evaluate whether alloreactive CD8+ effector T cells obtained stem cell-like properties, e.g. the ability to self-renew to continually generate effector cells, we adoptively transferred highly purified miHA H60-specific (H60+) CD8+ effector T cells into secondary allogeneic and congenic recipients, respectively. As compared to congenic recipients, allogeneic recipients had 80-fold more proliferating H60+CD8+ effector T cells. These donor H60+CD8+ effector T cells expressed high levels of CD122, CD69, CXCR3, PD1, IFNγ and Granzyme B, required miHA H60 stimulation to sustain their replication with effector function and expression of stem cell genes, and caused severe GVHD in secondary allogeneic recipients. These results indicate that stem cell transcriptional programs expressed in embryonic and neural stem cells may play important roles in effector T cells. Among these stem cell genes, Ezh2 emerges as an important therapeutic target in modulating alloreactive T cell-mediated GVHD. Disclosures:Zhang:University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding.

Mechanisms of Cross-Presentation in Graft-Vs-Host Disease.

Abstract 687Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD, while donor APCs promote GVHD in a MHC matched (C3H.SW (right arrow) B6; both H-2b) model (Shlomchik et al, Science 1999; Matte et al., Nat Med 2004). However, how donor APCs promote maximal GVHD was not addressed. The LTFNYRNL peptide from H60 is a dominant minor histocompatibility antigen (miHA) presented by H-2Kb. To study cross-presentation of H60, we crossed B6 mice congenic for H60 (CH60; hematopoietically restricted) or transgenic for H60 driven by an actin promoter (actH60; H60 is ubiquitously expressed) with B6 Kb-/- mice. These mice express H60 but cannot directly present it to donor CD8 cells as they do not express Kb. CH60C*Kb-/-and actH60*Kb-/- were irradiated and reconstituted with C3H.SW (H60-) bone marrow,106(6 superscript) CD8 T cells and 2*105( 5 superscript) CD4 (to promote the CD8 response to H60). Using H60-MHC tetramers, we detected H60-specific CD8 T cell expansion as early as day 8, with a peak at day14, demonstrating cross-priming by donor C3H.SW APCs. Intracellular IFN-γ staining and an in vivo CTL assay showed that these cross-primed CD8 T cells had effector functions. Surprisingly, accumulation of H60-tetramer+ cells was greater when it was exclusively cross-presented. SIINFEKL, a peptide derived from ovalbumin (OVA), is also presented by Kb. Therefore to confirm our findings we used B6 mice transgenic for ovalbumin crossed to Kb-/- mice (ova*Kb-/-) as recipients in the same model. SIINFEKL-tetramer+ T cells expansion was also observed in ova*Kb-/- recipients, demonstrating cross-priming. The source of miHA did not affect the cross-priming as similar SIINFEKL-reactive T cell expansion occurred in retransplanted (right arrow)ova*Kb-/-, ova*Kb-/-(right arrow) Kb-/- bone marrowγKb-/- chimeras. Cross-priming of SIINFEKL-reactive CD8 cells even occurred when BALB/c mice transgenic for OVA (BALB/c-ova; (H-2d)) were transplanted with B6 BM and a mix of B6 CD4 and CD8 cells. SIINFEKL-reactive cells produced IFN-γ and killed SIINFEKL-pulsed B6 cells in vivo. Because of the availability of knockout/transgenic mice backcrossed to B6, we used this system to explore mechanisms of cross-presentation. Donor CD11c+ dendritic cells (DCs) were required as cross-priming was abrogated when BALB/c-OVA mice were transplanted with BM from mice constitutively lacking CD11c+ DCs (Birnberg et al, Immunity 2008). CD4 help has been reported to be important for cross-priming. Surprisingly, however, cross-priming by donor APCs was unaffected when BALB/c-OVA mice were transplanted with B6 MHCII-/- BM but was greatly reduced in recipients of B6 CD40-/- BM. Thus, while CD40L activation of cross-priming DCs is important, CD4 cells which are likely the source of the CD40L need not actually make T cell receptor:MHC contacts with the cross-presenting DC. CD40L-conditioning of donor APCs is not required to cross-prime memory cells, as sort-purified memory CD8 cells from SIINFEKL-vaccinated mice expanded robustly in actOVA*Kb-/- but not Kb-/- mice. Cross-priming also occurred in recipients of IFNAR1-/- BM, indicating that Type I IFN activation of donor APCs is not required as has been reported in nontransplant settings. Taken together, our data demonstrate that cross-presentation by donor DCs occurs in MHC-matched and -mismatched transplants, and this cross-presentation likely explains the reduced GVHD we observed in recipients of MHCI- donor bone marrow. That T cells can be cross-primed to nonhematopoietic antigens provides a basis for persistent GVHD and for the generation of CD8 responses against antigens not initially targeted. We also found transplantation to be a permissive environment for cross-priming in that CD4 help could be delivered in trans, type I IFN APC activation was not required and memory cells could be activated without CD4 help. These data provide further rationale for targeting donor DCs and pathways required for cross-presentation to prevent and treat GVHD. Disclosures:No relevant conflicts of interest to declare.

