Unraveling graft-versus-host disease and graft-versus-leukemia responses using TCR Vß spectratype analysis in a murine bone marrow transplantation model.

The optimum use of allogeneic blood and marrow transplantation (BMT) as a curative therapy for hematological malignancies lies in the successful separation of mature donor T cells that are host reactive and induce graft-versus-host disease (GVHD) from those that are tumor reactive and mediate graft-versus-leukemia (GVL) effects. To study whether this separation was possible in an MHC-matched murine BMT model (B10.BR→CBA) with a CBA-derived myeloid leukemia line, MMC6, we used TCR Vß CDR3-size spectratype analysis to first show that the Vß13 family was highly skewed in the B10.BR anti-MMC6 CD8(+) T cell response but not in the alloresponse against recipient cells alone. Transplantation of CD8(+)Vß13(+) T cells at the dose equivalent of their constituency in 1 × 10(7) CD8(+) T cells, a dose that had been shown to mediate lethal GVHD in recipient mice, induced a slight GVL response with no concomitant GVHD. Increasing doses of CD8(+)Vß13(+) T cells led to more significant GVL responses but also increased GVHD symptoms and associated mortality. Subsequent spectratype analysis of GVHD target tissues revealed involvement of gut-infiltrating CD8(+)Vß13(+) T cells accounting for the observed in vivo effects. When BMT recipients were given MMC6-presensitized CD8(+)Vß13(+) T cells, they displayed a significant GVL response with minimal GVHD. Spectratype analysis of tumor-presensitized, gut-infiltrating CD8(+)Vß13(+) T cells showed preferential usage of tumor-reactive CDR3-size lengths, and these cells expressed increased effector memory phenotype (CD44(+)CD62L(-/lo)). Thus, Vß spectratyping can identify T cells involved in antihost and antitumor reactivity and tumor presensitization can aid in the separation of GVHD and GVL responses.

Induction of acute GVHD by sex-mismatched H-Y antigens in the absence of functional radiosensitive host hematopoietic-derived antigen-presenting cells.

It is currently thought that acute GVHD cannot be elicited in the absence of Ag presentation by radiosensitive host hematopoietic-derived APCs after allogeneic BM transplantation. Because clinical data suggest that sex-mismatched H-Y Ags may be important minor histocompatibility Ags for GVH responses, we directly tested their relevance and ability to initiate GVHD when presented by either the hematopoietic- (host or donor) or the nonhematopoietic-derived APCs. H-Y minor Ag incompatibility elicited both CD4(+) and CD8(+) T-cell driven GVHD lethality. Studies with various well-established BM chimera recipients, in contrast to the current views, have reported that in the absence of functional radiosensitive host hematopoietic-derived APCs, H-Y Ag presentation by either the donor hematopoietic-derived or the host nonhematopoietic-derived APCs is sufficient for inducing GVHD. Our data further suggest that infusion of sufficient numbers of alloreactive donor T cells will induce GVHD in the absence of radiosensitive host hematopoietic-derived APCs.

Treatment with GM-CSF secreting myeloid leukemia cell vaccine prior to autologous-BMT improves the survival of leukemia-challenged mice.

Vaccination with irradiated autologous tumor cells, engineered to secrete granulocyte macrophage-colony stimulating factor (GM-CSF) (GM tumor), can generate potent antitumor effects when combined with autologous bone marrow transplantation (BMT). That notwithstanding, the post-BMT milieu, characterized by marked cytopenia, can pose a challenge to the implementation of vaccine immunotherapies. To bypass this problem, partial post-BMT immune reconstitution has been allowed to develop prior to vaccination. However, delaying vaccination can also potentially allow the expansion of residual tumor cells. Other approaches have used reinfusion of "primed" autologous lymphocytes and multiple administrations of GM tumor cells, which required the processing of large amounts of tumor. Utilizing the MMB3.19 murine myeloid leukemia model, we tested whether a single dose of GM tumor cells, 7 days prior to syngeneic BMT, could be a curative treatment in MMB3.19-challenged recipient mice. This vaccination protocol significantly improved survival of mice by eliciting long-lasting host immune responses that survived lethal irradiation, and were even protective against post-BMT tumor rechallenge. Furthermore, we demonstrated that mature donor lymphocytes can also play a limited role in mounting the antitumor response, but our pre-BMT vaccination strategy obviated the need for either established de novo immune reconstitution or the use of multiple post-BMT immunizations.

