Peripheral deletion after bone marrow transplantation with costimulatory blockade has features of both activation-induced cell death and passive cell death

Two major pathways of death of previously activated T cells have been described: activation-induced cell death can be triggered by restimulating activated T cells with high concentrations of Ag, is Fas-dependent, is not influenced by proteins of the Bcl family, and is blocked by cyclosporin A; in contrast, passive cell death is induced by the withdrawal of growth factors and activation stimuli, is Fas-independent, and is blocked by Bcl family proteins. We examined the role of these two forms of cell death in the peripheral deletion of donor-reactive host T cells after allogeneic bone marrow transplantation and costimulatory blockade with anti-CD154 plus CTLA4Ig in two murine models. The substantial decline in donor-reactive CD4 cells seen in wild-type recipients 1 wk after bone marrow transplantation with costimulatory blockade was largely inhibited in Fas-deficient recipients and in Bcl-x(L)-transgenic recipients. We observed these effects both in a model involving low-dose total body irradiation and a conventional dose of bone marrow, and in a radiation-free regimen using high-dose bone marrow transplantation. Furthermore, cyclosporin A did not completely block the deletion of donor-reactive CD4(+) T cells in recipients of bone marrow transplantation with costimulatory blockade. Thus, the deletion of donor-reactive T cells occurring early after bone marrow transplantation with costimulatory blockade has features of both activation-induced cell death and passive cell death. Furthermore, these in vivo data demonstrate for the first time the significance of in vitro results indicating that proteins of the Bcl family can prevent Fas-mediated apoptosis under certain circumstances.     

CD4 T cell-mediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40-CD40 ligand interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.     

Combinations of anti-LFA-1, everolimus, anti-CD40 ligand, and allogeneic bone marrow induce central transplantation tolerance through hemopoietic chimerism, including protection from chronic heart allograft rejection

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vbeta11(+) T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening ("chronic rejection").     

Alloreactive memory T cells are responsible for the persistence of graft-versus-host disease

Graft-vs-host disease (GVHD) is caused by a donor T cell anti-host reaction that evolves over several weeks to months, suggesting a requirement for persistent alloreactive T cells. Using the C3H.SW anti-C57BL/6 (B6) mouse model of human GVHD directed against minor histocompatibility Ags, we found that donor CD8(+) T cells secreting high levels of IFN-gamma in GVHD B6 mice receiving C3H.SW naive CD8(+) T cells peaked by day 14, declined by day 28 after transplantation, and persisted thereafter, corresponding to the kinetics of a memory T cell response. Donor CD8(+) T cells recovered on day 42 after allogeneic bone marrow transplantation expressed the phenotype of CD44(high)CD122(high)CD25(low), were able to homeostatically survive in response to IL-2, IL-7, and IL-15 and rapidly proliferated upon restimulation with host dendritic cells. Both allogeneic effector memory (CD44(high)CD62L(low)) and central memory (CD44(high)CD62L(high)) CD8(+) T cells were identified in B6 mice with ongoing GVHD, with effector memory CD8(+) T cells as the dominant (>80%) population. Administration of these allogeneic memory CD8(+) T cells into secondary B6 recipients caused virulent GVHD. A similar allogeneic memory CD4(+) T cell population with the ability to mediate persistent GVHD was also identified in BALB/b mice receiving minor histocompatibility Ag-mismatched B6 T cell-replete bone marrow transplantation. These results indicate that allogeneic memory T cells are generated in vivo during GVH reactions and are able to cause GVHD, resulting in persistent host tissue injury. Thus, in vivo blockade of both alloreactive effector and memory T cell-mediated host tissue injury may prove to be valuable for GVHD prevention and treatment.     

Allogeneic bone marrow grafts with high levels of CD4(+) CD25(+) FoxP3(+) T cells can lead to engraftment failure

