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A new immunosuppressive agent makes its debut in primate models of kidney transplantation. The drug has the potential to join the ranks of immunosuppressants currently used in transplantation.

To the Editor: Xenotransplantation provides a potentially promising solution to the shortage of human organs and tissues and offers advantages over other approaches. The development of genetically ...

Abstract 1911 A major limitation of adoptive T cell therapies for cancer is the failure to maintain durable anti-tumor immunity. Graft-versus-tumor responses following bone marrow transplantation (BMT) may only be short-lived due to 1) defects in memory precursor generation and 2) exhaustion of surviving CTL that results from direct recognition of alloantigen upon non-hematopoietic cells {Flutter et al. JCI 2010}. In this study, we have explored the potential for enhancing co-stimulatory signals either alone, or in combination with co-inhibitory PD-1-PD-L1 blockade to improve the long term CTL response. Signalling through OX40, a TNF-receptor family member, has been shown to have an important role in long-term immunity, including an enhancement in the generation of CD8 T cell memory precursors. The mechanisms of action are complex and may include both direct effects on CD8 cells and indirect effects on CD4 helper cells or via inhibition of Treg. In initial experiments, we evaluated the effects of early enforced OX40 co-stimulation following delayed transfer of donor T cells to haplo MHC-mismatched chimeras, 10 weeks following nonmyeloablative BMT. OX40 expression peaked on transferred CD4 and CD8 T cells in the first 1–2 weeks following transfer and was sustained thereafter, especially in the CD4 subset. 48 hours after T cell transfer, recipient mice were treated with agonistic anti-OX40 antibody (OX86) or isotype control. OX86 treatment led to a 9-fold increase in the expansion of CTL in comparison to isotype control treated mice, enhanced production of Granzyme B and IFNγ and led to more rapid eradication of host hematopoietic targets or host tumor cells. Moreover, OX86 antibody acted directly on CD8 T cells and bypassed the requirement for help from donor CD4 cells. However, although enforced OX40 co-stimulation boosted the primary effector response, it did not increase numbers of memory precursor cells, as assessed by survival and recall responses following transfer to antigen free hosts, and was unable to prevent eventual exhaustion of surviving donor CTL as tested at 60 days following transfer. Similarly, OX86 was unable to prevent exhaustion of CD8 cells transgenic for the male antigen-specific Matahari (Mh) TCR following adoptive transfer to male BMT recipients reconstituted with female BM. We have shown previously that the functions of exhausted donor CD8 cells are partially restored by blockade of the co-inhibitory PD-1 pathway in both haplo mismatched and MHC-matched mHAg mismatch models. We hypothesized that provision of co-stimulatory signals when exhaustion had become established would increase the effectiveness of co-inhibitory blockade. Therefore, 6 weeks after Mh CD8 T cell transfer to male BMT recipients, we examined the effect of OX86, with or without additional blockade of the PD-1 pathway. Only a minority of Mh CD8 cells from animals receiving isotype control antibody were proliferating in vivo as measured by BrdU incorporation over a 7 day pulse (20 +/−3% BrdU+) and few cells were able to produce IFNγ following antigen stimulation in vitro (3.5+/−1.4 x104 IFNγ+ cells/spleen). OX86 alone offered no restoration of function (15 +/− 2% BrdU+; 3.3+/−0.4 x104 IFNγ+ cells; p=ns). Blockade of PD-L1 modestly increased turnover of cells (37 +/− 6 % BrdU+; p In conclusion, forced co-stimulation via OX40 alone is unable either to prevent CTL exhaustion or restore CD8 T cell function when exhaustion has become established. In contrast, the marked synergy observed when agonistic OX40 signals are combined with co-inhibitory blockade, is consistent with a model in which the PD-1 pathway acts at a critical checkpoint that regulates the response to co-stimulation. Thus, these data suggest a novel approach to restoring the functions of exhausted anti-tumor CTL by modulating co-stimulatory and co-inhibitory pathways simultaneously. Disclosures: No relevant conflicts of interest to declare.

Graft versus host disease (GVHD remains the major obstacle to the application of bone marrow transplantation across HLA barriers. Recent advances in our understanding of GVHD pathophysiology have resulted in the evaluation in animal models and in clinical trials of some novel approaches to avoiding and treating GVHD. Continued advances in our knowledge are likely to result in the clinical application of biological therapies to maximize graft versus leukemia effects and alloengraftment, while avoiding GVHD.

Radioresistant host elements mediate positive selection of developing thymocytes, whereas bone marrow-derived cells induce clonal deletion of T cells with receptors that are strongly autoreactive. In contrast to T cell development, little is known about the elements governing the natural killer (NK) cell repertoire, which, similar to the T cell repertoire, differs between individuals bearing different major histocompatibility complex (MHC) phenotypes. We have used murine bone marrow transplantation models to analyze the influence of donor and host MHC on an NK cell subset. We examined the expression of Ly-49, which is strongly expressed on a subpopulation of NK cells of H-2b mice, but not by NK cells of H-2a mice, probably because of a negative effect induced by the interaction of Ly-49 with Dd. To evaluate the effect of hematopoietic cell H-2a expression on Ly-49 expression of H-2b NK cells, we prepared mixed allogeneic chimeras by administering T cell-depleted allogeneic (B10.A, H-2a) and host-type (B10, H-2b) marrow to lethally irradiated B10 mice, or by administering B10. A marrow to B10 recipients conditioned by a nonmyeloablative regimen. Expression of H-2a on bone marrow-derived cells was sufficient to downregulate Ly-49 expression on both H-2a and H-2b NK cells. This downregulation was thymus independent. To examine the effect of H-2a expressed only on radioresistant host elements, we prepared fully allogeneic chimeras by administering B10 bone marrow to lethally irradiated B10.A recipients. B10 NK cells of these fully allogeneic chimeras also showed downregulation of Ly-49 expression. The lower level of H-2a expressed on H-2b x H-2a F1 cells induced more marked downregulation of Ly-49 expression on B10 NK cells when presented on donor marrow in mixed chimeras than when expressed only on radioresistant host cells. Our studies show that differentiation of NK cells is determined by interactions with MHC molecules expressed on bone marrow-derived cells and, to a lesser extent, by MHC antigens expressed on radioresistant host elements.

Engrafted allogeneic hematopoietic cells have a unique capacity to induce a state of donor-specific transplantation tolerance across major histocompatibility complex barriers. This state allows permanent acceptance of donor-type organ grafts, with otherwise normal immunocompetence. We hypothesized that introduction of allogeneic MHC genes into autologous bone marrow which is then returned to recipient mice might similarly induce specific tolerance to products of the introduced MHC genes, without the risk of graft-vs-host disease. We demonstrate here that the introduction of MHC class I Kb cDNA by retrovirus-mediated gene transfer into B10.AKM (Kk) hematopoietic cells confers specific hyporesponsiveness to allogeneic skin grafts expressing Kb.

Objective: To characterize deletional tolerance of peripheral alloreactive CD8 T cells induced by anti-CD40L and allogeneic BMT. Methods: B6 mice received 3Gy TBI on Day -1 and 2mg anti-CD40L ip prior to B10.A BMT on Day 0. For CD8 transfer, KbDb KO mice were given 6-9x10^6 purified, recipient-type CD8 cells on Day 0. For deletion and microarray studies, synchimeras containing 10-20% 2C TCR Tg (donor-reactive) cells among CD8 T cells were used. Results: Using adoptive transfer, we determined that the inhibitory PD-1 receptor is required on CD8 T cells for tolerance (6/7 vs. 0/8 chimeras with WT vs. KO CD8s). 2C cells in animals treated with blocking anti-PD-1 initially disappeared at a similar rate to those in tolerized mice. On Day 6 the frequency of apoptotic 2C cells in tolerant controls was equivalent to that in anti-PD-1-treated mice (p = 0.2) and greater than that in mice with no mAb (p = 0.03). Following complete rejection of donor BM, 2C cells in anti-PD-1-treated mice expanded (p = 0.004 vs. tolerant at Day 42). Microarray analysis on sorted 2C+ CD8 T cells isolated on Day 6 post-BMT revealed significantly higher transcript levels for the apoptosis-associated Nr4a family members Nur77 (7.3 fold, p = 0.01), Nurr1 (23 fold, p = 0.01), and Nor1 (9 fold, p = 0.007) and the tolerance-associated Egr family members Egr-1 (4 fold, p = 0.01), Egr-2 (15 fold, p = 0.001), and Egr-3 (8 fold, p = 0.04) in tolerant compared to rejecting mice.