Generation of tumor-specific T lymphocytes using dendritic cell/tumor fusions and anti-CD3/CD28.

Adoptive immunotherapy with tumor-specific T cells represents a promising treatment strategy for patients with malignancy. However, the efficacy of T-cell therapy has been limited by the ability to expand tumor-reactive cells with an activated phenotype that effectively target malignant cells. We have developed an anticancer vaccine in which patient-derived tumor cells are fused with autologous dendritic cells (DCs), such that a wide array of tumor antigens are presented in the context of DC-mediated costimulation. In this study, we demonstrate that DC/tumor fusions induce T cells that react with tumor and are dramatically expanded by subsequent ligation of the CD3/CD28 costimulatory complex. These T cells exhibit a predominantly activated phenotype as manifested by an increase in the percentage of cells expressing CD69 and interferon gamma. In addition, the T cells upregulate granzyme B expression and are highly effective in lysing autologous tumor targets. Targeting of tumor-specific antigen was demonstrated by the expansion of T cells with specificity for the MUC1 tetramer. Stimulation with anti-CD3/CD28 followed by DC/tumor fusions or either agent alone failed to result in a similar expansion of tumor-reactive T cells. Consistent with these findings, spectratyping analysis demonstrates selective expansion of T-cell clones as manifested by considerable skewing of the Vbeta repertoire following sequential stimulation with DC/tumor fusions and anti-CD3/CD28. Gene expression analysis was notable for the upregulation of inflammatory pathways. These findings indicate that stimulation with DC/tumor fusions provides a unique platform for subsequent expansion with anti-CD3/CD28 in adoptive T-cell therapy of cancer.

The immunological impact of genetic drift in the B10.BR congenic inbred mouse strain.

The MHC-matched, minor histocompatibility Ag (miHA)-mismatched B10.BR-->CBA strain combination has been used to elucidate the immunobiology of graft-vs-host disease (GVHD) following allogeneic bone marrow transplantation. Studies conducted in the 1980s had established that B10.BR CD8+ T cells were capable of mediating GVHD in the absence of CD4+ T cells, and that CD4+ T cells were unable to induce lethal disease. In more recent studies with this GVHD model, we detected etiological discrepancies with the previously published results, which suggested that genetic drift might have occurred within the B10.BR strain. In particular, there was increased allorecognition of CBA miHA by B10.BR CD4+ T cells, as determined by both TCR Vbeta spectratype analysis and the induction of lethal GVHD in CBA recipients. Additionally, alloreactivity was observed between the genetically drifted mice (B10.BR/Jdrif) and mice rederived from frozen embryos of the original strain (B10.BR/Jrep) using Vbeta spectratype analysis and IFN-gamma ELISPOT assays, suggesting that new miHA differences had arisen between the mice. Furthermore, T cell-depleted B10.BR/Jdrif bone marrow cells were unable to provide long-term survival following either allogeneic or syngeneic bone marrow transplantation. Gene expression analysis revealed several genes involved in hematopoiesis that were overexpressed in the lineage-negative fraction of B10.BR/Jdrif bone marrow, as compared with B10.BR/Jrep mice. Taken together, these results suggest that genetic drift in the B10.BR strain has significantly impacted the immune alloreactive response in the GVHD model by causing altered expression of miHA and diminished capacity for survival following transplantation into lethally irradiated recipients.

Antiviral responses following L-leucyl-L-leucine methyl esther (LLME)-treated lymphocyte infusions: graft-versus-infection without graft-versus-host disease.

Although allogeneic hematopoietic progenitor cell transplant (HPCT) is curative therapy for many disorders, it is associated with significant morbidity and mortality, which can be related to graft-versus-host disease (GVHD) and the immunosuppressive measures required for its prevention and/or treatment. Whether the immunosuppression is pharmacologic or secondary to graft manipulation, the graft recipient is left at increased risk of the threatening opportunistic infection. Refractory viral diseases in the immunocompromised host have been treated by infusion of virus-specific lymphotyces and by unmanipulated donor lymphocyte infusion (DLI) therapy. L-leucyl-L-leucine methyl ester (LLME) is a compound that induces programmed cell death of natural killer (NK) cells, monocytes, granulocytes, most CD8(+) T cells, and a small fraction of CD4(+) T cells. We have undertaken a study of the use of LLME-treated DLI following T cell-depleted allogeneic HPCT, specifically to aid with immune reconstitution. In this ongoing clinical trial, we have demonstrated the rapid emergence of virus-specific responses following LLME DLI with minimal associated GVHD. This paper examines the pace of immune recovery and the rapid development of antiviral responses in 6 patients who developed viral infections during the time period immediately preceding or coincident with the administration of the LLME DLI.

