Alloreactive Lymphocytes Research Articles

Clinical Evidence of Autologous Graft versus Tumor Effect

Problem statement: The infused alloreactive lymphocytes in allogeneic stem cell transplantation (Allo-SCT) lead to an unspecific immune response causing Graft-Versus-Tumor (GVT) effect and Graft-Versus-Host Disease (GVHD). The current dogma regarding the anti-tumor effect of GVT is only observed in the Allo-SCT and not in the autologous hematopoietic stem cell transplantation (AHSCT). Approach: This article reviewed the medical literature to show clinical evidence of an autologous graft versus tumor effect. Results: Our group is the first one to publish that patients achieving a higher Absolute Lymphocyte Count (ALC), as a surrogate marker of immune recovery, recovery post-AHSCT experienced superior survival across multiple hematologic malignancies and solid tumors. Moreover, the ALC recovery post-AHSCT depended on the amount of infused ALC collected at the same time stem cells were collected in the autograft. Conclusion: The superior survival observed based on ALC recovery post-AHSCT provided the first clinical evidence of an autologous graft versus tumor effect. In addition, the association between the infused autograft ALC and ALC-recovery post-AHSCT suggested that the stem cell autograft should not be viewed only as the means to collect enough stem cells for hematologic engraftment, but also as an adoptive immunotherapeutic strategy to enhance immune recovery, transcelating into better clinical outcomes.

Adoptive T Cell Therapy Using Educated T Cells Generated by the Sequential Stimulation with DC/Tumor Fusion Cells and Anti-CD3CD28

Hematological malignancies demonstrate susceptibility to T cell mediated killing as evidenced by the potent graft versus disease effect mediated by alloreactive lymphocytes following allogeneic transplantation. A major area of investigation concerns the development of adoptive immunotherapy that would more selectively target and eliminate tumor cells. This strategy is dependent on the expansion of tumor reactive effector cells while minimizing the presence of regulatory T cells and other contributing elements to tumor mediate immunosuppression. Prior studies have demonstrated that ligation of the CD3/CD28 complex results in expansion of T cells ex vivo with phenotypic characteristics that are dependent on the immunologic milieu at the time of stimulation. We have developed a promising cancer vaccine in which autologous tumor cells are fused with dendritic cells (DCs) resulting in the presentation of a wide spectrum of tumor antigens in the context of DC mediated costimulation. We postulated that stimulation with DC/tumor fusions followed by anti-CD3/CD28 would result in the expansion of activated T cells targeting tumor antigens. Tumor cells were obtained from peripheral blood of patients with AML, or bone marrow aspirates of patients with AML and multiple myeloma. DCs were generated from adherent mononuclear cells cultured with rhIL-4, GM-CSF and TNF and fused with tumor cells by coculture in 50% solution of polyethylene glycol. T cells were stimulated by DC/tumor fusions prior to or following exposure to antiCD3/CD28 antibody coated plates for 48 hours. Stimulation by fusions followed by anti-CD3/CD28 resulted in the synergistic expansion of T cells as manifested by the stimulation index (SI) in excess of that seen when cells were stimulated by either pathway alone or first stimulated by anti-CD3/CD28 then fusions. DC/AML fusions induced autologous T cell proliferation with an SI of 3.3 with memory effector cells (CD45RO+) comprising 10% of the total T cell population. In contrast, sequential stimulation with DC/AML fusions followed by anti-CD3/CD28 resulted in a rise in T cell proliferation (SI 8.2) of which 39% of the resultant populations expressed CD45RO. Similarly a rise in CD4+/CD25+ cells was observed following sequential stimulation with DC/AML fusions followed by anti-CD3/CD28 (9.3% vs. 2.7% following stimulation with DC/AML fusions alone). In addition, an increased percentage of CD4+/CD25+ cells expressed IFNγ when exposed to anti-CD3/CD28 following coculture with fusion cells (7% compared to 2% with fusions alone). A significant rise in the percent of Foxp3+ cells was not seen. Expression of granzyme B is up regulated in activated cytolytic CD8+ T cells that confer perforinmediated killing of target cells. As compared to un-stimulated T cells, stimulation with DC/tumor fusions or anti-CD3/CD28 alone resulted in a 3.3 and 3.8 fold increase in CD8+ T cells expressing granzyme B, respectively. In contrast, sequential stimulation with DC/tumor fusions and anti-CD3/CD28 induced a 19-fold expansion of granzyme B+ cells consistent with their enhanced cytolytic capacity. In addition, sequential stimulation with DC/tumor fusions and anti-CD3/CD28 results in heightened capacity to lyse autologous tumor targets as compared to T cells stimulated by fusions alone in a cytotoxicity assay. In spectratyping analysis, T cells undergoing sequential stimulation with DC/tumor fusion cells and anti-CD3CD28 demonstrate greater skewing of CDR3-size usage in the T cell receptor as compared to T cells stimulated by fusions or anti-CD3/CD28 alone consistent with the expansion of a defined clonal population. The pattern of gene expression in T cells stimulated sequentially by DC/AML fusions and anti-CD3CD28 is being assessed by gene arrays to further define the unique nature of this population. In conclusion, we have demonstrated that stimulation of T cells by DC/tumor fusions followed by exposure to anti-CD3/CD28 antibodies results in the expansion of tumor reactive activated T cell populations. A clinical trial evaluating the safety and efficacy of adoptive immunotherapy using T cells generated by sequential stimulation with DC/tumor fusions and anti- CD3CD28 for patients with multiple myeloma following autologous transplantation is planned.