Mixed hematopoietic chimerism can be induced in mice receiving allogeneic BMT with non-myeloablative conditioning with T cell-depleting mAbs, low dose TBI, and thymic irradiation (TI) (7 Gy). Chimeras are tolerant to donor and host antigens. We have reduced the amount of conditioning used to achieve mixed chimerism by using high stem cell doses in place of TBI, and by replacing both recipient T cell depletion and TI with costimulatory blockade. We have now achieved lasting chimerism and tolerance without host pre-conditioning, by giving a high dose of marrow followed by two costimulatory blockers. Donor cells seed the thymus life-long and mediate clonal deletion of donor-reactive T cells. Host APC in the thymus delete host-reactive thymocytes. In the regimens involving costimulatory blockade, peripheral mechanisms, including deletion, tolerize the pre-existing T cell repertoire. The low toxicity of these regimens suggest an approach to using BMT for the induction of donor-specific transplantation tolerance. In these murine studies, GVHD is avoided because the reagents used to deplete or tolerize recipient T cells are still present when donor marrow is given, so donor T cells are also depleted or inactivated. Mixed chimerism can also be used to exploit the graft-vs-leukemia (GVL) effect of GVH-reactive donor T cells while avoiding GVHD. Donor lymphocyte infusions convert established mixed chimeras to full donor chimeras, but GVHD does not occur. Based on this observation, we initiated a clinical trial of mixed chimerism induction followed by DLI at 5 weeks for the treatment of hematologic malignancies. We have seen that sustained mixed chimerism can be achieved, that delayed DLI can convert mixed chimeras to full donor chimeras without causing severe GVHD, and that powerful anti-tumor effects are achievable. Donor-specific allograft tolerance has been achieved in a multiple myeloma patient simultaneously receiving non-myeloablative BMT and a kidney transplant from the same donor.

Abstract 3368 Poster Board III-256 Specific tolerance following combined kidney and bone marrow transplantation (Kd/BMT)for patients with end stage renal disease (ESRD) with or without an underlying malignancy has been accomplished, as evidenced by prolonged normal renal function without ongoing immunosuppression (IS)and the demonstration of in vitro donor specific hyporesponsiveness (N Engl J Med 2008; 358:353-361; Am J Transplant 2006;6:2121-2133). In order to achieve potent anti-myeloma responses and induce tolerance through the induction of mixed chimerism (MC) for the renal allograft, 7 patients (median age 48 (34-55) yrs with multiple myeloma (MM) and ESRD received a combined HLA-matched Kd/BMT, with longest follow-up time of almost 11 years. An eighth pt developed cyclophosphamide (CY) cardiotoxicity on day -5 and did not receive a transplant. Preparative therapy for the transplants consisted of CY 60 mg/kg (days -5, -4) with hemodialysis 14 hrs after each CY dose, equine anti-thymocyte globulin, 15-20 mg/kg on days -1, +1, +3, and +5 and thymic irradiation (700 cGy) on day -1. Cyclosporine (CSP) was begun on day -1, with combined Kd/BMT on day 0. Nine additional donor lymphocyte infusions were given to 5 pts (5 for chimerism conversion, 4 for persistent/progressive disease (PD)). Acute (A) and chronic (C) GVHD developed in one patient following DLI for early PD, while chronic GVHD developed in two pts (one after a second stem cell transplant). Characteristics and outcomes of the 7 combined Kd/BMT recipients are as follows: # after 2nd transplant (myeloablative) from the same donor ; FDC: full donor chimerism In summary, 5 of 7 pts are alive, 4 without evidence of MM from 2.7 to 10.9 yrs post-Kd /BMT. Three pts have normal renal function without IS, while two pts have normal renal function on IS for chronic GVHD. Sustained renal allograft tolerance and prolonged anti-myeloma responses are achievable following nonmyeloablative HLA-matched kidney and BMT and the induction of mixed chimerism. This study was supported by the Immune Tolerance Network, National Institute of Allergy and Infectious Diseases. Disclosures: Off Label Use: Equine anti-thymocyte globulin: in vivo T cell depletion Cyclophosphamide:conditioning therapy for transplantation.

Objective: To determine the mechanism by which a widely used anti-mouse CD40L mAb (clone MR1) given with allogeneic bone marrow cells (BMCs) tolerizes peripheral CD8 T cells. Methods: B6 mice received 3Gy total body irradiation one day prior to ip injection of 2mg a-CD40L & iv infusion of 25x106 MHC-mismatched B10.A BMCs. This regimen induces long-term mixed chimerism, as measured by durable presence of donor leukocytes, without depletion of recipient CD4 or CD8 T cells. Results: Although genetic deficiency of CD40L alone allowed graft acceptance in recipients depleted of CD8 cells, it did not prevent rejection in mice replete with CD8 T cells. Thus, Fc-dependent functions of a-CD40L were studied. A role for complement was excluded, as 4/5 C3 KO recipients became chimeric. CD8 T cell-intrinsic CD40L was dispensable (3/5 chimeras each in mice with WT or KO CD8 cells), indicating that a-CD40L need not act directly on CD8 cells. However, FcgRIIB KO recipients promptly rejected donor BM unless CD8 T cells were depleted (5/7, 0/5, 7/8, & 5/7 chimeras for WT, KO, WT + a-CD8, & KO + a-CD8 groups, respectively). Recipient B cells are the likely source of FcgRIIB, as DC-derived FcgRIIB was not essential (4/6 chimeras each in mice with WT or KO DC). Conclusions: A-CD40L with allogeneic BMCs generates immune complexes that ligate FcgRIIB, inducing as yet undefined downstream effects essential for CD8 T cell tolerance. Supported by NIH Grant RO1 49915 & NDSEG Fellowship.

NKT cells from naïve mice are mainly CD4+ or CD4−CD8−. However, it has been reported that CD8+ NKT cells can be expanded in vitro from splenocytes, bone marrow cells and thymocytes of C57BL/6 (B6) mice by stimulation with anti-CD3 mAb and cytokines, and that the expanded CD8+ NKT cells mediate strong graft-vs.-leukemic (GVL) effects without severe GVHD after adoptive transfer into allogeneic mice. We now describe the presence of CD8+NK1.1+ cells in recipient livers (approximately 2–6%), but not in other tissues (spleen, lung, bone marrow, thymus and PBMC), in various allogeneic hematopoietic cell transplantation (allo-HCT) models. The generation of CD8+NK1.1+ cells is likely a consequence of alloresponses, as these cells were not detected in the liver of syngeneic HCT controls. Flow cytometric analysis confirmed that these cells are CD1d-independent, TCRαβ+ T cells with a memory phenotype (CD44+ and CD62L−), and do not express CD49b, Ly-49C/I, Ly-49G2, or Ly-49D. In a sublethally (6 Gy)-irradiated B6-to-B6D2F1 allo-HCT model, NK1.1+ CD8 T cells became detectable by week 2, increased in number until approximately week 8, and gradually declined thereafter but were still detectable in the liver at day 100 after allo-HCT. By using CD45.1 and CD45.2 congeneic donors, we determined that the majority of NK1.1+ CD8 T cells were derived from the donor splenocytes. Furthermore, depletion of CD8+, but not NK1.1+, cells from the donor splenocytes prior to transplantation prevented the generation of NK1.1+ CD8 T cells, indicating that these cells were derived from donor NK1.1−CD8+ splenic T cells. Our data demonstrate that donor CD8 T cells can acquire NK1.1 expression upon activation in allo-HCT recipients, and that these NK1.1+ CD8 T cells maintain a memory phenotype and persist in the recipients with preferential accumulation in the liver. Studies are currently in progress to determine the role of activated donor NK1.1+ CD8 T cells in GVHD and GVL effects.