Overlap between in vitro donor antihost and in vivo posttransplantation TCR Vbeta use: a new paradigm for designer allogeneic blood and marrow transplantation.

Following allogeneic blood and marrow transplantation (BMT), mature donor T cells can enhance engraftment, counteract opportunistic infections, and mount graft-versus-tumor (GVT) responses, but at the risk of developing graft-versus-host disease (GVHD). With the aim of separating the beneficial effects of donor T cells from GVHD, one approach would be to selectively deplete subsets of alloreactive T cells in the hematopoietic cell inoculum. In this regard, TCR Vbeta repertoire analysis by CDR3-size spectratyping can be a powerful tool for the characterization of alloreactive T-cell responses. We investigated the potential of this spectratype approach by comparing the donor T-cell alloresponses generated in vitro against patient peripheral blood lymphocytes (PBLs) with those detected in vivo posttransplantation. The results indicated that for most Vbeta families that exhibited alloreactive CDR3-size skewing, there was a robust overlap between the in vitro antipatient and in vivo spectratype histograms. Thus, in vitro spectratype analysis may be useful for determining the alloreactive T-cell response involved in GVHD development and, thereby, could serve to guide select Vbeta family depletion for designer transplants to improve outcomes.

Inter-strain tissue-infiltrating T cell responses to minor histocompatibility antigens involved in graft-versus-host disease as determined by Vbeta spectratype analysis.

Lethal graft-vs-host disease (GVHD) can be induced between MHC-matched murine strains expressing multiple minor histocompatibility Ag differences. In the B6->BALB.B model, both CD4(+) and CD8(+) donor T cells can mediate lethal GVHD, whereas in the B6->CXB-2 model, only CD8(+) T cells are lethal. TCR Vbeta CDR3-size spectratyping was previously used to analyze CD8(+) and CD4(+) T cell responses in lethally irradiated BALB.B and CXB-2 recipients, which showed significant overlap in the reacting repertoires. However, CD4(+) T cells exhibited unique skewing of the Vbeta2 and 11 families in only BALB.B recipients. These Vbeta family reactivities were confirmed by immunohistochemical staining of lingual epithelial infiltrates, and by positive and negative selection Vbeta family transfer experiments for GVHD induction in BALB.B recipients. We have now extended these studies to examine the T cell repertoire responses involved in target tissue damage. Infiltrating B6 host-presensitized CD8(+) and CD4(+) T cells were isolated 8-10 days post-transplant from the spleens, intestines and livers of CXB-2 and BALB.B transplant recipients. For both T cell subsets, the results indicated overlapping tissue skewings between the recipients, also between the tissues sampled within the respective recipients as well as tissue specific responses unique to both the BALB.B and CXB-2 infiltrates. Most notably, the CD4(+) Vbeta 11(+) family was skewed in the intestines of BALB.B but not CXB-2 recipients. Taken together, these data suggest that there are likely to be target tissue-related anti-multiple minor histocompatibility Ag-specific responses in each of the strain recipients, which may also differ from those found in peripheral lymphoid organs.

T cell repertoire complexity is conserved after LLME treatment of donor lymphocyte infusions.