Immunomodulatory Effects of Vitamin D: Implications for the Prevention and Treatment of Graft Versus Host Disease

Allogeneic bone marrow transplantation is a uniquely curative therapy for a subset of patients with hematologic malignancies. However, morbidity and mortality related to graft versus host disease remain substantial. Dendritic cells (DCs) are highly potent antigen presenting cells that play a crucial role in maintaining the balance of immune activation and tolerance as well as the nature of immune reconstitution post-transplant. Persistence of host DCs in the early post transplant period has been shown to play an important role in the activation and expansion of alloreactive lymphocytes and the concomitant risk of GVHD. A major focus of research to enhance outcomes following allogeneic transplantation involves the manipulation of DC recovery post-transplant to minimize activation of alloreactive lymphocytes while preserving anti-tumor immunity responsible for the graft versus disease effect. Vitamin D is a hormone involved in bone metabolism and calcium homeostasis. More recently, vitamin D has been shown to have potent immunomodulatory effects. We have evaluated the effect of vitamin D on the phenotypic and functional characteristics of DC and T cell populations. We have demonstrated that vitamin D hinders the maturation of DCs such that cells that are differentiated in its presence exhibit decreased expression of costimulatory molecules. Peripheral blood mononuclear cells (PBMCs) were isolated from leukopaks obtained from normal donors by ficoll density centrifugation. DCs were generated by culturing the monocyte enriched adherent fraction with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) for 5 days, followed by maturation in the presence of TNFa (25 ng/ml) for 48 hours. DCs were generated in the presence and absence of 10nM of 1,25 hydroxyvitamin D. Mean expression of the costimulatory molecule CD80 and the maturation marker CD83 decreased from 60% to 37% and 53% to 27% respectively in the presence of vitamin D (N=3). To assess the effect of vitamin D on the functional potency of DCs as antigen presenting cells, the capacity of mature DCs to stimulate allogeneic T cell proliferation in the presence of vitamin D was determined. Mature DCs were generated from peripheral blood precursors and then cultured with allogeneic T cells isolated by T cell separation column at a ratio of 1:10. After 5 days, cocultures were pulsed with tritiated thymidine overnight and proliferation was determined by measuring the stimulation index (SI) as defined by uptake of tritiated thymidine of cocultured cells/uptake of tritiated thymidine of T cells alone. Results of 10 serial studies demonstrated the addition of vitamin D resulted in a blunted T cell proliferative response, with mean SI that decreased from 13 to 5. Similarly, the addition of vitamin D to a coculture of DCs and autologous T cells resulted in a 50% reduction in the T cell proliferative response to tetanus toxoid, a recall antigen. In addition, T cells stimulated by allogeneic DCs in the presence of vitamin D were polarized to secrete Th2 cytokines. In three experiments, the mean percentage of T cells secreting IL-10 increased from 2.7% to 4.4%, while expression of IFNγ decreased from a mean of 2.6% to 1.9% in the presence of vitamin D. The presence of vitamin D did not induce FOXP3 expressing regulatory T cell populations, which accounted for 8% and 7% of the T cell population following stimulation by allogeneic DCs in the presence and absence of vitamin D respectively. These data suggest that exposure to vitamin D exerts a tolerizing influence on T cells mediated by its impact on antigen presenting cells. Vitamin D may therefore have a role in the prevention and treatment of graft versus host disease. A clinical trial evaluating the use of vitamin D in the early post-transplant period for the prevention of GVHD is planned.