TCR Diversity of H60-Specific CD8 T Cells During the Response Evolution and Influence of CD4 Help

H60 is a hematopoietic cell-specific dominant minor histocompatibility antigen that is considered to be ideal for modeling leukemia treatment after bone-marrow transplantation. We characterized the H60-specific CD8 T-cell response as CD4 help dependent. This study investigated the T-cell receptor (TCR) repertoires during the evolution of H60-specific CD8 T-cell responses and influence of CD4 help on the diversity. Ex vivo TCR V beta and complementarity-determining region 3 length spectratypic and clonotypic analyses were performed using H60-tetramer-binding CD8 T cells purified from the mice undergoing the primary, secondary, and tertiary responses with cognate help, and the secondary response with noncognate separate CD4 help. Involvement of a broad spectrum of TCRs was observed in the H60-specific primary response. With the involvement of diverse V beta families in the secondary and tertiary responses, complementarity-determining region 3 length and clonotypic diversities within the V beta subfamilies gradually decreased throughout the response evolution. In tertiary repertoires, the usage of V beta 8.3 and focused clonal usage within each V beta subfamily were prominent. When noncognate separate CD4 help was provided during the induction of H60-specific secondary responses, extremely limited TCRs constituted the repertoire of reactive CD8 T cells, and most of these TCRs coincided with those observed in the secondary or tertiary repertoires provided with cognate help. This study is the first to characterize the diversity of TCRs specific for hematopoietic cell-specific mouse minor H antigens and demonstrate the effect of CD4 help on CD8 TCR repertoire diversity. Our data provide a basis for modeling therapeutic applications.

Cognate CD4 help is essential for the reactivation and expansion of CD8 memory T cells directed against the hematopoietic cell–specific dominant minor histocompatibility antigen, H60

AbstractIn contrast to previous notions of the help-independency of memory CD8 T cells during secondary expansion, here we show that CD4 help is indispensable for the re-expansion of once-helped memory CD8 T cells, using a hematopoietic cell–specific dominant minor histocompatibility (H) antigen, H60, as a model antigen. H60-specific memory CD8 T cells generated during a helped primary response vigorously expanded only when rechallenged under helped conditions. The help requirement for an optimal secondary response was confirmed by a reduction in peak size by CD4 depletion, and was reproduced after skin transplantation. Helpless conditions or noncognate separate help during the secondary response resulted in a significant reduction in the peak size and different response kinetics. Providing CD4 help again during a tertiary challenge restored robust memory expansion; however, the repeated deprivation of help further reduced clonal expansion. Adoptively transferred memory CD8 T cells did not proliferate in CD40L−/− hosts. In the CD40−/− hosts, marginal memory expansion was detected after priming with male H60 cells but was completely abolished by priming with peptide-loaded CD40−/− cells, suggesting the essential role of CD40 and CD40L in memory responses. These results provide insight into the control of minor H antigen-specific CD8 T-cell responses, to maximize the graft-versus-leukemia response.

Altered Distribution of H60 Minor H Antigen-Specific CD8 T Cells and Attenuated Chronic Vasculopathy in Minor Histocompatibility Antigen Mismatched Heart Transplantation in Cxcr3−/− Mouse Recipients

Chemokine-chemokine receptor interactions and the subsequent recruitment of T lymphocytes to the graft are believed to be among the initial events in the development of acute and chronic rejection of heart transplants. We sought to determine the role of chemokine receptor Cxcr3 on the development of acute and chronic rejection in a multiple minor Ag mismatched mouse heart transplant model. The frequencies and kinetics of immunodominant H60 (LTFNYRNL) miHA-specific CD8 T cells in wild-type or Cxcr3-/- C57BL/6 recipients were monitored using MHC class I tetramer after BALB/b donor hearts were transplanted. Acceptance of grafts, severity of rejection, and infiltration of T cells were not altered in Cxcr3-/- recipients. However, graft survival was moderately prolonged in Cxcr3-/- recipient mice undergoing acute rejection. Analyses of splenocytes, PBLs, and graft-infiltrating cells revealed increased alloreactive T cells (H60-specific CD8 T cells) in the peripheral blood and spleen but not in the graft. Adoptively transferred Cxcr3-/- CD8 T cells in the BALB/b heart-bearing B6 scid mice showed retention of alloreactive CD8 T cells in the blood but less infiltration into the graft. Cxcr3-/- recipients with long-term graft survival also showed a marked decrease of CD8+ T cell infiltration and reduced neo-intimal hyperplasia. These data indicate that Cxcr3 plays a critical role in the trafficking as well as activation of alloreactive T cells. This role is most eminent in a transplant model when a less complex inflammatory milieu is involved such as a well-matched graft and chronic rejection.

