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Host antigen presenting cells (APCs), particularly DCs have a critical role in mediating allogeneic T-cell responses, however, the mechanisms that influence the donor DC reconstitution and persistence of residual host APCs after alloBMT are not characterized. We hypothesize that MHC disparity, pre-transplant conditioning and donor T cells directly influence donor DC reconstitution and amount of residual host DCs after alloBMT. To determine the role of major and minor histocompatibility antigens, syngeneic, B6Ly5.2 (H2b, CD45.1+)C57BL/6 (H2b, CD45.2+), MHC-matched B6Ly5.2 (H2b, CD45.1+)C3H.SW (H2b, CD45.2+) and MHC-mismatched C57BL/6 (H2b)BALB/c (H2d) chimeras were constructed after lethal TBI (900 cGy) and transplantation of 1 x 107 bone marrow (BM) cells from corresponding donors. Six weeks later DC chimerism was determined in the spleen and subcutaneous axilary lymph nodes (LNs) based on the differential expression of CD45 marker. In all three sets of chimeras full reconstitution with donor-derived CD11c+ DCs was found in the spleen but significant amount of residual host DCs was found in the subcutaneous LNs of syngeneic (15%) and MHC-matched (10%) but not MHC-mismatched chimeras (

To probe the mechanisms that regulate the fate of DLI-derived CD4 + and CD8 + T cells and their responses to host antigens, we utilized a MHC-matched minor antigen mismatched model in which DLI were administered to stable allogeneic chimeras expressing differential levels of model antigen. Transgenic mice with ubiquitous (HA104) or pancreas-restricted (Ins-HA) expression of hemagglutinin (HA) as model antigen served as recipients, while the MHC-matched minor-antigen-mismatched T cell receptor (TCR) transgenic mice containing H-2K d restricted CD8 + (CL-4) or I-E d restricted CD4 + T cells (6.5) specific for HA peptide served as a source of antigen-specific lymphocytes. After lethal conditioning, HA104, Ins-HA on BALB/c (H2 d) background and control BALB/c mice received bone marrow from MHC-compatible B10.D2 (H-2 d) donors, followed by DLI three weeks later. DLI consisted of 2 x 10 7 B10.D2 splenocytes spiked with 3 x 10 6 TCR transgenic CL-4 or 6.5 T cells. Differential expression of the Thy 1.1 antigen on transgenic T cells and CFSE labeling were used for in vivo monitoring of CL-4 and 6.5 T cell fate and proliferation pattern. In vivo findings were also correlated with functional testing of the transgenic T cell capacity to respond to their cognate antigen at designated time points. CL-4 T cells adoptively transferred to the HA104 chimeras divided rapidly and by day 6, their level of expansion in the spleen (52x10 6 ; n=3/mice per group) was 12 and 7 folds higher than in the Ins-HA and non-transgenic control BALB/c chimera, respectively. Following that time point, the total amount of CL-4 T cells in all three groups of chimeras progressively declined. In vitro studies with cognate peptide at days 19 and 24 post-transfer revealed that the remaining CL-4 T cells in the HA104 chimeras were anergic in comparison to cells from the other two groups that all vigorously proliferated and produced interferon, although in Ins-HA mice decreased amounts were present on a per cell basis. 6.5 cells also underwent rapid proliferation and expansion in the HA104 chimeras (42.7x10 6 cells/spleen) by day 6 post-transfer with a level of expansion 21 and 22 fold higher than in the Ins-HA and non-transgenic control BALB/c chimeras, respectively. In vitro assessment of 6.5 cell function retrieved from the HA104 chimeras revealed that they were profoundly anergic and had acquired a regulatory function as determined by their ability to suppress in vitro proliferation of naive 6.5 cells. Results from the other two groups show that 6.5 T cells had undergone homeostatic proliferation with minimal expansion. However these cells exhibited in vitro hypoproliferative responses to the nominal antigen and at all time points secreted very low levels of IL-2, on a per cell basis. These data suggest the following: a) DLI-derived CD4 + and CD8 + T cells transferred to the allogeneic host have an intrinsically different fate as implied by the functional responses of Cl-4 and 6.5 T cells regardless of in vivo antigen recognition ; b) the ubiquitously expressed agonist antigen on hematopoietic cells has a direct role in inducing CD4 + cells with regulatory phenotype ; c) the distribution pattern of targeted antigens should be considered in selecting the targets for adoptive immunotherapy with antigen-specific CD8 + clones.