Slow reconstitution of the T cell repertoire after allogeneic blood or bone marrow stem cell transplantation is a major risk factor for patient mortality. The delivery of immunocompetent T cells as delayed donor lymphocyte infusions (DLIs) is a potential way of counteracting this problem. The development of graft-versus-host disease (GVHD) is a potential complication of this procedure, however. We previously found that in P-->F1 haploidentical murine models, the ex-vivo treatment of donor lymphocytes with L-leucyl-L-leucine methyl ester (LLME) can prevent the onset of GVHD after DLI, likely by inducing cell death in most of the perforin-positive CD8(+) T cells and in a fraction of CD4(+) T cells. Our previous preclinical studies have formed the basis of an ongoing phase I clinical trial in which patients received LLME-treated DLI from their original donor in an attempt to accelerate T cell reconstitution. To understand how this treatment strategy might affect the complexity of the DLI T cell repertoire, we used T cell receptor Vbeta spectratype analysis to evaluate the DLI product pre-LLME and post-LLME treatment. The results indicated that the LLME-treated DLI product exhibited CDR3-size distribution complexities similar to those of its untreated donor sample. In addition, comparisons of the CD4(+) and CD8(+) T cell repertoire from the donor before LLME treatment with that of the recipient post-DLI demonstrated equal complexity for most of the resolvable Vbeta families. Finally, the in vitro proliferative capacity of LLME-treated DLI product in response to allo-stimulation in a one-way mixed lymphocyte reaction was comparable to that of the untreated product.

T-cell receptor V(alpha) usage by effector CD4+Vbeta11+ T cells mediating graft-versus-host disease directed to minor histocompatibility antigens.

T-cell receptor (TCR) Valpha (TRAV) and Vbeta (TRBV) chains provide the T-cell specificity for recognition of major histocompatibility complex (MHC)-bound antigens. However, there is limited information on the diversity of TRAV use within an antigen response. Previous investigation of CD4(+) T-cell-mediated graft-versus-host disease (GVHD) in the minor histocompatibility antigen-mismatched C57BL/6 (B6)-->BALB.B irradiated murine model determined that Vbeta11(+) T cells were associated with disease severity. Polymerase chain reaction (PCR)-based complementarity-determining region 3 (CDR3)-sized spectratype analysis of B6 Vbeta11(+) T cells from the spleens of recipient BALB.B mice undergoing GVHD indicated biased use within the V(alpha)6, 9, 13, 14, 18, and 22 families. To probe deeper into this limited V(alpha) response, the current study was undertaken to further define TRAV-Jalpha (TRAJ) nucleotide sequences found in host-presensitized B6 Vbeta11(+) T cells proliferating in response to in vitro stimulation with BALB.B splenocytes. Using the nonpalindromic adaptor PCR method, we found dominant use of the TRAV13-TRAJ16 transcript combination. Then, using laser capture microdissection, we found use of the identical TRAV-TRAJ nucleotide sequence in areas dominated by infiltrating Vbeta11(+) CD4(+) T cells during the development of GVHD in both the rete-like prominences of the dorsal lingual epithelium and the ileal crypts of the small intestine.

Cytokeratin15-positive basal epithelial cells targeted in graft-versus-host disease express a constitutive antiapoptotic phenotype.

The normal gene expression profile of rete-tip keratinocytes targeted in human graft-versus-host disease (GVHD) remains unexplored. Murine lingual epithelium, unlike murine skin, consists of a basal layer that resembles human cutaneous rete ridges and harbors rete tip-associated cells that express cytokeratin 15 (K15), a marker for epithelial stem cells. Target cell apoptosis in murine GVHD preferentially involves subpopulations of basal cells that (1) reside at tips of lingual rete ridge-like prominences (RLPs), (2) constitutively express K15 protein, (3) express the proapoptotic protein Bax early in disease progression, and (4) coincide spatially with putative epithelial stem cells. Here, we show by real-time reverse transcription-PCR that immunohistochemistry-guided laser-captured K15-positive (K15+) murine basal cells constitutively express quantitatively higher mRNA levels for K15 but lower mRNA levels of Bax than do K15- basal cells, consistent with the presumed stem cell nature of K15+ basal cells. Moreover, apoptosis gene array screening of K15+ microdissected basal cells demonstrated a dominant trend toward the preferential expression of genes associated with protection from apoptosis. Accordingly, genes that regulate apoptotic vulnerability are differentially expressed in basal layer subpopulations distinguishable by K15 expression.

T-cell receptor Valpha spectratype analysis of a CD4-mediated T-cell response against minor histocompatibility antigens involved in severe graft-versus-host disease.