Previous studies have shown that essentially all donor CD4 T cells adopt the activated/memory phenotype in the setting of GVHD induced in freshly conditioned hosts. However, it is unknown whether this activation is all antigen-driven or might reflect lymphopenia-induced activation and/or “bystander” activation by the abundant pro-inflammatory stimuli present in the GVHD environment. We compared the activation and accumulation of GVH-reactive CD4 T cells specific for recipient superantigens, minor (mHA) and MHC antigens with that of GVH non-reactive TCR transgenic CD4 T cells in lethally irradiated BMT recipients across a full haplotype barrier (B6→B6D2F1). Wild-type B6D2F1 or transgenic B6D2F1 mice expressing membrane-bound chicken ovalbumin (OVA-B6D2F1) received lethal total body irradiation (TBI), B6 bone marrow cells (BMC) to prevent death from aplasia and a donor lymphocyte inoculum of 106 OTII CD4 T cells (specific for OVA peptide 323-339/IAb, representing a mHA) and 107 polyclonal B6 splenocytes, to preserve the strong polyclonal T cell response. On days 3 and 6 post-transplant, evidence for activation of OTII CD4 T cells was detected only in OVA-B6D2F1 recipients and not in wild-type recipients, as shown by upregulation of CD44 (average median fluorescence intensity 250 vs 92; p

T cell immunotherapies for cancer should ideally generate high levels of anti-tumor activity, with minimal host injury and permit the prolonged survival of functional memory/effector cells to prevent tumor recurrence. Following allogeneic stem cell transplantation, delayed donor leukocyte infusion (DLI) is one strategy employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease. However, patients remain at significant risk of relapse following DLI and murine models of delayed DLI indicate that this results from the eventual loss of functional, alloreactive cytotoxic T lymphocytes (CTL) [Mapara et al. Transplantation 2003]. We hypothesised that the loss of functional CTL is driven by persistent stimulation of donor CD8 cells by alloantigen expressed by peripheral tissues. In order to follow and characterise an alloreactive CD8 response under conditions in which alloantigen was present or absent in peripheral tissues, we employed a model in which either parental B6 (H2b) or B6 x DBA-2 F1 (BDF1, H2dxb) mice were lethally irradiated and reconstituted with a mixture of B6 and BDF1 T cell depleted bone marrow. 8-10 weeks later congenic CD45.1 B6 splenocytes were transferred into the established mixed chimeras. This allowed us to test the importance of peripheral antigen in the loss of alloreactive CTL responses, since alloantigen was either restricted to the hematopoietic system (B6 +BDF1 → B6) or was ubiquitously expressed (B6 +BDF1 → BDF1). Following transfer of CD45.1 B6 splenocytes, the ensuing alloantigenspecific T cell response in both groups led to the elimination of alloantigen-positive (BDF1-derived) hematopoietic elements. Thereafter, alloreactive CD8 cells resided in an environment in which peripheral alloantigen was present (PA+) or absent (PA-). We observed similar kinetics of initial CD45.1+ CD8 cell proliferation and expansion and similar acquisition of a CD44highCD62Llow phenotype. However, by day 60, there were striking differences in the phenotype and function of transferred CD8 cells. In PA- hosts, CD45.1+ CD8 cells killed allogeneic target cells effectively both in vitro and in vivo, underwent rapid proliferation in a mixed leukocyte reaction and produced the effector cytokine, IFN-γ. In contrast CD45.1+ CD8 cells from PA+ hosts had little or no cytotoxic activity, did not proliferate to alloantigen and were IFN-γlow. Moreover, CD45.1+ CD8 cells from PA+ hosts displayed high levels of the co-inhibitory receptor PD-1, low levels of the IL-7Rα chain and responded poorly to IL-7 and IL-15 in vitro, a phenotype typical of the ‘exhaustion’ signature observed in CTL following chronic antigen exposure. In comparison, CD45.1+ CD8 cells from PA- hosts expressed significantly lower levels of PD-1, higher levels of IL-7Rα and demonstrated better responsiveness to IL-7 and IL-15 in vitro. In vitro PD-1 or PD-L1 blockade restored IFN-γ generation to CD45.1+ CD8 cells from PA+ hosts, suggesting that the PD-1 pathway may play a functional role in driving exhaustion of these cells. Importantly we observed no loss of long-term alloreactive CD4 responses in either PA+ or PA- hosts. This finding is consistent with a model in which peripheral alloantigen drives exhaustion since the majority of cells expressing Class II alloantigens in PA+ and PA- hosts would be restricted to the hematopoietic system and thus, would have been cleared in the initial alloresponse. The full exhausted phenotype of alloreactive CD8 cells described above was not seen until at least 30 days after transfer to PA+ hosts. However, as early as day 14, CTL primed in PA+ hosts produced less IFN-γ in comparison to those primed in PA-hosts, even though they were still equivalent in terms of their cytotoxicity. Furthermore, when CD8 cells primed in PA+ hosts were transferred to secondary antigen-free hosts, they still displayed reduced ‘fitness’ compared to CTL originally primed in PA- hosts. These data show that peripheral alloantigen qualitatively affects donor CTL function during priming and drives their eventual exhaustion. Additionally they suggest that blockade of co-inhibitory signals may have potential in restoring function to such cells as has been demonstrated in models of chronic infection.

Transfer of small numbers of naïve allogeneic T cells into freshly irradiated (FI) mice leads to severe, lethal GVHD. In contrast, when large numbers of naïve donor T cells are transferred as delayed donor lymphocyte infusions (DLI) given to established mixed hematopoietic chimeras (MC), a GVH reaction limited to the lymphohematopoietic system (LHGVHR) occurs and does not lead to GVHD. A similar phenomenon can occur when DLI are given to patients in whom mixed chimerism is established with non-myeloablative conditioning. LHGVHR without GVHD across MHC barriers provides an approach to separating GVL and GVHD and an understanding of this phenomenon will be essential to optimize its clinical application. We have previously shown that directly GVH-alloreactive CD8 T cells injected into FI mice or in delayed DLI given to MC show rapid proliferation and accumulation in lymphoid organs and similar modification of homing receptors, but with different kinetics in each type of recipient. However, GVH-reactive CD8 T cells traffic to and accumulate in the epithelial GVHD target tissues only in FI mice and not in MC. In order to determine the influence of the host environment on CD4-mediated GVH responses, we compared the expansion and activation of GVH-reactive CD4 T cells specific for recipient minor (mHA) and MHC antigens in the presence of a full haplotype mismatch (B6→B6D2F1) in MC and FI mice. With a non-myeloablative protocol, we established B6→B6D2F1 MC in transgenic B6D2F1 recipients expressing membrane-bound chicken ovalbumin (OVA-B6D2F1). Six weeks later, a group of MC and a group of OVA-B6D2F1 mice lethally irradiated on the same day received 106 OTII CD4 T cells (specific for OVA peptide 323-339/IAb, representing a mHA) along with 107 polyclonal B6 splenocytes to preserve the strong polyclonal response. FI mice also received B6 bone marrow cells (BMC) to prevent death from aplasia. Activation of OTII CD4 T cells was similar in both MC and FI mice, with no statistically significant differences in the increase in CD44 (average median fluorescence intensity [MFI] 245 and 235, respectively), %CD69+ (average 52% in MC and 36% in FI) and %CD25+ cells (average 18% in MC and 20% in FI) and downregulation of CD62L (average 18% positive in MC and 10% in FI). These changes occurred as early as day 3 in FI mice and were delayed to day 6 in MC. On day 6, OTII CD4 T cells accumulated to a 5-fold greater extent in spleens of MC compared to FI mice (mean±SD of 1.47±0.12 × 106in MC vs 0.30±0.03×106in FI; p