Regulatory CD4(+) CD25(+) FoxP3(+) T cells (T(regs) ) suppress immunological reactions. However, the effect of adding T(regs) to hematopoietic stem cell grafts on recovery and graft versus host disease (GvHD) is unknown. T(regs) from splenocytes of C57Bl/6 and Balb/c wild-type mice were isolated by MACS separation and analyzed by flow cytometry. Using a murine syngeneic transplantation model that clearly distinguishes between donor and host hematopoiesis, we showed that co-transplantation of bone marrow cells (BMCs) with high levels of T(regs) leads to a 100% survival of the mice and accelerates the hematopoietic recovery significantly (full donor chimerism). In allogeneic transplantation, bone marrow and T(regs) co-transplantation were compared to allogeneic bone marrow transplantation with or without the addition of splenocytes. Survival, leukocyte recovery, chimerism at days -2, 19, 33, and 61 for murine CD4, human CD4, HLA-DR3, murine CD3, murine CD8, murine Balb/c-H2K(d) , murine C57Bl/6-H2K(b) , and GvHD appearance were analyzed. Allogeneic bone marrow transplantation requires the addition of splenocytes to reach engraftment. Mice receiving grafts with bone marrow, splenocytes and high levels of allogeneic T(regs) died within 28 days (hematopoietic failure). Here, we show also detailed flow cytometric data reagarding analysis of chimerism after transplantation in unique murine hematopoietic stem cell transplantation models. Our findings showed that the syngeneic co-transplantation of CD4(+) , CD25(+) , FoxP3(+) T-cells and BMCs induced a stimulating effect on reconstitution of hematopoiesis after irradiation. However, in the allogeneic setting the co-transplantation of T(regs) aggravates the engraftment of transplanted cells.     

Activation of alloreactive CD8+ T cells operates via CD4-dependent and CD4-independent mechanisms and is CD154 blockade sensitive

CD154, one of the most extensively studied T cell costimulation molecules, represents a promising therapeutic target in organ transplantation. However, the immunological mechanisms of CD154 blockade that result in allograft protection, particularly in the context of alloreactive CD4/CD8 T cell activation, remain to be elucidated. We now report on the profound inhibition of alloreactive CD8(+) T cells by CD154 blockade via both CD4-dependent and CD4-independent activation pathways. Using CD154 KO recipients that are defective in alloreactive CD8(+) T cell activation and unable to reject cardiac allografts, we were able to restore CD8 activation and graft rejection by adoptively transferring CD4(+) or CD8(+) T cells from wild-type syngeneic donor mice. CD4-independent activation of alloreactive CD8(+) T cells was confirmed following treatment of wild-type recipients with CD4-depleting mAb, and by using CD4 KO mice. Comparable levels of alloreactive CD8(+) T cell activation was induced by allogenic skin engraftment in both animal groups. CD154 blockade inhibited CD4-independent alloreactive CD8(+) T cell activation. Furthermore, we analyzed whether disruption of CD154 signaling affects cardiac allograft survival in skin-sensitized CD4 KO and CD8 KO recipients. A better survival rate was observed consistently in CD4 KO, as compared with CD8 KO recipients. Our results document CD4-dependent and CD4-independent activation pathways for alloreactive CD8(+) T cells that are both sensitive to CD154 blockade. Indeed, CD154 blockade was effective in preventing CD8(+) T cell-mediated cardiac allograft rejection.     

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.     

Human cytomegalovirus infection of bone marrow myofibroblasts enhances myeloid progenitor adhesion and elicits viral transmission

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.     

TLR agonists abrogate costimulation blockade-induced prolongation of skin allografts

Costimulation blockade protocols are effective in prolonging allograft survival in animal models and are entering clinical trials, but how environmental perturbants affect graft survival remains largely unstudied. We used a costimulation blockade protocol consisting of a donor-specific transfusion and anti-CD154 mAb to address this question. We observed that lymphocytic choriomeningitis virus infection at the time of donor-specific transfusion and anti-CD154 mAb shortens allograft survival. Lymphocytic choriomeningitis virus 1) activates innate immunity, 2) induces allo-cross-reactive T cells, and 3) generates virus-specific responses, all of which may adversely affect allograft survival. To investigate the role of innate immunity, mice given costimulation blockade and skin allografts were coinjected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid), TLR4 (LPS), or TLR9 (CpG) agonists. Costimulation blockade prolonged skin allograft survival that was shortened after coinjection by TLR agonists. To investigate underlying mechanisms, we used "synchimeric" mice which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, coadministration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells and shortened skin allograft survival. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) up-regulated CD44, and 3) failed to undergo apoptosis. B6.TNFR2-/- and B6.IL-12R-/- mice treated with costimulation blockade plus LPS also exhibited short skin allograft survival whereas similarly treated B6.CD8alpha-/- and TLR4-/- mice exhibited prolonged allograft survival. We conclude that TLR signaling abrogates the effects of costimulation blockade by preventing alloreactive CD8+ T cell apoptosis through a mechanism not dependent on TNFR2 or IL-12R signaling.     