Although CD4(+) T cells can have an important role in mediating lethal graft-versus-host disease (GVHD) directed to multiple minor histocompatibility antigens (miHA) after bone marrow transplantation, their precise characterization and effector function remains elusive. In this regard, T cell receptor (TCR) Vbeta spectratype analysis has been a powerful tool for identifying donor CD4(+) T cell populations expanding to host miHA after bone marrow transplantation in the major histocompatibility complex-matched C57BL/6 (B6) --> C.B10-H2(b) (BALB.B) model of lethal GVHD. Removal of all of the Vbeta(+) T cell families containing these responding cells from the donor inoculum has proven to be an effective means of preventing the development of GVHD. Previous studies have also found that of the 11 miHA-responsive B6 CD4(+) Vbeta(+) T cell families, transplantation of Vbeta2(+) and Vbeta11(+) T cells together into lethally irradiated BALB.B mice appeared to be primarily responsible for the severity of resultant GVHD. Further focusing on these critical CD4 responses, in this study we demonstrate that B6 CD4(+)Vbeta11(+) T cells alone can induce lethal GVHD in BALB.B recipients. In addition, immunohistochemical staining of host lingual and intestinal epithelial tissues supported the capacity of Vbeta11(+) T cells to infiltrate typical GVHD-associated target areas. To further characterize the specific CD4(+)Vbeta11(+) T cells involved in this anti-miHA response, TCR Valpha spectratype analysis was performed and indicated that 6 Valpha chains were used by this reactive population. These results provide further evidence that a restricted repertoire of T cell specificities, presumably recognizing a correspondingly low number of miHA, is sufficient for the induction of severe GVHD.

A CD4 domain 1 CC' loop peptide analogue enhances engraftment in a murine model of bone marrow transplantation with sublethal conditioning.

Host CD4(+) T cells that survive sublethal or even lethal preconditioning regimens can participate in the process of hematopoietic stem cell graft rejection, particularly when the transplantations are performed across a major histocompatibility complex (MHC) class II barrier. To enhance donor marrow engraftment, we tested the efficacy of a small synthetic cyclic heptapeptide, 802-2 (CNSNQIC), which was designed to closely mimic the CD4 domain 1 CC' surface loop, theoretically involved in CD4/MHC class II complex oligomerization and subsequent CD4(+) T-cell activation. Previously, this peptide was found to have inhibitory activity in murine models for CD4(+) T cell-dependent graft-versus-host disease and skin allograft rejection. Herein, we used the MHC class II--disparate bm12 --> B6-CD45.1 sublethal irradiation transplantation model to test the possibility that the 802-2 peptide could enhance the engraftment of donor T cell-depleted bone marrow (ATBM). Sublethally irradiated B6-CD45.1 mice that received bm12 ATBM in combination with the 802-2 peptide demonstrated increased donor marrow cell engraftment as compared with mice that received ATBM alone; this suggests that the 802-2 peptide may be useful as an immunomodulating agent to overcome MHC class II mismatch barriers in hematopoietic stem cell transplantation.

A CD8 DE loop peptide analog prevents graft-versus-host disease in a multiple minor histocompatibility antigen-mismatched bone marrow transplantation model.

Donor CD8(+) T cells can be potent mediators of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation to either major histocompatibility complex (MHC) class I-or multiple minor histocompatibility antigen-mismatched recipients. To develop small molecular inhibitors of CD8(+) T-cell activity, theoretical structural analysis of the human CD8 alpha molecule was previously used to identify potential functional surface epitopes that interact with the MHC class I molecule. The DE loop (p71-78) was identified as such a target region, and a panel of synthetic cyclized peptide mimics of this region were tested for their inhibitory effects on cytotoxic T lymphocyte activity in human cell-mediated lympholysis assays. Peptide 1109 (CKRLGDTFVC) was most effective at inhibiting specific target cell lysis. Accordingly, studies were conducted to determine whether there was sufficient cross-species homology in the DE loop region and its nonpolymorphic interactive site on the beta(2)-microglobulin domain of the MHC class I molecule to allow similar inhibition of murine CD8(+) cytotoxic T lymphocyte activity. On the basis of strong in vitro inhibitory activity of 1109 in the murine system, the capacity of the peptide to inhibit in vivo CD8(+) T-cell effector functions in skin and hematopoietic stem cell transplantation models was examined. In the C57BL/6 anti-bm1 skin allograft rejection model, across an MHC class I barrier, a single injection of 1109 at the time of transplantation significantly prolonged graft survival. Moreover, 1109 administered at the time of transplantation in the multiple minor histocompatibility antigen-disparate B10.BR-->CBA GVHD model significantly prolonged the survival of lethally irradiated mice that underwent transplantation with donor bone marrow cells and CD8(+) T cells. Histopathologic analysis confirmed that mice treated with the synthetic peptide exhibited diminution of epithelial target cell injury. Specificity of the peptide effect was evidenced by draining lymph node cells from B10.BR mice that had been challenged with CBA lymphocytes and simultaneously treated with 1109. These cells could not generate secondary proliferative responses in vitro upon stimulation with CBA splenocytes but could respond to third-party C57BL/6 stimulation. Thus, the 1109 peptide has potential application in the prevention of CD8-mediated GVHD development.