The immunologic properties of undifferentiated and osteogenic differentiated mouse mesenchymal stem cells and its potential application in bone regeneration

The concept of using mesenchymal stem cells (MSCs) in bone repair has progressively evolved, and the goal of cell-mediated therapy is to prolong the natural physiological abilities of healing, or substitute them, when these are lacking, failing, or progressing too slowly. The future application of MSCs in human therapies depends on the establishment of preclinical studies with other mammals, such as mouse. Surprisingly, the immunologic properties of murine MSCs remain poorly documented. In the present study, flow cytometry revealed that undifferentiated murine MSCs and osteogenic cells differentiated from MSCs (DOCs) express major histocompatibility complex (MHC) class I (H-2 b), but not class II (I-A b). After exposure to interferon-gamma (IFN- γ) for 48 h, MHC class II and costimulatory molecules (B7-1 and B7-2) on the cell surface showed evident up-regulation. Undifferentiated MSCs and DOCs proved to be poor stimulators of T cell proliferation, eliciting alloreactive lymphocyte proliferative responses as low-allogenic stimulators. Initial results show that the expression of MHC class I, MHC class II, B7-1 and B7-2 was similar on human bone morphogenetic protein 2 (BMP2)-expressing recombinant adenoviral vector (AdBMP2) transduced MSCs (30 MOI) when compared with non-transduced cells. However, AdBMP2 gene transfected MSCs elicited significant stimulatory responses. The findings will be important for studying the in vivo behaviour and the fate of MSCs after grafting in mouse pathology models in bone regeneration.

Effect of Seliciclib (CYC202, R-Roscovitine) on Lymphocyte Alloreactivity and Acute Kidney Allograft Rejection in Rat

T cell stimulation by alloantigens is followed by cell cycle progression, an event that is critically dependent on cyclin-dependent kinases. We conducted a study to evaluate whether the cyclin-dependent kinase inhibitor seliciclib affected rat lymph node cells (LNc) activation and proliferation induced by either concanavalin A or allogeneic splenocytes in vitro and studied the mechanisms underlying the suppressive effect. We also investigated the immunosuppressive properties of seliciclib in vivo. Seliciclib completely inhibited in vitro proliferation of LNc and CD8 T cells, in response to either concanavalin A or allogeneic splenocytes. The percentage of activated LNc was lower in mixed leukocyte reactions (MLR) added with seliciclib than in MLR added with vehicle. The percentages of viable and apoptotic cells at the end of MLR with seliciclib were comparable to those of MLR with vehicle. LNc pre-exposed in MLR to seliciclib did not respond to further stimulation with alloantigens, and neither IL-2 nor IL-15 restored proliferation. These data indicate that the inhibitory effect of seliciclib on T cell alloreactivity is not because of cytotoxic effect but is associated with induction of profound T cell anergy. LNc harvested at the end of the primary MLR with seliciclib did not suppress the proliferation of syngeneic LNc cells toward allogeneic splenocytes, thus excluding that seliciclib induced the formation of regulatory cells. Finally, seliciclib partially prolonged grafted animal survival in a rat model of fully major histocompatibility complex-mismatched kidney transplantation. Altogether these results document that seliciclib regulates lymphocyte reactivity and may exert an immunosuppressive effect in vivo in the setting of transplantation.

A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique

AbstractSelective allodepletion is a strategy to eliminate host-reactive donor T cells from hematopoietic stem cell allografts to prevent graft-versus-host disease while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were cocultured with responder cells from HLA-matched or -mismatched donors. Optimal results were achieved when cocultured cells were incubated with 7.5 μM TH9402, followed by dye extrusion and exposure to 5 Joule/cm2 light energy at 5 × 106 cells/mL. In mismatched stimulator-responder pairs, the median reduction of alloreactivity was 474-fold (range, 43-fold to 864-fold) compared with the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9-fold to 3.3-fold) reduction. In matched pairs, alloreactive helper T-lymphocyte precursors were reduced to lower than 1:100 000, while third-party responses remained higher than 1:10 000. This establishes a clinical-scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current Good Manufacturing Practice. This procedure is currently being investigated in a clinical trial of allotransplantation.

Evidence for a Selective Migration of Fetus-Specific CD4+CD25bright Regulatory T Cells from the Peripheral Blood to the Decidua in Human Pregnancy

During pregnancy, the maternal immune system has to tolerate the persistence of fetal alloantigens. Many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. Murine studies suggest that CD4(+)CD25(+) T cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. Previous studies by our group demonstrate that a significantly higher percentage of activated T cells and CD4(+)CD25(bright) T cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy. In this study, we examined the phenotypic and functional properties of CD4(+)CD25(bright) T cells derived from maternal peripheral blood and decidual tissue. Depletion of CD4(+)CD25(bright) T cells from maternal peripheral blood demonstrates regulation to third party umbilical cord blood cells comparable to nonpregnant controls, whereas the suppressive capacity to umbilical cord blood cells of her own child is absent. Furthermore, maternal peripheral blood shows a reduced percentage of CD4(+)CD25(bright)FOXP3(+) and CD4(+)CD25(bright)HLA-DR(+) cells compared with peripheral blood of nonpregnant controls. In contrast, decidual lymphocyte isolates contain high percentages of CD4(+)CD25(bright) T cells with a regulatory phenotype that is able to down-regulate fetus-specific and fetus-nonspecific immune responses. These data suggest a preferential recruitment of fetus-specific regulatory T cells from maternal peripheral blood to the fetal-maternal interface, where they may contribute to the local regulation of fetus-specific responses.