Host and donor immune responses contribute to antiviral effects of amotosalen-treated donor lymphocytes following early posttransplant cytomegalovirus infection

We have previously shown that amotosalen-treated splenocytes rescued allorecipients from a lethal dose of mouse CMV (MCMV) administered on day 0 in experimental parent C57BL/6-->CB6F1 allogeneic bone marrow transplant. In this study, we investigated the mechanism of antiviral activity of amotosalen-treated donor splenocytes when sublethal MCMV infections were administered 7 days posttransplant. Recipients of 3 x 10(6) untreated splenocytes were used as control. Following MCMV infection, recipients of untreated splenocytes had 40% early mortality due to acute graft-vs-host disease compared with no deaths among recipients of 10 x 10(6) treated splenocytes. However, recipients of both types of donor splenocytes effectively cleared MCMV from their liver. Like the untreated CD8(+) T cells, amotosalen-treated CD8(+) T cells equally retained their in vivo CTL activity against MCMV early peptide-pulsed targets and expressed similar levels of granzyme B within 11 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with both donor spleen- and host-derived anti-MCMV CD8(+) T cells in their blood and lymphoid organs, with significantly higher numbers of host-derived CD4(-)CD8(-) (double negative) T cells in the spleens of recipients of treated splenocytes compared with the recipients of untreated splenocytes. Additionally, recipients of amotosalen-treated splenocytes had lower levels of serum IFN-gamma and TNF-alpha in response to MCMV infection compared with untreated recipients. Thus, adoptive immunotherapy with treated T cells is a novel therapeutic approach that facilitates hematopoietic engraftment and permits antiviral immunity of both donor and host T cells without graft-vs-host disease.     

Allograft rejection by primed/memory CD8+ T cells is CD154 blockade resistant: therapeutic implications for sensitized transplant recipients

We have shown that CD8(+) CTLs are the key mediators of accelerated rejection, and that CD8(+) T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8(+) T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8(+) T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8(+) T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8(+) T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8(+) T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8(+) T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance.     

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.     

Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.     

Flt3 ligand expands lymphoid progenitors prior to recovery of thymopoiesis and accelerates T cell reconstitution after bone marrow transplantation

Deficient thymopoiesis and retarded recovery of newly developed CD4(+) T cells is one of the most important determinants of impaired immunocompetence after hemopoietic stem cell transplantation. Here we evaluated whether Fms-like tyrosine kinase 3 (Flt3) ligand (FL) alone or combined with IL-7 affects T cell recovery, thymopoiesis, and lymphoid progenitor expansion following bone marrow transplantation in immunodeficient mice. FL strongly accelerated and enhanced the recovery of peripheral T cells after transplantation of a low number of bone marrow cells. An additive effect on T cell recovery was not observed after coadministration of IL-7. Lineage(-)sca-1(+)c-kit(+)flt3(+) lymphoid progenitor cell numbers were significantly increased in bone marrow of FL-treated mice before recovery of thymopoiesis. Thymocyte differentiation was advanced to more mature stages after FL treatment. Improved T cell recovery resulted in better immunocompetence against a post-bone marrow transplantation murine CMV infection. Collectively, our data suggest that FL promotes T cell recovery by enhanced thymopoiesis and by expansion of lymphoid progenitors.     

Analysis of the role of negative T cell costimulatory pathways in CD4 and CD8 T cell-mediated alloimmune responses in vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.     

Virotherapy Using Myxoma Virus Prevents Lethal Graft-versus-Host Disease following Xeno-Transplantation with Primary Human Hematopoietic Stem Cells

Graft-versus-host disease (GVHD) is a potentially lethal clinical complication arising from the transfer of alloreactive T lymphocytes into immunocompromised recipients. Despite conventional methods of T cell depletion, GVHD remains a major challenge in allogeneic hematopoietic cell transplant. Here, we demonstrate a novel method of preventing GVHD by ex vivo treatment of primary human hematopoietic cell sources with myxoma virus, a rabbit specific poxvirus currently under development for oncolytic virotherapy. This pretreatment dramatically increases post-transplant survival of immunocompromised mice injected with primary human bone marrow or peripheral blood cells and prevents the expansion of human CD3(+) lymphocytes in major recipient organs. Similar viral treatment also prevents human-human mixed alloreactive T lymphocyte reactions in vitro. Our data suggest that ex vivo virotherapy with myxoma virus can be a simple and effective method for preventing GVHD following infusion of hematopoietic products containing alloreactive T lymphocytes such as: allogeneic hematopoietic stem and progenitor cells, donor leukocyte infusions and blood transfusions.     