In murine models of hematopoeitic cell transplantation (HCT), a graft-versus-leukemia (GVL) effect can be effectively separated from graft-versus-host disease (GVHD) by giving delayed donor-leukocyte infusions (DLI) to established mixed hematopoietic chimeras (MC) (Mapara, Transplantation 2003). In these models, the absence of conditioning-induced inflammation at the time of DLI prevents trafficking to GVHD target tissues, limiting the GVH response to the lymphohematopoeitic system (Chakraverty, J Exp Med 2006). While clinical trials have shown that the same outcome is possible in humans, some patients develop severe GVHD following delayed DLI (Dey, Blood Biol Marrow Transplant 2003). In contrast to mice, who recover T cell numbers quickly after HCT through de novo thymopoiesis, humans remain lymphopenic for many months. The current study investigates the role of recipient lymphopenia in the development of GVHD following delayed DLI to MC. Allogeneic (B10.A) or syngeneic (B6) lymphocytes (30 × 106 whole splenocytes) and bone marrow cells (10 × 106) were administered to unconditioned RAG–1 knockout (KO) B6 recipients or to established MC, and animals were followed for development of GVHD. By day 7 after injection, the RAG KO recipients of allogeneic lymphocytes (allogeneic group) had lost significantly more weight (p

Following an in vivo T cell depleting non-myeloablative conditioning regimen, 5 patients, aged 22–49, received combined kidney and bone marrow transplantation from a haploidentical related donor. Rituximab was included in the conditioning for patients 4 and 5. All patients developed initial mixed chimerism but lost it by day 21; no patient developed GVHD. Four patients discontinued immunosuppression from 240 to 422 days after BMT and have remained off immunosuppression for 9 to 52 months with no evidence of allograft rejection. Flow cytometry was used to assess lymphocyte subsets recovering after transplant. CD3 counts recovered slowly, exceeding 500 cells/μl at days +271, +365, +640 and +450. While memory CD45RO+ cells were most prevalent among CD4+ cells, naïve-type CD4+CD45RA+ cells, presumably arising from the recipient thymus, ranged from 8% to 56% at the time when total CD4 counts recovered to >100 cells/μl (days +165, +21, +352, +240). Notably, a very high proportion of initially recovering T cells were CD3+CD4+ expressing CD25 in all patients as early as day 7 and persisted over 1 year in 2 patients. At approximately day +120 and +365, we further characterized these cells for CD127, FOXP3, CD45RO, CD45RA, HLA-DR and CD62L expression. At Day +120, all 4 patients showed increased frequencies (10.7±4.6%) of CD25+CD127-FOXP3+ regulatory T cells (Treg) within the CD4 population compared to healthy subjects (3.8±0.4%). Expression of CD45RO, CD45RA, CD62L and HLA-DR was variable. By 1 year post-transplant, frequencies of Treg had decreased to levels similar to those in normal subjects. In vitro assays for CD8 and CD4 T cell-mediated alloreactivity (CML/MLR) showed development of long-lasting donor-specific unresponsiveness by 3 months after transplant in Patients 2, 4 and 5, and by 9 months in Patient 1. Responses to 3rd party recovered in all patients after a period of unresponsiveness. In Patient 1, in whom anti-donor CML reactivity declined gradually to become unresponsive by 9 months, depletion of CD4+CD25+ cells revealed a residual anti-donor CML and MLR response at 1year but not at 18 months. In 2 other patients, depletion of CD4+CD25+ cells did not reveal an anti-donor response at time points analyzed from day +122 to 2 years. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, loss of killing activity against donor RTEC was observed post-transplant. The high percentage of Treg recovering early after transplant suggests that they may play a role in initial tolerance induction. This regulatory mechanism may be followed by later deletion of donor-reactive T cells. The variable ability to detect regulation of anti-donor reactivity may reflect the strength of the initial response, as patients with weak pre-transplant anti-donor responses and rapid post-transplant development of donor unresponsiveness did not reveal anti-donor response when Treg were depleted. In addition, infiltration of Treg at the graft site, not revealed by the assays described, might be responsible for tolerance in these patients.

Extrinsic factors within the host environment are crucial in determining recruitment of graft-versus-host (GVH)-reactive T cells to peripheral tissues and the capacity of these cells to induce graft-versus-host disease (GVHD). In this study, we have examined how the host environment influences graft-versus-leukemia (GVL) activity. Transfer of small numbers of allogeneic T cells to freshly irradiated (TBI allo) mice induces both GVL and GVHD, whereas transfer of much higher numbers of T cells to established (>8 weeks) mixed chimeras (MC) can induce GVL without GVHD. Using an EL4 tumor protection assay and low doses of B10.A splenocytes (3 × 10e6, a dose 1 log lower than normally transferred to MC to induce GVL), we observed that tumor-free survival of recipient mice was greater following transfer to TBI allo B6 recipients than in B6 + B10.A → B6 MC. To determine the mechanisms for this disparity, we used a BALB/c recipient/B6 donor strain combination, in which we could track the distribution of donor T cells within secondary lymphoid organs and bone marrow (BM) following transfer to TBI allo or MC recipients. Despite similar expansions within the spleen, there was a significant delay in the accumulation of polyclonal donor T cells (B6 CD45.1) and transgenic 2C GVH-reactive CD8+ T-cells (bearing TCR specific for recipient antigen) in the BM of MC compared to TBI allo mice. Moreover, in vivo cytotoxicity of host B cell targets occurred rapidly and was virtually complete in TBI allo recipients, but was absent in MC even at late time points. To evaluate the acquisition of effector functions in a clonal GVH-reactive CD8+ T cell population, we sorted 2C T cells at intervals following transfer and performed quantitative RT-PCR of molecules linked to effector differentiation. Strikingly, transcription of IFN-γ, granzyme B and TNF-α was significantly higher in cells derived from TBI allo hosts compared to MC. Similar differences in IFN-γ and granzyme B protein expression were confirmed in the polyclonal donor CD8+ T cell population. Since, full GVL activity might also depend upon the survival of GVH-reactive CTL, we also examined the viability of donor T cells during the initial response in both environments. We observed higher rates of sustained 2C CD8+ T cell apoptosis (as indicated by annexin V staining) following T cell transfer to MC. Furthermore, we also detected lower expression of common γ chain cytokine receptors that mediate responsiveness to IL-2, IL-7 and IL-15, upon donor T cells from MC. However, following secondary co-transfer to syngeneic recipients for 21 days, memory phenotype polyclonal donor CD8+ T cells derived from established MC (CD45.1+) were recovered to a greater extent than T cells initially derived from TBI allo mice (Thy1.1+), arguing against any intrinsic defect in the viability of GVH-reactive T cell populations emerging in the former setting. Indeed, when co-cultured in the presence of individual cytokines, MC-derived CD8+ T cells maintained viability to a greater (IL-2, IL-7) or equivalent (IL-15) extent as TBI allo-derived cells. Taken together, these data suggest that disparity in GVL activity following to TBI allo recipients and MC can be explained by differences in the effector function and survival of anti-host CTL in quiescent MC environment. Reduced GVL activity of donor T cells on a per-cell basis in MC can be compensated for by transferring greater numbers of cells.