Successful Translation of a GMP-Based, Clinical Scale Selective Allodepletion Approach for Matched Donor-Recipient Pairs from Bench-to-Bedside.

Selective allodepletion (SD) is a strategy to eliminate host-reactive donor T-cells from hematopoietic stem cell allografts to prevent graft versus host disease (GvHD) while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion (PD) process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the rhodamide-based photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were co-cultured with responder cells from HLA-matched or mismatched donors. Optimal results were achieved when co-cultured cells were incubated with 7.5 μM TH9402, followed by dye-extrusion and exposure to 5 Joule/cm2 light energy at 5x106 cells/ml. [Display omitted] In six mismatched stimulator-responder pairs, the median reduction of allo-reactivity was 474-fold (range, 43–864) when compared to the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9–3.3) reduction. In three matched pairs alloreactive helper T-lymphocyte precursors (HTLps) were reduced below 1:100,000 while third party responses remained above 1:10,000. CMV-specific T cells and responses to SEB were preserved. The intracellular TH9402 amount after extrusion correlated significantly with the depletion efficacy obtained against the original stimulator (r2=0.76; p=0.0235) but not against the 3rd party (r2=0.05; p=0.68). This establishes a clinical scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current good manufacturing practice (cGMP) being reliably capable of generating at least 107/kg SD T cells for transfusion using a single PD session. In establishing this SD approach, we set out to design a technique that could ultimately be adopted in many cell transplant processing centers. Recent review of our Investigational New Drug (IND) application by the Food and Drug Administration (FDA) and review of our clinical protocol by our Institutional Review Board have been successful, and a clinical trial is in progress where recipients with hematological malignancies are given a myeloablative preparative regimen followed by a transplant of selected CD34+ cells together with 5 x 106 SD T cells/kg from HLA-matched sibling donors.

Interleukin-7 induced facilitation of immunological reconstitution of sublethally irradiated mice following treatment with alloreactive spleen cells in a murine model of B-cell leukemia/lymphoma (BCL1)

Interleukin-7 (IL-7) plays a key role in maturation and function of both T and B cells. We investigate the potential use of recombinant human IL-7 for facilitation of graft-versus-leukemia (GVL) effects mediated by T cells following transplantation in a murine model. Administration of IL-7 in vivo to allogeneic-transplanted mice improved disease-free survival: 67% of mice treated with IL-7 remained alive and disease free for more than 60 days, in comparison to 17% of the controls (P<0.05). Similar results were obtained when C57BL/6 spleen cells sensitized against irradiated B-cell leukemia (BCL(1)) cells in the presence of IL-7 were transplanted to F(1) mice, followed by IL-7 treatment in vivo. Of the BALB/c mice that received spleen cells from F(1) mice treated with IL-7 following transplantation of C57BL/6 spleen cells sensitized with irradiated BCL(1) in the presence of IL-7, only 29% developed leukemia, as compared to 79% in the control group (P<0.05). Mice treated with IL-7 showed increased splenic and thymic cellularity and improved T cell-dependent proliferative responses compared to the controls (P<0.05). IL-7 may provide a novel tool to enhance immune reconstitution following transplantation of mismatched stem cells and for enhancement of GVL effects mediated by alloreactive lymphocytes.

39. CD3/CD28 Activation and Culture with IL-7 and IL-15 Permit Efficient Transfer of a Suicide Gene in Highly Alloreactive Central Memory Lymphocytes

39. CD3/CD28 Activation and Culture with IL-7 and IL-15 Permit Efficient Transfer of a Suicide Gene in Highly Alloreactive Central Memory Lymphocytes

Experimental Study on Prevention of GVHD by Selective Elimination of Alloreactive Donor Lymphocytes Prior to Stem Cell Transplantation.

Objective: To investigate a new concept aiming for induction of graft-vs-leukemia (GVL) effect prior to stem cell transplantation (SCT). Mismatched lymphocytes given pre-SCT will be followed by selective elimination of alloreactive donor lymphocytes, thus avoiding lethal graft-vs-host disease (GVHD).Methods: Female (BALB/c×C57BL/6)F1 mice (H-2d/b) as recipients received sublethal total body irradiation (TBI) of 4 Gy (60Coγ-ray) on day 0 followed by being inoculated with 0.5×107 P388D1 leukemia cell line on day 1, injection of 1.5×107 allogeneic splenocytes supplied by C57BL/6 male mice(H-2b)for induction of GVHD, intraperitoneally injection of cyclophosphamide (Cy) (200 mg/kg) or TBI (9 Gy) were given on day 7, one day later, treated mice were rescued with 3×107 syngeneic bone marrow cells supplied by (BALB/c×C57BL/6)F1 male mice(H-2d/b). Recipients were observed clinical manifestation, phenotype, re-establishment of haematogenesis, histopathologic changes of internal organs suffered from GVHD and investigated donor chimerism by the semi-quantitate analyses of polymerase chain reaction (PCR). Data was analyzed by SPSS 10.0 software and expressed as mean ± SD.Results: Recipients had no occurrence of leukemia and GVHD by selective elimination of alloreactive donor lymphocytes by Cy and TBI, survived more than 210 days, to become complete-donor chimerism on day +21. The ratio of chimerism descended subsequently, but still displayed mixed-chimerism on day +90. Control mice died of evident GVHD, leukemia or other death-related-transplantation within 20 to 36 days(p<0.01). Attempting to induce GVL effects by mismatched lymphocytes given before stem cell transplantation followed by selective elimination of alloreactive donor lymphocytes, thus avoiding graft-vs-host disease (GVHD) was feasible.