Both gamma delta T cells and NK cells inhibit the engraftment of xenogeneic rat bone marrow cells and the induction of xenograft tolerance in mice

In murine allogeneic bone marrow transplantation recipients, treatment of the hosts with a nonmyeloablative regimen, including depleting anti-CD4 and anti-CD8 mAbs, allows establishment of long-term mixed chimerism and donor-specific tolerance. However, in the xenogeneic rat-to-mouse combination, additional anti-Thy1.2 and anti-NK1.1 mAbs are required. We have now attempted to identify the xenoresistant mouse cell populations that are targeted by anti-NK1.1 and anti-Thy1.2 mAbs. C57BL/6 (B6) wild-type, B6 TCRbeta(-/-), and B6 TCRdelta(-/-) mice received anti-CD4 and anti-CD8 mAbs, followed by 3 Gy of whole body irradiation, 7 Gy of thymic irradiation, and transplantation of T cell-depleted rat bone marrow cells. Anti-NK1.1 and anti-Thy1.2 mAbs were additionally administered to some groups. Increased rat chimerism was observed in TCRdelta(-/-) mice treated with anti-CD4, anti-CD8, and anti-NK1.1 mAbs compared with similarly treated TCRbeta(-/-) mice. In TCRbeta(-/-) mice, but not in TCR delta(-/-) mice, donor chimerism was increased by treatment with anti-Thy1.2 mAb, indicating that CD4(-)CD8(-)TCRgammadelta(+)Thy1. 2(+)NK1.1(-) cells (gammadelta T cells) are involved in the rejection of rat marrow. In addition, chimerism was enhanced in both TCRbeta(-/-) and TCRdelta(-/-) mice treated with anti-CD4, anti-CD8, and anti-Thy1.2 mAbs by the addition of anti-NK1.1 mAb to the conditioning regimen. Donor-specific skin graft prolongation was enhanced by anti-Thy1.2 and anti-NK1.1 mAbs in TCRdelta(-/-) mice. Therefore, in addition to CD4 and CD8 T cells, gammadelta T cells and NK cells play a role in resisting engraftment of rat marrow and the induction of xenograft tolerance in mice.     

Genetic characterization of strain differences in the ability to mediate CD40/CD28-independent rejection of skin allografts

Simultaneous blockade of the CD40 and CD28 T cell costimulatory pathways effectively promotes skin allograft survival in C3H/HeJ mice, extending median survival times (MSTs) beyond 100 days. This strategy is markedly less effective in C57BL/6 mice, with MSTs ranging between 20 and 30 days. In this study, we investigate the underlying genetic causes of these distinct phenotypes. Using H-2 congenic mice, we show that the genetic basis for the varied responses between these two strains is independent of the H-2 locus and T cell precursor frequency. C57BL/6 mice treated with costimulation blockade are able to generate allospecific CTL- and IFN-gamma-producing T cells within 3-4 wk posttransplant, whereas mice with a C3H background generate neither CTL- nor IFN-gamma-producing cells. Thus, differences appear to be in the generation of the immune response and not T cell homing. Strain differences in costimulation blockade-induced hyporesponsiveness persist in the absence of CD4(+) T cells, implying a direct effect on CD8(+) T cells. We demonstrate that genetic differences are important in cells of hemopoietic origin and that the costimulation blockade-resistant phenotype is dominant. Analysis of BXH recombinant inbred strains indicates that multiple loci contribute to the phenotype, and that the blockade resistance loci are preliminarily linked to 17 markers on four chromosomes. We conclude that strain variation in allograft MSTs following CD40/CD28 blockade results from the ability of CD8(+) T cells in some strains to use alternative modes of costimulation to mount an effective alloresponse.     

Costimulation requirements for antiviral CD8+ T cells differ for acute and persistent phases of polyoma virus infection

The requirement for costimulation in antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections, but it is less defined for CD8+ T cell responses to persistent virus infection. Using mouse polyoma virus (PyV) as a model of low-level persistent virus infection, we asked whether blockade of the CD40 ligand (CD40L) and CD28 costimulatory pathways impacts the magnitude and function of the PyV-specific CD8+ T response, as well as the humoral response and viral control during acute and persistent phases of infection. Costimulation blockade or gene knockout of either CD28 or CD40L substantially dampened the magnitude of the acute CD8+ T cell response; simultaneous CD28 and CD40L blockade severely depressed the acute T cell response, altered the cell surface phenotype of PyV-specific CD8+ T cells, decreased PyV VP1-specific serum IgG titers, and resulted in an increase in viral DNA levels in multiple organs. CD28 and CD40L costimulation blockade during acute infection also diminished the memory PyV-specific CD8+ T cell response and serum IgG titer, but control of viral persistence varied between mouse strains and among organs. Interestingly, we found that CD28 and CD40L costimulation is dispensable for generating and/or maintaining PyV-specific CD8+ T cells during persistent infection; however, blockade of CD27 and CD28 costimulation in persistently infected mice caused a reduction in PyV-specific CD8+ T cells. Taken together, these data indicate that CD8+ T cells primed within the distinct microenvironments of acute vs persistent virus infection differ in their costimulation requirements.