Role for CD40 and CD40L Expression in Generating CD8 T Cell Response to Minor Histcompatibility Antigen, H60

We studied the role for expression of CD40 and CD40L by CD4 and CD8 T cells in the generation of CD8 T cell response to minor histocompatibility antigen, H60. H60 is a cellular antigen to which CD8 responses require CD4 T cell help. Methods: CD40- or CD40L-deficient mice were adoptively transferred with normal CD4 or CD8 T cells or with memory CD4 or CD8 T cells, and were immunized with male H60 congenic splenocytes to induce CD8 T cell response to H60. Peripheral blood CD8 T cell from the immunized mice were stained with the H60 tetramer. Results: CD8 T cell response to H60 was not induced in both CD40- and CD40L-deficient mice. Adoptive transfer of CD40 +/+ CD8 T cells into CD40-deficient mice did not compensate the defect in inducing CD8 T cell response to H60, while the H60-specific CD8 T cells were activated in the CD40-deficient mice that were adoptively transferred with CD40 +/+ CD4 T cells. Adoptive transfer of CD40L +/+ CD4 T cells into CD40L-deficient mice induced primary CD8 T cell response for H60 and the presence of CD40L +/+ CD4 T cells was required even for memory CD8 T cells response to H60. Conclusion: Our results suggest that the CD40-CD40L interaction mediates the delivery of CD4 T cell help to naive and memory H60-specific CD8 T cells. While the expression of CD40L by CD4 T cells is essential, signaling through CD40 on CD8 T cells is not required for the induction of CD8 T cell response to H60.

A Murine Model of Bone Marrow Failure Mediated by Disparity in Minor Histocompatibility Antigens.

Destruction of hematopoietic cells in aplastic anemia and other hematologic diseases is mediated in most patients by effector cells of the immune system. We have developed a mouse model of immune-mediated bone marrow failure which employs parental lymph node cell infusion into F1 recipients, resulting in bone marrow hypercellularity, pancytopenia, and death from infection and bleeding, without other organ involvement by graft-versus-host disease (Bloom, ML et al. Exp. Hematol . 32:1163, 2004; Chen, J et al. Blood 104:1671, 2004). Because major histocompatibility antigens are not the targets in acquired aplastic anemia, we now have developed a model of murine marrow failure based on disparity of minor histocompatibility antigens. Lymph node cells from C57BL/6 mice were infused into sublethally-irradiated, MHC-matched, C.B10-H2b/LilMce recipients. Animals developed severe pancytopenia and marrow hypoplasia within two-three weeks. CD8+ T lymphocytes were expanded in the blood and infiltrated bone marrow, becoming detectable at day 7 and reaching peak levels at days 10–12. There were no inflammatory responses observed in the skin, intestines, or other visceral organs by gross or microscopic pathological examination. In our experiments we observed a time-dependent expansion followed by contraction of CD8+ T cells specific for a minor histocompatibility antigen H60 as measured by flowcytometry using an H60-specific tetramer. H60 is an antigen peptide derived from a glycoprotein, a known ligand for stimulatory NKG2D receptors, and is immunodominant over other minor antigens in stem cell transplantation. The proportion of H60-specific CD8+ T cells was strongly negatively correlated with peripheral blood white cell, neutrophil, and platelet counts. Isolated H60-specific T-cells from bone marrow of affected animals induced apoptosis in vitro of normal C57BL/6 bone marrow cells in co-culture. The degree of apoptosis was further increased by addition of CD4 T-cells from same affected donors, suggesting a helper lymphocyte effect. The role of H60-specific T cells was demonstrated in further transplant experiments. Infusion of 5 × 106 C57BL/6 lymph node cells that had been depleted of H60-specific T cells was unable to induce marrow failure in C.B10 recipients, while the same number of cells with the addition of 20–90 × 103 H60-specific CD8+ T cells led to thrombocytopenia and leucopenia in recipients. H60-specific T cells thus appear to be key effectors, responsible at least for the initiation of marrow cell destruction. Mice could be treated with cyclosporine at 50 mg/g/day for five days, if treatments were begun at the time of lymph node cell infusion. Immunosuppressive therapy abolished H60-specific CD8+ T cell expansion and attenuated the development of peripheral pancytopenia, effectively rescuing animals. In conclusion, we demonstrate in this model of immune-mediated bone marrow failure that a single clone of peptide-specific T-cells is capable of efficient marrow destruction and the production of aplastic anemia.

Antigen-Specific CD8+ Memory T Cells Survive, Function and Populate the Host Marrow Compartment Following Ablative TBI and Allogeneic BMT.

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 (<30%) compartments was observed 2 weeks after allogeneic HCT. Such findings may reflect that activation-induced cell death in the spleen induced by antigen in the allogeneic HCT was greater than in the bone marrow. Thus, reduced apoptosis in the marrow microenvironment could reflect survival advantage for CD8+ TM in this compartment. Current studies are determining the effects of ablative conditioning on ex vivo effector function activity of MiHA-specific CD8+ TM.

Contrasting Effects of Post-Transplant Lymphopenia on Proliferation and Degranulation in Antigen-Specific CD8 Memory T Cells.

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.