We developed a haploidentical HCT protocol based on MHC-mismatched murine models, in which mixed chimerism (MC) is induced as a platform for adoptive immunotherapy via delayed donor leukocyte infusions (DLI). While the scientific concept has remained the same, this haplo-HCT protocol has undergone multiple revisions since its inception, such as substitution of MEDI-507 (a CD2 monoclonal antibody) for ATG, addition of ex vivo TCD of donor graft (via CD34+ cell selection), changes in MEDI-507 doses/schedules and finally addition of fludarabine-all based on evolving clinical experience and new discoveries from murine models. A total of 14 patients with advanced, mostly therapy-refractory disease (NHL, n=7; HD, n=3; t-AML, n=2; ALL, n=1; MDS, n=1), received our most recent nonmyeloablative strategy consisting of cyclophosphamide, fludarabine, MEDI-507, thymic irradiation on day -1 and cyclosporine for GVHD prophylaxis (with taper and discontinuation by day 35 for patients with MC and no evidence of GVHD) followed by GCSF-mobilized ex vivo TCD haplo-HCT (HLA 1/6 mismatched, n=7; HLA 2/6 mismatched, n=7). Median number/kg (range) of infused CD34+ and CD3+ cells were 6.84 x106 (3.19 x 106 – 14.7 x 106) and 4.73 x 104 (0.06 x 104 – 11.8 x 104), respectively. One patient died shortly after the transplantation due to intracranial hemorrhage. Of 13 evaluable patients, all initially achieved MC without GVHD, but 3 lost detectable donor graft despite multiple DLIs. Split lineage MC occurred in 10 patients, with a predominance of early donor granulocyte chimerism (mean 91%, 87%, 96%) and a lower percentage of early donor T-cell chimerism (57%, 60%, 97%) at 30, 90 and 150 days post-HCT. Ten out of 10 patients ultimately achieved full donor chimerism (FDC) in myeloid lineage and 9 achieved FDC in T-cell lineage (spontaneously, n=8; following DLI, n=2). Natural killer cells recovered relatively early, despite the presence of circulating MEDI-507. Eight patients developed acute GVHD: grade I, n=3; grade II, n=4; grade IV, n=1. One died from GVHD-related complications, 1 died of late idiopathic pneumonitis and 5 died from progressive disease. Median overall survival (OS) of all 14 patients is 195 days (range, 31–1028) post-HCT; at 1 year the OS is 39.2% for all 14 patients. Six patients are alive at a median of 604 days (range, 180–1028) post-HCT, including 3 in complete remission of whom 2 had therapy-related MDS/AML. HLA mismatches in the GVH or HVG direction did not influence graft outcome. We also assessed inhibitory killer immunoglobulin-like receptor (KIR)-HLA epitope mismatches (missing ligand) in the GVH and HVG (host versus graft) directions based on HLA and KIR genotyping of patients and their donors. There was a trend in the association between spontaneous FDC and fewer missing donor HLA ligands for host KIR receptor (

Graft versus host disease (GvHD) remains a frequent and severe complication of allogeneic stem cell transplantation (SCT) and is mediated by recognition of recipient alloantigens by donor T cells. Numerous interventions have been employed against GvHD which target the number and function of T cells including pharmacological non-specific immunosuppression, depletion of donor T cells or positive selection of donor CD34+ stem cells. While these strategies reduce the risk of GvHD, the benefits are often offset by prolonged immunodeficiency and increased risk of infection and disease relapse. Thus, research efforts have focused on developing a method to selectively deplete alloreactive T cells without compromising other immune responses. One approach to reducing alloreactivity is to remove donor T cells that are activated after ex-vivo co-culture with recipient antigen-presenting cells (APC). Clinical studies have been performed using anti-CD25-conjugated immunotoxin to deplete alloreactive T cells after incubation with haplo-identical PBMC ex-vivo. Although this approach is promising, post-transplant infections, disease relapse and residual GvHD remain a problem. These observations mandate further experimentation to improve our strategies for allodepletion. We undertook studies to identify an inducible surface activation marker expressed selectively on alloreactive effector T cells following culture with HLA-mismatched allostimulators. Costimulatory molecules play a major role in activation of the effector T cells involved in GvHD. In contrast to CD28, which is constitutively expressed, TNF receptor (TNFR) superfamily members are induced and therefore their expression can also be used as an indicator of T cell activation during GvHD, in experimental animal models and allogeneic human transplantation. Here, using peripheral blood mononuclear cells as responders and HLA-mismatched dendritic cells as allostimulators, we observed that compared to other activation markers the TNFR superfamily member, CD134, was superior because of its completely negative base-line expression and rapid upregulation upon activation. Depletion of CD134+ cells from responder populations dramatically reduced specific alloreactivity as determined by reduction of helper T cell precursor (HTLp) frequencies below the threshold predicting development of clinical GvHD and reduction of cytolytic T cell precursor (CTLp) frequencies by 92% against original allostimulators, while retaining responses to third party alloantigens. However, effector CD8+ T cells expressing perforin and granzyme B were preserved in the CD134 allo-depleted populations. Detailed analysis revealed that, by this approach, functional CMV-specific effectors were retained as determined by their identification with CMV-specific pentamers and assessment of their functional responses by CMV-specific ELISpot. Effectors specific for WT1 leukemia antigen were also retained. In addition, Foxp3+ Treg cells were preserved in the allodepleted populations. These observations suggest that CD134-based selective allodepletion may allow allogeneic SCT without requirement for global T cell depletion or non-specific immunosuppression and without loss of pathogen-specific and leukemia-specific immunity.

Mixed hematopoietic chimerism provides a powerful means of inducing robust, donor-specific tolerance. In this article, the minimal requirements for achieving mixed chimerism, the development of new reagents that promote its achievement, and the mechanisms by which peripheral and intrathymic tolerance are achieved via mixed chimerism are discussed. An emerging understanding of these mechanisms, along with the development of new immunosuppressive reagents, is allowing advancement toward clinical application of this approach.

BACKGROUND Surprisingly, some patients who reject donor marrow grafts following nonmyeloablative HCT sustain remissions of advanced hematologic malignancies (Br J Haematol2005, 128:351). In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism and anti-tumor responses against host-type tumors (Blood2003, 102:2300) and that both CD4+ and CD8+ T cells are necessary to achieve maximum anti-tumor effects (J Immunol2005, 175:665). We now examined whether or not NK and/or NKT cells are required in the anti-tumor effect. METHODS Mixed chimerism was achieved in BALB/c (H-2d) mice conditioned with depleting anti-CD4 and CD8 mAbs on Day -5, cyclophosphamide 200 mg/kg on Day -1 and 7 Gy thymic irradiation on Day 0 prior to transplantation of 25x106 B10.BR (H-2k) or B10.RIII (H-2r) bone marrow cells. Some groups received RLI (3x107 BALB/c spleen cells) seven weeks post-BMT. A20 cells (5x105) were given i.v. one week after RLI. Some groups included Jα281-deficient BALB/c (Jα281 KO) mice that lack invariant NKT (iNKT) cells or NK1.1+ BALB/c mice whose NK and NKT cells can be depleted by anti-NK1.1 Ab, PK136. Some groups received a depleting dose of anti-NK1.1 antibody before and after RLI administration. RESULTS Untreated recipients of NK1.1-depleted RLI (n=9) showed similar tumor survival compared to recipients of NK1.1-sufficient RLI (n=9) (median survival time [MST] 52 versus 52 days respectively, p=0.68). NK1.1-depleted (with mAb in vivo) chimeras that received RLI (n=8) had shorter survival (MST 35.5 days) than NK1.1-sufficient chimeras that received RLI (n=10) (MST 68 days, P CONCLUSION When NK and NKT cells were depleted from NK1.1+ BALB/c recipients, no anti-tumor effect was observed. In contrast, no reduction in anti-tumor effect was observed in chimeras given NK1.1-depleted RLI. Invariant NKT cells are required to achieve anti-tumor effects in this model. Therefore, NKT cells and possibly NK cells in the recipient are essential for the anti-tumor effect of RLI, but NK and NKT cells in the RLI do not play a role. The requirement for both T cells and NK/NKT cells suggests an essential interaction between the innate and acquired immune systems.