A growing body of evidence suggests that functional unresponsiveness of the immune system to growing solid tumors may be due to tolerance in tumor-specific CD4+ T cells, whereas tumor-specific CD8+ T cells exist in a state of partial activation with limited effector function. We therefore hypothesized that an allogeneic graft-versus-host reaction may provide a helper effect that augments the anti-tumor immune response of host CD8+ T cells in mixed hematopoietic chimeras. To test this hypothesis, we analyzed host versus donor T cell responses to AH1, an H-2Ldrestricted tumor antigen expressed by CT-26, a colon cancer of BALB/c mice. BALB/c mice with pre-established subcutaneous tumor underwent NST from MHC-compatible B10.D2 donors. Two weeks later, groups of chimeras received nothing, B10.D2 splenocytes IV, CT-26 tumor vaccine (irradiated CT-26 cells mixed with a GM-CSF secreting bystander cell line), or both. By staining with H-2Ld tetramers loaded with the AH1 423-431 a.a. peptide, AH1-speci.c CD8+ T cells were found to comprise 2-3% of total spleen CD8+ T cells three weeks after vaccination. In contrast, AH1-speci.c T cells could not be detected in the non-transplanted, tumor bearing mice. To further characterize AH1-specific T cells we characterized T cell avidity for antigen, as measured by binding of AH1 peptide-loaded, Ld IgG dimmers. AH1 specific T cells were easily expanded in vitro using AH-1 peptide pulsed irradiated BALB/c splenocytes. After four weeks of stimulation the highest percentages of cultures containing AH1-specific T cells were derived from tumor-bearing chimeras that received B10.D2 splenocytes and a CT-26 vaccine. Expanded cultures contained AH-1 specific CD8+ T cells derived from the host and the donor, based on the differential expression of Ly9.1 marker on the host lymphocytes and consistent with our recent observations in short-term cultures (Luznik, L. et al: Blood ; in press). Interestingly, AH1-specific allogeneic and host CD8+ T cells, expanded from the mixed chimeras had a higher affinity than the T cells derived from the vaccinated non-transplanted mice. In conclusion, mixed chimerism provides an optimal platform for immunotherapy of solid tumors not only because of allogeneic effect but also through augmentation of host tumor-specific immunity.

Donor lymphocyte infusions (DLI) and allogeneic T cells specific for defined minor histocompatibility antigen (mHAg) are increasingly used in the treatment of relapsed malignancies after allogeneic BMT. However, the fate and function of adoptively transferred T cells are poorly understood. To evaluate the role of host chimerism, complete and mixed chimeras were constructed by transplanting DBA/2 mice (H-2d) with bone marrow (BM) from MHC-mismatched C56BL/6 (H-2b) donors after myeloablative and non-myeloablative conditioning, respectively. Three weeks later, both sets of chimeras received CFSE-labeled B6.SJL T cells, which uniquely express the CD45.1 allele. On day 6 and 10 after DLI administration, 86% and 93% of CD45.1+ T cells in mixed chimeras, respectively, and 41% and 63% of CD45.1+ T cells in full donor chimeras, respectively, underwent more than 7 divisions. Serial monitoring of CD45.1+ T cells in full donor chimeras revealed that 28% of these cells remain undivided for more than 20 days. To define the effect of targeted antigen tissue expression pattern on the fate of allogeneic antigen-specific T cells, we used transgenic mice with wide (HA104) or pancreas restricted (Ins-HA) expression of hemaglutinin (HA). TCR transgenic Clone 4 (CL4) mice, which contain H-2Kd restricted CD8+ T cells specific for HA peptide were used as a source of antigen specific T cells. HA104 or Ins-HA mice on BALB/c background underwent BMT from MHC-compatible B10.D2 donors, followed by DLI from TCR transgenic B10.D2-CL4 mice three weeks later. Differential expression of the Thy 1.1+ antigen on transgenic T cells was used for in vivo monitoring of their fate. Adoptively transferred TCR-specific transgenic CD8+ T-cells engrafted, expanded, and then sharply declined and became undetectable in the mice with wide distribution of targeted HA antigen. In contrast, transgenic T cells expanded but remained detectable for at least 30 days in the animals with pancreas restricted HA expression without causing diabetes. However, co-administration of vaccinia virus expressing HA together with transgenic T cells resulted in diabetes. In conclusion, residual host chimerism, targeted antigens pattern of expression and antigen-specific vaccinations have a potential to influence the fate of adoptively transferred allogeneic T-cells.