Evolution of responding CD4+ and CD8+ T-cell repertoires during the development of graft-versus-host disease directed to minor histocompatibility antigens.

Graft-versus-host disease (GVHD) can be induced in lethally irradiated mice after allogeneic bone marrow transplantation between major histocompatibility complex-matched strains expressing multiple minor histocompatibility antigen differences. In the B6 --> BALB.B irradiation model, both CD4(+) and CD8(+) donor T cells have the capacity to mediate lethal GVHD. Previously, CDR3-size spectratyping was used to analyze these T-cell responses at a single early time point (day 5) after transplantation and revealed clonal or oligoclonal expansions of the V beta 2, 4, and 6 to 14 families for the CD4(+) response and of the V beta 4, 6, 8 to 11, and 14 families for the B6 CD8(+) response. Appropriate positive selection of these T-cell receptor V beta-skewed CD4(+) and CD8(+) T-cell subsets and their subsequent transfer into lethally irradiated BALB.B recipients resulted in fatal GVHD induction. In contrast, BALB.B mice transplanted with nonskewed V beta CD4(+) T cells survived, with minimal symptoms of GVHD. This study was undertaken to investigate the evolution of the donor/antihost minor histocompatibility antigen T-cell repertoire responses throughout the course of GVHD development. The results indicated that a number of V beta families were consistently involved throughout the course of GVHD, whereas some V beta families exhibited skewed expansions only in either the early or late stages of disease. In addition, sequence analysis of relevant representative skewed CDR3 bands from the CD4(+) V beta 11(+) and the CD8(+) V beta 14(+) families, both of which exhibited strong consistent responses, demonstrated increased use of the J beta 2.5 and J beta 2.4 segments, respectively, thus identifying the T-cell receptor specificities involved.

Specific donor Vbeta-associated CD4 T-cell responses correlate with severe acute graft-versus-host disease directed to multiple minor histocompatibility antigens.

CXB-2/By (CXB-2) recombinant inbred mice express a subset of the minor histocompatibility antigen (miHA) repertoire expressed by C.B10-H2(b)/LiMcdJ (BALB.B) mice. On lethal irradiation and the transplantation of H2(b)-matched C57BL/6 (B6) T cell-depleted bone marrow cells, along with naive unfractionated T cells, both strains succumb to acute graft-versus-host disease (GVHD). Although alloreactive B6 CD4(+) T cells are a necessary source of T-cell help for the B6 CD8(+) component of the GVHD response in both recipient strains, they are capable of mediating severe GVHD by themselves only in BALB.B mice. Previous CD4(+) T-cell receptor repertoire analysis demonstrated overlapping oligoclonal Vbeta use between the CD4(+) B6 anti-BALB.B and B6 anti-CXB-2 responses, with indications of additional BALB.B unique T-cell responses (Vbeta2 and Vbeta11). We report here that the more severe B6 anti-BALB.B response is not due to a quantitative difference in the responding cells, because the frequency of alloreactive donor CD4(+) T cells over time was equivalent in the spleens of BALB.B versus CXB-2 recipients. The responses were also similar in the number of infiltrating B6 CD4(+) T cells in the lingual epithelium of the 2 recipients. In contrast, a significantly greater degree of infiltration and injury of BALB.B intestinal epithelium correlated with the increased level of clinical GVHD severity. Of most significance, despite the involvement of at least 11 Vbeta-associated CD4(+) T-cell families in the overall B6 anti-BALB.B response, the development of severe GVHD correlated with the presence of Vbeta2- and Vbeta11-positive donor T cells. Transplantation of donor CD4(+) T cells from Vbeta-associated families that were shared between the B6 anti-BALB.B and anti-CXB-2 responses resulted in minimal GVHD potential. These data suggest that severe GVHD across miHA barriers depends on the involvement of a restricted number of potent T-cell specificities and implies that there are only a limited number of corresponding responsible miHAs.