Background: The induction of stable hematopoietic cell chimerism through bone marrow transplantation (BMT) has been demonstrated to induce donor-specific tolerance in rodent, porcine, nonhuman primate, and human clinical allogenic models, and has also been successful in concordant rodent and nonhuman primate xenogeneic models, as well as in the pig-to-NOD/SCID humanized mouse xenogenic model. However, stable chimerism and tolerance has been difficult to achieve in the discordant pig-to-baboon xenotransplantation model, possibly due in part to the presence in baboons of pre-formed natural xeno-reactive antibodies to a1,3-galactose (Gal) determinants expressed in pigs, but not in Old World primates and humans. The recent availability of miniature swine homozygous for a disruption in the gene encoding a1,3-galactosyltransferase (GalT-KO pigs) has now made it possible to study pig-to-baboon xenografts in the absence of effects of anti-Gal antibodies. We have investigated the GalT-KO pig-to-baboon model further by modifying the conditioning and immunosuppression regimen to facilitate engraftment and tolerance through bone marrow transplantation. Methods: BM was harvested from GalT-KO swine (n=3). Baboons (n=3) were pre-treated with whole body (3 Gy) and thymic (7 Gy) irradiation, Sangstat rabbit anti-thymocyte globulin (ATG), LoCD2b (rat IgG2b anti-primate CD2) and splenectomy, and received FK506 immunosuppressive and supportive therapy for 28 days. The baboons were monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells in the bone marrow by porcine cytochrome b specific PCR of colony-forming units (CFUs), and for cellular reactivity to pig cells by MLR and CML. Antibody formation to LoCD2b and ATG was tested by enzyme-linked immunosorbent assay (ELISA), and antibody reactivity to GalT-KO pig cells was tested by flow cytometry and antibody mediated cytotoxicity assay. Results: A mean of 1.4 × 109 BM cells/kg was infused into each baboon. Although pig cells were undetectable in the peripheral blood of the baboons by flow cytometry, porcine progenitor cell engraftment as well as chimerism in the bone marrow and thymus was detected by PCR in the first baboon on day 28. ELISA results indicated the presence of antibodies to rat (LoCD2b) and rabbit (ATG) immunoglobulin within two weeks; however, no antibodies to pig cells could be detected by flow cytometry or cytotoxicity assay. The second baboon had undetectable serum antibodies to pig cells for 60 days despite the presence of induced antibodies to rat LoCD2b and rabbit ATG. Porcine progenitor cell engraftment was confirmed by PCR of CFUs on day 60 and MLR showed no response to pig although the animal regained alloresponses by this time. The third baboon, in contrast, had detectable induced serum antibodies to pig cells as well as rat and rabbit immunoglobulin by day 14 following BMT. Conclusions: Engraftment has been achieved following GalT-KO pig-to-baboon BMT with evidence of specific humoral and cellular non-responsiveness to pig cells (2/3 baboons), suggesting the possibility that this protocol may facilitate xenograft tolerance.

ES may accompany hematologic recovery following SCT and is characterized by fever and rash, in addition to one of the following clinical syndromes: noncardiogenic pulmonary edema, hepatic dysfunction, renal dysfunction, weight gain, or transient encephalopathy. A retrospective analysis was performed to evaluate the incidence of ES using a published clinical definition (Spitzer TR, Bone Marrow Transplant2001; 27:893–898), the use of corticosteroids in the peri-engraftment period, the relationship of ES to the development of GVHD, and the fate of the patient’s chimerism. Seventy-three patients with a HM (NHL, n=45; HD, n=9; AML, n=6; ALL, n=1; CLL, n=6; MDS, n=1; MM, n=4) who were treated with a nonmyeloablative conditioning regimen consisting of cyclophosphamide, antithymocyte globulin, thymic irradiation, cyclosporine, and hematopoietic SCT (bone marrow, n =57; peripheral blood stem cells, n =15) were analyzed. Chimerism was assessed weekly for the first 100 days, then q6 months by VNTR/STR analysis and/or flow cytometry. In the absence of full donor chimerism (FDC) or evidence of acute GVHD, donor leukocyte infusions (DLI) were given beginning 5 weeks post-transplant to convert mixed chimerism (MC) to FDC and thus maximize a graft-versus-tumor effect. Thirty-five (50%) patients met the clinical definition of ES, presenting with fever and rash (100% of ES patients), hepatic dysfunction (74.3%), fluid retention/weight gain (60%), noncardiogenic pulmonary edema (22.9%), renal dysfunction (22.9%), and transient encephalopathy (5.7%). Three (4.1%) patients never engrafted and thus were censored from the population. Median time to engraftment was 13 (range 9 to 18) days in ES patients and 14 (range 9 to 21) days in non-ES patients. The incidence of significant (≥ grade II) aGVHD was 45.7% in ES patients versus 22.9% in non-ES patients. The incidence of chronic GVHD was similar in both groups (63.6% in ES patients vs. 68.0% in non-ES patients). Loss of donor chimerism (LDC) was seen at a lower rate in ES patients (15.2%) vs. non-ES patients (36.4%). Conversely, full donor chimerism (FDC) was higher in ES patients (48.5%) versus non-ES patients (36.4%). Corticosteroids were administered based on clinical suspicion of ES. Thirty two (91.4%) of ES patients and nineteen (54.3%) of non-ES patients received steroids. Resolution of clinical abnormalities occurred in twenty seven (84.4%) of ES patients and eighteen (94.7%) of patients who did not fulfill the criteria for ES. In conclusion, patients with ES had a higher propensity to develop clinically significant acute GVHD, suggesting that ES may represent early GVHD. However, those who developed LDC and ES had indistinguishable clinical presentations of their ES. Thus, ES represents heterogeneous populations of patients with diverse immunologic alloreactivity (either GVH or HVG reactivity). The high incidence of ES (and of both GVHD and LDC) in this population of patients suggests that competing GVH and HVG alloreactivity following nonmyeloablative conditioning (and in all likelihood the occurrence of a “cytokine storm”) might be responsible for the clinical manifestations of this syndrome.

BACKGROUND Surprisingly, some patients who reject donor marrow grafts following nonmyeloablative HCT sustain remissions of advanced hematological malignancies (Br J Haematol2005, 128:351). In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced loss of donor chimerism and anti-tumor responses against host-type tumors (Blood2003, 102:2300) and that T cells in the RLI are necessary to achieve maximum anti-tumor effects (J Immunol2005, 175:665). We now examined whether or not this anti-tumor response demonstrates long-term memory. We attempted to enhance anti-tumor effects by adding exogenous IL-15, which promotes CD8 memory T cell survival. METHODS Mixed chimerism was achieved in BALB/c (H-2d) mice conditioned with depleting anti-CD4 and CD8 mAbs on Day -5, cyclophosphamide 200 mg/kg on Day -1 and 7 Gy thymic irradiation on Day 0 prior to transplantation of 25x106 B10.BR (H-2k) or B10.RIII (H-2r) bone marrow cells. Some groups received RLI (3x107 BALB/c spleen cells) seven weeks post-BMT. A20 cells (5x105) were given i.v. one week after RLI. Some groups received a secondary A20 challenge (105) 100 days after the first challenge. Some groups received 5 μg of IL-15 intraperitoneally, starting one week after RLI injection and every 12 h thereafter for a total of 10 doses. Spleen cells were isolated and anti-tumor cytotoxicity was evaluated after 5 days’ coculture with irradiated A20 cells. RESULTS We rechallenged RLI-treated long-term tumor survivors with a second lethal dose of A20 tumor cells 100 days after the first tumor challenge. One third of long-term survivors (n=20) rejected the lethal dose of A20 (median survival time [MST] 72 days). In contrast, all mice in the control group that did not previously receive RLI and received their first tumor challenge concurrently with the rechallenge group died, with MST 56 days (n=23) (p=0.02). Thus, 1/3 of long-term tumor survivors retained sufficiently strong anti-tumor immunity to reject secondary tumor. We have previously demonstrated in vitro tumor-specific cytotoxic responses 11 weeks after first tumor challenge. We now examined anti-tumor cytotoxicity at a later time point and observed such responses even 11 months after first tumor challenge in some of the tumor survivors. From these results, we speculated that memory CTL may play an important role in the anti-tumor effect of RLI. We hypothesized that exogenous IL-15 administration might enhance the anti-tumor effect of RLI, since it promotes generation and maintenance of memory T cells. Indeed, we observed increased tumor-free survival in mice that received RLI and IL-15 (n=14) compared to recipients of RLI alone (n=14) (MST 64 versus 48 days respectively, p=0.03). CONCLUSION Together, these data suggest that RLI therapy can evoke long-term anti-tumor immunity and that the anti-tumor response may be enhanced by administration of exogenous IL-15, reinforcing the potential of RLI therapy to provide a new HCT strategy lacking the risk of graft-versus-host disease.