Amplification of Graft vs Malignancy (GVM) Effects by Using Intentionally Mismatched Donor Lymphocytes While Avoiding Graft-vs-Host Disease in Conjunction with Haploidentically Mismatched Stem Cell Allografts in Patients with Hematological Malignances.

Alloreactive donor lymphocytes can induce potent graft vs malignancy (GVM) effects in conjunction with allogenenic stem cell transplantation or when given as donor lymphocyte infusion (DLI) following allogeneic stem cell transplantation. Unfortunately GVM effects are frequently accompanied by graft vs host disease (GVHD) and in many cases, despite severe GVHD, no adequate control of tumor progression can be achieved. In order to improve GVM effects while avoiding GVHD, we have attempted to use natural killer (NK) cells activated with rIL-2 isolated from the blood of haploidentically mismatched donors following engraftment of T-cell depleted donor stem cells. A pilot trial was conducted in patients with hematological malignancies 3 MDS; 2 AML; 1 ALL; 1 biphenotypic leukemia; 5 lymphoma (2 Burkitt's; 1 anaplastic (ALK) & 1 Hodgkin's), all resistant to chemotherapy, 4 failing autologous and 1 allogeneic transplant. Eleven received haploidentically mismatched graft. Prevention of rejection was accomplished by activation-induced clonal deletion following infusion of donor buffy coat 24 hours before administration of cytoxan 120–180mg/kg to eliminate host anti-donor T lymphocytes or by using total body irradiation 750cGy following conditioning that was fludarabine based. Positively selected CD34+ cells or CD3 depleted stem cells mobilized with G-CSF were infused on day 0. Based on our murine data, mismatched rIL-2 activated NK cells cause no GVHD. Consequently, 22 procedures were done involving administration of purified NK cells 1–15 months post transplantation. Donor mononuclear cells were activated 4 days at 37°C in 5% CO2 in air with rIL-2 (6,000 IU/ml) and administered on day 4. NK cells were positively selected with anti-CD56, or by negative selection of anti-CD3 using Miltenyi's immunomagnetic beads. Using this procedure engraftment of mismatched allografts can be accomplished with no or minimal GVHD. Of 11 treated recipients, 5 entered CR. The first patient failing 2 prior transplants from a matched sibling is alive and well 4 years out. Out of 10 patients, 3 died from unrelated causes with no evidence of disease, but no GVHD developed. In conclusion, following engraftment of haploidentically mismatched related stem cell, donor-derived rIL-2 activated NK cells positively selected with anti-CD56 or negatively selected anti-CD3 immunomagnetic beads can be used to induce GVM effects while avoiding GVHD.

Induction of Anti-Cancer Effects by Targeting Alloreactive Intentionally Mismatched T Cells and NK Cells to Tumor Cells Using Bi-Specific Trifunctional Antibodies.