Patients undergoing non-myeloablative allogeneic stem cell transplantation (NST) often convert quickly to full donor hematopoietic chimerism, develop GVHD and are unable to receive donor lymphocyte infusions (DLI). We hypothesized that MC could be achieved and GVL effect of DLI could be preserved through the rational integration of post-transplantation immunosuppression based on their biochemical and molecular mechanisms of action. We were especially interested in the effects of Cyclosporine A (CSA) and Rapamycin (RAPA), which either prevent or promote tolerance induction, respectively, as well as post-transplantation cyclophosphamide (Cy), which selectively depletes lymphocytes proliferating in response to recent antigen stimulation. To test our hypothesis we developed a stringent model of NST in which BALB/c mice (H-2d) received 500 cGy total body irradiation, followed by transplantation of bone marrow cells and 10 million splenocytes from C57BL/6 (H-2b) donors resulting in potent lymphohematopoietic-graft-versus-host reaction (LH-GVHR) and fast conversion to complete donor chimerism. Groups of five mice each were treated with RAPA (1.5 mg/kg), CSA (15 mg/kg), or vehicle, daily for 14 days, and then three times weekly for an additional two weeks. Administration of RAPA or CSA from the day of NST was unable to diminish a potent LH-GVHR, resulting in conversion to full donor chimerism at the time of completion of drug treatment. The administration of a single dose of Cy on day 3 alone or addition of CSA from day 4 also resulted in complete donor chimerism, however, the combination of Cy on day 3 and RAPA from day 4, 10, or 20 post-NST resulted in the preservation of MC (61.7± ; 4.14, 53.83± ; 9.05 and 54.62± ; 10), respectively. Subsequent experiments were designed to measure the outcome of CSA and RAPA administration on LH-GVHR and GVL effect of DLI administered to mixed allogeneic chimeras constructed using the previously described NST conditioning regimen. C57BL-10 mice (H-2b) were transplanted with marrow from MHC-incompatible B10.BR (H-2k) donors and, three weeks later, received 2 x 107 B10.BR splenocytes followed by vehicle, CSA or RAPA in the same schedule as above. The conversion to full donor chimerism in the vehicle treated group and groups receiving CSA from day 0 or 20 after DLI was indicative of a potent LH-GVHR. In contrast, early administration of RAPA from day 0 or day 10 after DLI prevented LH-GVHR and preserved MC (40.9± ; 4.2 and 60.9± ; 9.1) while drug administration from day 20 after DLI administration resulted in conversion to full donor chimerism, respectively. In a similar experimental design, groups of animals treated with RAPA or CSA from day 0 or 20 after DLI, received lethal challenge of 1 x 105 C1498 leukemia cells IV on day 10. RAPA administration from day 0 interfered with the GVL effect in comparison to vehicle treated control group (0/10 vs 7/10 ; p