Importance of minor histocompatibility antigen expression by nonhematopoietic tissues in a CD4+ T cell-mediated graft-versus-host disease model.

Minor histocompatibility antigens with expression restricted to the recipient hematopoietic compartment represent prospective immunological targets for graft-versus-leukemia therapy. It remains unclear, however, whether donor T cell recognition of these hematopoietically derived minor histocompatibility antigens will induce significant graft-versus-host disease (GVHD). Using established bone marrow irradiation chimeras across the multiple minor histocompatibility antigen-disparate, C57BL/6-->BALB.B combination, we studied the occurrence of lethal GVHD mediated by CD4+ T cells in recipient mice expressing only hematopoietically derived alloantigens. Even substantial dosages of donor C57BL/6 CD4+ T cells were unable to elicit lethal GVHD when transplanted into [BALB.B-->C57BL/6] chimeras. Instead, chimeric mice displayed transient cachexia with reduced target-tissue injury over time, reflecting an early, limited, graft-versus-host response. On the other hand, the importance of minor histocompatibility antigens derived from nonhematopoietic tissues was demonstrated by the finding that [C57BL/6-->BALB.B] chimeric mice succumbed to C57BL/6 CD4+ T cell-mediated GVHD. These data suggest that severe acute CD4+ T cell-mediated GVHD across this minor histocompatibility antigen barrier depends on the expression of nonhematopoietically rather than hematopoietically derived alloantigens for maximal target-tissue infiltration and injury.

An epithelial target site in experimental graft-versus-host disease and cytokine-mediated cytotoxicity is defined by cytokeratin 15 expression.

The identity of cells within squamous epithelia that represent primary targets in acute graft-versus-host disease (GVHD) has been an enigma. Murine effector T cells implicated in the alloresponse by Vbeta complementarity-determining region-3 spectratype analysis were detected with a Vbeta-specific monoclonal antibody within discrete microdomains of tongue (lingual) squamous epithelium. These microdomains, termed rete-like prominences (RLPs), are similar to the rete ridges of human skin. Cells forming the basal layer of RLPs and of human skin rete ridges were shown to express a distinctive pattern of keratin expression defined by antibodies to cytokeratin 15 (K15). In experimental murine GVHD elicited across minor histocompatibility antigen barriers (miHA), early lesions involved selective apoptosis and loss of K15(+) staining within lingual RLPs. An in vitro organ culture model designed to investigate target cell injury by short-term exposure to tumor necrosis factor-alpha and interleukin-1beta, mediators relevant to GVHD, showed a similar pattern of apoptosis and loss of K15(+) reactivity within RLPs. In aggregate, these findings establish a novel cytoskeletal marker for target epithelial subpopulations that should facilitate evaluation of mechanisms of host cell injury in GVHD. These data may also enable the development of therapeutic approaches to abrogate disease at the level of target cell blockade.

Tolerance induction of alloreactive T cells via ex vivo blockade of the CD40:CD40L costimulatory pathway results in the generation of a potent immune regulatory cell.

We previously reported that ex vivo blockade of the CD40:CD40L costimulatory pathway in primary mixed lymphocyte reaction cultures resulted in profound in vitro secondary hyporesponsiveness and 30-fold or greater protection from graft-versus-host-disease (GVHD) lethality. Present studies demonstrate that tolerance induction via costimulatory blockade also results in the generation of a potent immunoregulatory cell that inhibits both naive and primed alloresponses. The immunoregulatory capacity was dependent upon cell-to-cell contact that prevented the full activation of the naive or primed cells. The inhibitory effect of tolerized cells did not preclude the response of naive T cells to nominal protein antigen if antigen was present at high concentration. However, under suboptimal antigen concentration, nonspecific inhibition of responses occurred. The tolerized regulatory cell population maintained a polyclonal T-cell receptor V beta repertoire that was broader than in control primed cultures. These data, the first to demonstrate that tolerance induction via CD40:CD40L costimulatory blockade results in potent regulatory function, are relevant to bone-marrow and solid-organ transplantation. The generation of potent immunoregulatory capacity during tolerization via CD40:CD40L blockade provides a fail-safe mechanism to control alloreactive T cells that may have escaped tolerization. These potent regulatory cells may be clinically exploitable for the treatment and prevention of GVHD or autoimmunity.