Engraftment of donor stem cells induces host vs graft unresponsiveness thus allowing durable engraftment of donor lymphocytes which can induce graft vs malignancy (GVM) effects following transplantation of MHC compatible donor stem cells. Unfortunately, graft vs host disease (GVHD) frequently results in major toxicity and mortality and limits the clinical efficacy of cell therapy. Although the intensity of GVM effects can be improved by using mismatched donors GVM effects cannot be induced in recipients of haploidentically mismatched allografts because of anticipated lethal GVHD. We have attempted to induce GVM effects by intentionally mismatched donor lymphocytes by targeting donor derived T cells and NK cells activated by rIL-2 to cancer cells, in order to improve the efficacy of the GVL effects on the one hand and prevent or minimize GVHD by targeting alloreactive cells to the tumor cells that need to be eliminated using bispecific tri-functional antibodies, on the other. A trifunctional bispecific antibody (BiLu) directed against murine CD3 and human epithelial-cell adhesion molecule (EpCAM), was administered concomitantly with naïve or IL-2 activated donor splenocytes to mice inoculated with a murine model of melanoma cells transfected with human EpCAM (B16-EpCAM). A total of 10/20 and 32/38 mice treated with BiLu and naïve or IL-2 activated donor splenocytes, respectively, were tumor-free survivors without GVHD for >250 days following tumor inoculation. Out of 28 of the disease free survivors (DFS) without GVHD, treated with IL-2 activated splenocytes and BiLu, 24 mice were resistant to a second tumor inoculum (104) given >250 days following the first tumor inoculation. In contrast, only 8 of the 18 DFS mice treated with naïve splenocytes and BiLu, and 5 of the 10 DFS controls treated with BiLu only, resisted the second tumor challenge. Induction of anti-tumor immunity by IL-2 activated cells and BiLu was more efficient and long-lasting in mice previously injected with a lethal first tumor cell dose (5×104) than in mice inoculated with a sub-lethal tumor cell dose (5×103). Interestingly, a similar infusion of donor cells labeled with bi-specific anti-EpCAM and anti-CD3 following allogeneic stem cell transplantation for metastatic breast cancer did not result in augmentation of GVHD. In conclusion, concomitant inoculation of alloreactive donor lymphocytes and trifunctional bispecific antibody can be effective for targeting of killer T and NK cells to tumor cells while avoiding GVHD, as well as for induction of long-lasting anti-cancer immunity, most likely due to presentation of tumor antigen to T cells by antigen presenting cells (dendritic cells) expressing Fc receptor binding to the Fc portion of the bi-specific antibody.

High Efficiency Clinical Scale Selective Depletion of Alloreacting T Cells Using Expanded T Lymphocytes as Antigen-Presenting Cells and a TH9402-Based Photodepletion Technique in HLA-Mismatched and Matched Donor-Recipient Pairs.

Selective depletion (SD) is a strategy to eliminate host-reactive donor lymphocytes from blood stem cell allografts to prevent GVHD and maintain GVL-effects. As an alternative to CD25-immunotoxin-based SD, we investigated a photodepletion (PD) process, whereby allo-activated donor cells are labeled with a photosensitizing rhodamine-based dye, 4,5-dibromorhodamide 123 (TH9402), and exposed to visible light, which preferentially eliminates allo-activated dye-retaining cells. Stimulator cells were prepared from recipient-derived leukapheresis mononuclear cells (MNCs) and cultured using anti-CD3 and 100 IU IL-2/ml. Responder cells (leukapheresis MNCs) from 3 random HLA-mismatched volunteers and 3 HLA-matched sibling donors were cocultured 1:1 with irradiated stimulators for 3 days. Cultured cells were incubated with 7.5 μM TH9402, followed by dye-extrusion and exposure to 5 Joule light energy in the PD light source (Celmed Bioscience Inc., Canada) at 5x106 cells/ml in FEP plastic bags. Flow cytometry was performed before and after PD and included surface phenotyping for CD25 and intracellular staining for forkhead protein3 (foxp3). Depletion efficacy was studied by mixed lymphocyte reactions (MLR) in mismatched pairs and by helper-T-lymphocyte precursor (HTLp) frequency assay in matched pairs. All six clinical-scale experiments provided sufficient reduction of allo-reactivity and retention of third party responses as measured against a pool of 5 donors. In mismatched pairs mean reduction of allo-reactivity was 703-fold (± 141) when compared to unmanipulated donors. Third-party responses were maintained, with a mean reduction of only 1.3 ± 0.15-fold (Figure A). In matched pairs alloreactivity was reduced below the “GVHD-threshold” of 1/100.000 whilst third party responses remained above 1/10.000 precursors (Figure B). Effective depletion was observed despite that fact that a small fraction of CD25+ T cells remained after PD. These were mainly CD4+CD25+ T cells (1.5 ± 1.1 % of CD4+) and very residual CD8+CD25+ T cells (0.3 ± 0.2 % of CD8+), and represented predominantly CD4+foxp3+ regulatory T cells (Tregs) which persisted after PD (1.4 ± 1.7 % of CD4+). This establishes a clinical scale PD process capable of highly efficient removal of alloreactive lymphocytes from mismatched and matched MLRs while maintaining desirable third party responses. As PD targets activation-based changes in MDR-1 that result in an altered dye efflux, the mechanism of action is distinct from surface-marker-based allodepletion (e.g. CD69, CD25). Thus, PD may overcome instability of activation-based surface marker expression resulting in more consistent and effective depletion. PD may further mitigate the risk of GVHD by conserving Tregs. This approach will now be tested in a clinical SD trial. [Display omitted]