Mixed chimerism (MC) can be induced in mice after non-myeloablative conditioning and allogeneic HSCT. Full donor hematopoiesis conversion from MC can occur when nontolerant donor lymphocyte infusions (DLI) are given weeks following the transplant as a result of lymphohematopoietic GVH reactions that eradicate leukemia/lymphoma without GVHD. Based on this murine model, we initiated a trial in patients with advanced hematologic malignancies of non-myeloablative therapy with HLA-matched related donor HSCT with intentional induction of MC followed by prophylactic DLI (pDLI) at day +35 post-HSCT in those without clinical GVHD. This analysis examines the influence of the kinetics of sustained donor lymphoid (CD3+ cells) chimerism on transplant outcomes. Fifty-four patients with advanced hematologic malignancies received nonmyeloablative preparative therapy (cyclophosphamide, equine antithymocyte globulin, thymic irradiation and a brief course of cyclosporine) and HLA-matched HSCT (bone marrow, BM, n=42; GCSF-mobilized peripheral blood stem cells, PBSC, n=12). Chimerism analyses were performed weekly until day +100 post-HSCT and then once monthly for a year. Twenty-nine patients with aggressive malignant lymphomas (NHL, n=21; HD, n=8) were selected out of 50 evaluable patients in an effort to analyze a relatively uniform group in terms of their disease and sustained chimerism; patients who lost the allograft were removed from this analysis. Twenty five patients attained stable FDC, defined as > or = 90% donor T cells, and 4 maintained stable MC. Using 42 days post-HSCT as the defining point for chimerism conversion 13 patients achieved early FDC and 16 patients achieved late FDC. Median time to achieve FDC in the early and late groups was 14 days (range, 7–42) and 70 days (range, 49–91) respectively. Separation produced fairly equal distribution by disease characteristics as follows (early to late): NHL, 10 to 11 and HD, 3 to 5. The early group included 10 PBSC recipients who did not receive pDLI because of the spontaneous conversion to FDC; in contrast, the majority of the late group received BMT and 13 of them received DLI (pDLI, n=10, therapeutic DLI, n=3). The incidence of acute GVHD (grades II–IV) was similar between the early and late groups with rates of 77% (n=10 of 13) and 69% (n=11 of 16) respectively; for chronic GVHD, there was a tendency toward an increased rate in the early group of 75% (3/4) compared to the late group rate of 63% (5/8). Overall disease response rates in the early and late groups were 62% and 69%, but there was a trend toward improved complete remission rates in the late group relative to the early group (50% vs 31%). Progression-free survival (PFS) and overall survival (OS) at 1 year were improved in the late compared to the early group [31% (5/16) vs 15% (2/13) and 50% (8/16) vs 38% (5/13), respectively]. At 5 years, there was a statistically significant improved PFS and OS rate in the late group over the early group [31% (5/16) vs 0% (0/13), p =0.037, for both PFS and OS]. In conclusion, these data support the observation from our mouse models that the most potent graft-vs-tumor effect occurs in mixed chimeras with late chimerism conversion, often after delayed pDLI.

Mixed chimerism (MC) can be induced in mice across MHC barriers using nonmyeloablative conditioning and T cell depletion. Delayed donor lymphocyte infusions (DLI) convert this MC state to full donor hematopoiesis and impart a powerful graft versus tumor effect without inducing GVHD. Based on this principle, we developed a clinical trial with a goal of inducing a GVH-free mixed chimeric platform for delayed cellular immunotherapy via DLI following nonmyeloablative haploidentical HSCT. This study analyzed graft outcomes in 13 patients who received in vivo and ex vivo TCD, haploidentical (HLA 1–3 Ag mismatched) HSCT for refractory hematologic malignancies (NHL, n=7; CLL, n=1; HD, n=3; CML, n=1; MDS, n=1). Conditioning consisted of MEDI-507 (siplizumab, an anti-CD2 mAb) for in vivo T cell depletion of both the host and donor, cyclophosphamide, fludarabine, thymic irradiation, and a brief course of cyclosporine. An earlier cohort of 4 patients did not receive fludarabine. All patients received ex vivo TCD of G-CSF-mobilized peripheral blood stem cells using a CD34+ cell-selection device. A median of 7.38 x 106 (3.19 x 106 to 1.49 x 107) CD 34+ cells/kg and a median of 5.24 x 104 (6.31 x 102 to 1.61 x 105) CD3+ cells/kg were given. DLI was given as early as 5 weeks in patients without evidence of GVHD. Inhibitory killer immunoglobulin-like receptor (KIR)-HLA epitope mismatches (missing ligand) in the GVH and HVG (host versus graft) directions were assessed based on HLA and KIR genotyping of patients and their donors. Natural killer (NK) cells recovered relatively early, despite the presence of circulating MEDI-507. Split lineage MC was observed in each case, with a predominance of early donor myeloid chimerism and a lower percentage of donor T cell chimerism. Four of the 13 patients eventually lost their grafts despite DLI, including 2 who were not conditioned with fludarabine. Five patients spontaneously achieved full donor chimerism (FDC) and 4 converted to FDC following DLI. The spontaneous chimerism group had significantly fewer HLA mismatches in the HVG direction. Four out of the 5 had only one mismatch, whereas the patients who had graft loss or required DLI had at least two mismatches (p=0.007 by Fisher’s exact test). This finding remained significant when the subgroup of patients not conditioned with fludarabine was excluded from the analysis (p=0.048). Spontaneous FDC was also associated with the presence of graft HLA-C group 1 (HLA-CAsn80) ligand for host NK cell inhibitory receptor KIR2DL2/3 (KIR-L match/compatible, 4 out of 4 evaluable patients) while this graft HLA ligand tended to be absent in the group that lost the graft or required DLI to achieve chimerism (KIR-L mismatch, 5 out of 8 patients) (p=0.08 by Fisher’s exact test). There was no significant relationship between donor KIR and host HLA types in the GVH direction for predicting engraftment. While these observations are preliminary given the heterogeneity of the conditioning regimens and the small number of patients, the data suggest that the lesser degree of HLA mismatching and the presence of KIR ligand compatibility in the HVG direction may influence donor engraftment following nonmyeloablative TCD haploidentical HSCT and warrant further study.

Purpose: To evaluate T cell recovery and donor chimerism following haploidentical hematopoietic cell transplantation (HCT) with a non-myeloablative conditioning approach that includes T cell depletion of host and donor and delayed DLI. Methods: Eighteen patients, 3 cohorts of 4 patients each and 1 cohort of 6 evaluable patients/10 transplanted, with chemorefractory hematologic malignancies, received related HLA 1–3 of 6, A, B, or DR antigen mismatched donor HCT after non-myeloablative conditioning with Medi-507 (anti-CD2 humanized mAb; Biotransplant, Inc.), cyclophosphamide, thymic irradiation and peritransplant cyclosporine. The patients in Protocols A received a MEDI-507 test dose of 0.1 mg/kg on Day -2 followed by 0.6 mg/kg on Days −1, 0 and +1 and transplantation of unmanipulated bone marrow. In Protocol B, the timing and dose of Medi-507 was modified. The patients in Protocol C and D received the latter Medi-507 protocol, but were given Isolex ®-selected CD34+ cells from G-CSF mobilized PBSC. Protocol D differs from Protocol C with the addition of fludarabine to more reliably achieve sustained chimerism. Donor leukocyte infusions were administered in an effort to convert mixed to full donor chimerism and to achieve a graft-versus-tumor effect. Chimerism was measured by peripheral blood microsatellite markers or by flow cytometry using HLA-specific mAbs. T cell recovery and phenotype were followed by flow cytometry. Because a high percentage of CD4 T cells post- transplant were CD25high, we performed quantitative RTPCR for Foxp3 and CTLA-4 on sorted PBMC populations. Results: T cell depletion early post-HCT was detected in all patients. There was a marked difference in the percentage of graft acceptance/loss, GVHD prevalence, and T cell phenotype related to each protocol modification. The majority (>90%) of CD4 T cells appearing in the first 100 days post-SCT were CD45RO+/CD45RA- “memory” cells and CD8 T cells were CD45RO+CD45RA-/CD62L-. In addition, a remarkably high percentage (19.5–75%, mean 38.1%) of CD4 T cells expressed high levels of CD25 in recipients of Protocols A, B, and C early post-HCT. CD25 expression decreased as T cell counts increased. By quantitative RTPCR, we found that sorted CD25highCD4 T cells expressed Foxp3 and CTLA-4, consistent with a regulatory phenotype. The addition of fludarabine in Protocol D resulted in an overall reduction in the percentage of peripheral CD4CD25high T cells compared to Protocol C at 4 weeks post-HCT (C 23.43% +/−4.7% versus D 2.1% +/− 0.9%, p Conclusions: These data suggest that the addition of fludarabine may increase the incidence of sustained and full donor chimerism. Fludarabine efficiently depleted host CD4+CD25high Foxp3+ cells, which may have a regulatory role, preventing spontaneous chimerism conversion and associated GVHD.