Induction of Maternal Tolerance to Fetal Alloantigens by RANTES Production

Previous studies have demonstrated a requirement for RANTES (regulated on activated normal T-cell expressed, and secreted) at immune privileged sites; we have investigated the role of RANTES in the induction of maternal-fetal tolerance. Endometrial and peripheral T lymphocytes were obtained from women with recurrent pregnancy losses (RPLs) and fertile women. RANTES modulation by progesterone or paternal alloantigens was measured by enzyme-linked immunosorbent assay or flow cytometry analysis. Progesterone significantly increased intracellular RANTES expression in CD4+ and CD8+ endometrial T cells. Moreover, alloreactive lymphocytes from RPL patients produced lower RANTES levels when compared with those from fertile women. At the local level, treatment with recombinant RANTES induced a decrease in CCR5 and CXCR4 messenger RNA that correlated with an increase in T-bet expression. RPL patients and normally fertile women express RANTES similarly, but differ in their patterns of RANTES receptor expression. RANTES may be implicated in the local induction of a Th1-type response necessary for successful implantation. Altered response to RANTES stimulation among some RPL patients may be responsible for poor pregnancy outcomes.

Post transplant persistence of host cells augments the intensity of acute graft-versus-host disease and level of donor chimerism, an explanation for graft-versus-host disease and rapid displacement of host cells seen following non-myeloablative stem cell transplantation?

Although the use of non-myeloablative stem cell transplantation (NST) reduces the severity of graft-versus-host disease (GVHD), GVHD remains a major complication following allogeneic transplantation. Since following NST in comparison with myeloablative conditioning, higher proportions of host immunohematopoietic cells may persist while donor-derived alloreactive lymphocytes are being infused, thus possibly serving as host antigen presentation for continuous stimulation of donor T cells, we speculated that GVHD may be similarly amplified by conditioning followed by intentional administration of host cells. This hypothesis was tested in a preclinical animal model. Increased incidence of GVHD, higher mortality and increased levels of chimerism were observed in recipients reconstituted with host cells, particularly with non-irradiated spleen cells. Graft-versus-leukemia effect was not impaired by post transplant cell administration. These results suggest that GVHD may be amplified by recipient cell infusion using either irradiated or viable stimulatory host cells, thus possibly explaining in part higher than anticipated incidence of GVHD and rapid displacement of host cells and conversion to 100% donor type cells following NST. Administration of irradiated host antigen-presenting cells post transplantation may thus represent a potential approach for amplification of the alloreactive capacity of donor lymphocytes following stem cell transplantation.

A new strategy using autologous dendritic cells and lymphokine-activated killer cells for cancer immunotherapy: efficient maturation of DCs by co-culture with LAK cells in vitro

Among a variety of antigen presenting cells (APCs), accumulating results support that the mature dendritic cell (DC) has the potential to induce efficient cytotoxic T lymphocyte (CTL) responses in the context of peptide-based immunotherapy. DCs have been known to assume the mature form by signaling through the CD40-CD40 ligand (CD40L) interaction, which may be provided by activated CD4+ T cells expressing abundant CD40L molecules on their surfaces. Here, we report that DCs generated from peripheral blood monocytes obtained from patients with advanced cancer exhibit a mature phenotype after co-culturing with autologous lymphokine-activated killer (LAK) cells generated by the stimulation of peripheral blood mononuclear cells with anti-CD3 monoclonal antibody (mAb) and interleukin (IL)-2. Part of this process appeared to be dependent on the expression of CD40L on the surface of LAK cells, although it was also suggested that some other humoral factors produced by LAK cells may be involved in this effect as well. DCs derived from the donors, of which LAK cells demonstrated a higher Th1/Th2 ratio upon activation determined by the intracellular detection of interferon-gamma and IL-4, showed more efficient maturation upon co-culture with LAK cells than DCs from donors with a low Th1/Th2 ratio. Importantly, these matured DCs induced a two-times stronger antigen-presenting capacity measured by an allo-reactive mixed lymphocytes reaction assay as compared to immature DCs. These results imply the use of the combination of DCs and LAK cells for immunotherapy against cancer.

Modulation of Graft-versus-Host Disease: Role of Regulatory T Lymphocytes

Graft-versus-host (GVH) disease (GVHD) continues to be a major life-threatening complication after allogeneic bone marrow transplantation. Considerable progress has been made elucidating the pathophysiology of acute GVHD. Mature donor T cells transferred along with the marrow graft directly recognize antigenic differences on antigen-presenting cells of the host. Once activated, donor antihost-specific T cells can mediate tissue destruction. Interestingly, the failure to clonally delete autoreactive T cells in the thymus can also lead to an autoimmune syndrome mimicking the pathology of GVHD. Negative selection in the thymus may be compromised either by damage to the thymic epithelium (because of a direct attack by donor antihost alloreactive T cells) or by the use of immunosuppressive drugs that inhibit clonal deletion. An important component underlying GVHD mediated by either alloreactive or autoreactive T cells is the absence of a competent peripheral regulatory system. Studies in animal model systems clearly indicate that regulatory T cells play a vital role in down-regulating GVHD and are critically important for the establishment of active dominant tolerance to both allo- and self-major histocompatibility complex antigens. Although multiple populations of cells appear to participate in this process, CD4 + regulatory T cells that innately express CD25 + appear to orchestrate the regulatory control of the immune response. Evidence for regulatory T cells in clinical bone marrow transplantation, however, remains rudimentary. The recent identification that CD4 +CD25 + regulatory T cells preferentially express the Foxp3 nuclear transcription factor and the development of molecular reagents to isolate antigen-specific T cells have provided unique opportunities to explore immunoregulatory mechanisms after clinical marrow transplantation. Recent studies in recipients of clinical bone marrow transplantation suggest that antigen-specific CD4 +CD25 +Foxp3 + T cells play a vital role in the regulatory control of GVH reactions mediated by both alloreactive and autoreactive lymphocytes. These regulatory T cells also appear to facilitate the establishment of donor antihost and donor antidonor (self) tolerance.

765. Suicide Gene Therapy by Central Memory Human T Lymphocytes

Adoptive immunotherapy represents a powerful therapeutic approach for the treatment of cancer and infectious diseases. A compelling example is represented by donor lymphocytes infusions (DLI) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although alloreactive donor lymphocytes are very powerful in mediating malignant-cell killing (graft-versus-leukemia-GVL), recognition of host antigens by donor cells often results in a life-threatening event called graft versus host disease (GVHD). To solve this double bind, we investigated the therapeutic potential of donor lymphocytes retrovirally transduced to express the suicide gene thymidine kinase of Herpes Simplex virus (TK) in high-risk patients affected by hematologic malignancies. Donor TK+ lymphocytes are sensitive to the pro-drug ganciclovir (GCV) and can thus be safely infused to patients after allo-HSCT. Preclinical and clinical studies have substantiated the concept that a time-wise infusion of GCV can fully control life-threatening GVHD and may separate GVHD form GVL. The efficacy of the suicide machinery suggests that an increase in the alloreactive potential of TK+ cells may boost the anti-tumor effect, while allowing the selective control of GvHD. The clinical efficacy of T-cell based therapy relies not only on the ability of T cells to mediate an anti-tumor response (effector phase), but also in the ability of T cells to persist and expand in vivo, thus providing a long-term protection from disease relapse (memory phase). In this study, we tackled the rules governing the generation and expansion of memory T cells, by exploiting the positive effects of the IL-7 homeostatic cytokine on memory cells, to increase the potency of the TK-based T cell therapy. We showed that polyclonal stimulation with anti-CD3 antibodies (aCD3) and culture in the presence of high doses of IL-2 generates mainly CD45RA-CCR7-effector memory (EM) cells, with a mixed phenotype in regard to CD28/CD27 co-expression and a limited ability to engraft and persist in vivo. On the contrary, polyclonal stimulation by anti-CD3/CD28 conjugated cell sized beads and culture in the presence of low doses of IL-7 result in the generation of TK+ cells with a central memory (CM) phenotype, CD45RA+CCR7+, CD28+CD27+, that produce high levels of IL-2 upon restimulation, and express sustained high levels of IL-7R alpha, a receptor required for the generation and survival of memory T-cells. In mixed lymphocytes cultures performed with CFSE-labeled TK+ cells we observed that CM TK+ cells display a higher proliferative alloreactive activity than EM TK+ cells. Finally, when infused in conditioned NOD/scid mice EM TK+ cells displayed a limited ability to engraft and cause xenogeneic-GvHD, while CM TK+ lymphocytes persistently engrafted and caused lethal GvHD in a significant fraction of mice. GvHD was successfully treated by GCV administration.

Short-term homing assay reveals a critical role for lymphocyte function-associated antigen-1 in the hepatic recruitment of lymphocytes in graft-versus-host disease

The liver is a major target organ of graft versus host disease (GvHD) with massive infiltration of alloreactive lymphocytes resulting in hepatitis and hepatocyte injury. Although adhesive mechanisms have been implicated in the biology of GvHD hepatitis, the identity of homing receptors involved in the initial recruitment of cells from the blood is not known. We have developed a short-term homing assay in a model of murine GvHD. Splenocytes from donors at an active stage of GvHD were injected intravenously into adoptive recipients also undergoing GvHD. The recruitment of cells to the liver was assessed 6h after cell transfer. Activated donor CD8 and CD4 lymphocytes expressed lymphocyte function antigen-1 (LFA-1), alpha4-integrins, and P-selectin binding ligands, and localized more efficiently than naïve T cells. Immunoneutralization of LFA-1 reduced the recruitment of CD8 and CD4 lymphocytes to the liver by more than 60%. Anti-LFA-1 antibody also markedly reduced infiltration of lymphocytes in periportal areas and protected against hepatocellular damage. We demonstrate a critical role of LFA-1 in the recruitment of activated lymphocytes to the liver and in immune-cell mediated hepatitis. LFA-1 may be an effective therapeutic target for protecting the liver following bone marrow transplantation.