Donor leucocyte infusions (DLI) given to established mixed chimeras (MC) can eliminate normal and malignant hematopoietic cells without causing graft-versus-host disease (GVHD). Identical DLI transferred immediately following lethal irradiation lead to severe GVHD. In contrast to freshly irradiated mice developing GVHD, established MC receiving DLI show delayed kinetics of effector/memory T cell (TE/M) expansion and a distinct distribution with no accumulation of cells in the gut. Lethal irradiation is associated with tissue injury, altered chemokine expression and cytokine dysregulation, and resolution of these changes is important in limiting GVHD following delayed DLI. In an attempt to define the respective roles of ‘intrinsic’ T cell versus ‘extrinsic’ environmental factors in shaping the character of the GVH response elicited, TE/M developing following GVHD induction were transferred to established MC. Conversely, TE/M developing following delayed DLI to MC were transferred to lethally-irradiated 2° recipients. In the first series of experiments, we transferred 1 x 107 C57BL/6 (B6) splenocytes and 5 x 106 B6 T-cell depleted bone marrow (TCD BM) to lethally-irradiated BALB/c recipients. On day 4 following BMT, when GVH-reactive T cell activation and expansion was maximal, 3 x 106 nylon wool-passaged TE/M derived from recipient spleens (equivalent to 1 recipient spleen to 1 secondary recipient) were transferred to established B6 → BALB/c MC or to lethally irradiated BALB/c 2° recipients. The latter mice received B6 TCD BM to prevent death from aplasia. Secondary established MC recipients were resistant to GVHD whereas the lethally-irradiated 2° recipients developed severe, lethal GVHD (median survival time-MST- mixed chimeras >100d versus irradiated mice 52d, p10% weight and developed clinical signs of mild GVHD, but then showed almost complete recovery by day 60 post-BMT. In sharp contrast, after a similar lag period, 2° recipients of CD45.1+-selected lymphocytes developed severe lethal GVHD (MST CD45.1+ cells 55d vs >100d non-fractionated cells, p

BACKGROUND Surprisingly, anti-tumor responses can occur in patients who reject donor grafts following nonmyeloablative hematopoietic cell transplantation (Dey et al., Biol Blood Marrow Transplant 7:604). In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced anti-tumor responses against host-type tumors (Rubio et al. Blood 102:2300). To further investigate the clinical relevance of this RLI model, we: Evaluated the effect of RLI from tumor-bearing mice Compared RLI with allogeneic lymphocyte infusion in untreated mice METHODS Mixed chimerism was achieved in BALB/c (H-2d) mice conditioned with depleting anti-CD4 and CD8 mAbs on Day-5, cyclophosphamide 200 mg/kg on Day -1 and 7 Gy thymic irradiation on Day 0 prior to transplantation of 25x106 B10.BR (H-2k) bone marrow cells. Some groups received RLI (3x107 BALB/c spleen cells) seven weeks post-BMT. Some RLI donor mice received BALB/c A20 B cell lymphoma cells (1x105) two weeks before RLI. Some groups received RLI depleted of B cells using B220 negative selection by MACS column. A20 cells (5x105) were given i.v. one week after RLI in chimeras or after allogeneic lymphocyte infusion (3x107 B10.BR spleen cells) to untreated BALB/c mice. RESULTS In the clinical setting, RLI would be obtained from tumor-bearing hosts. We therefore examined whether RLI is still effective when the lymphocytes are obtained from the tumor-bearing mice. Contamination of B220+ cells in the RLI was less than 0.03%. RLI recipients from the tumor-bearing mice (n=10) showed similar tumor survival compared to recipients of RLI from naïve donors (n=10) (median survival time [MST] 53 versus 50 days respectively, p=0.48) (Figure 1). Thus, with a purging procedure, the same anti-tumor effect was achieved with RLI from tumor-bearing hosts as from non-tumor-bearing hosts. Allogeneic lymphocyte injection is a potentially feasible anti-tumor therapy. We therefore compared anti-tumor effects of allogeneic lymphocyte infusion into naïve mice with that of RLI given to mixed chimeras. Chimeric RLI recipients mice (n=13) had longer survival (MST 61 days) than naïve mice receiving allogeneic lymphocytes (n=10) (MST 39.5 days, P CONCLUSION Together, these data reinforce the potential of RLI therapy to be a new HSCT strategy which does not have the risk of graft-versus-host disease.

One potential approach for the effective disengagement of graft-versus-leukemia (GVL) effects from graft-versus-host disease (GVHD) following BMT is the use of nonmyeloablative conditioning as a platform for the adoptive transfer of donor T cells. In pre-clinical models, donor CD8+ T cells can induce powerful responses against tumors of host origin, but the effect lacks durability such that a re-challenge with tumor inevitability leads to tumor progression and death. This deficit is associated with the failure of functional CD8+ effector/memory T cells (TE/M) to survive long-term post-DLI. To examine the fate of GVH-reactive CD8+ T cells following DLI, we established mixed hematopoietic chimeras (MC) in a parent →F1 model using a nonmyeloablative protocol that incorporates co-stimulatory molecule blockade. B6D2F1 mice received 3Gy TBI and intra-peritoneal injections of anti-CD154 and anti-CD8 mAb on day 0 followed by infusion of 2 x 107 C57BL/6 bone marrow cells. 10 weeks later, when mAb had cleared from the circulation, MC received DLI that included CD8+ T cells from 2C transgenic mice that bear TCR specific for recipient class I MHC Ld. Using a clonotypic marker to monitor the response, we observed expansion of 2C CD8+ cells, peaking in the spleen on day 7 and then rapidly declining such that 2C CD8+ T cells were

Donor leucocyte infusions (DLI) given to established mixed chimeras (MC) can eliminate normal and malignant hematopoietic cells without causing graft-versus-host disease (GVHD). DLI given immediately following lethal irradiation lead to severe GVHD. We examined the proliferation, expansion, differentiation and survival of GVH-reactive T cells following delayed DLI and compared the outcomes to those observed when identical DLI were administered early following lethal irradiation (TBI). MC recipients were prepared by TBI of BALB/c mice and reconstitution with mixed BALB/c and CD45.2 C57BL/6 (B6) T-cell depleted bone marrow (TCD BM). 10 weeks later, we transferred 1 x 107 CD45.1 B6 and 5 x 106 2C transgenic (tg) splenocytes. CD8+ T cells from 2C tg mice bear TCR specific for recipient class I MHC Ld. Polyclonal DLI-derived CD4+/CD8+ T cell responses were monitored by gating on CD45.1+ events and clonal 2C CD8+ T cell responses tracked using a clonotypic marker. For comparison, identical DLI together with TCD BM was administered to freshly irradiated BALB/c or B6 CD45.2 syngeneic recipients. MC recipients of delayed DLI developed a GVH reaction, as indicated by increases in donor chimerism, but no GVHD. In contrast, allogeneic recipients receiving DLI immediately following TBI developed lethal GVHD (median survival 32d vs. >100d post-delayed DLI, p50% by day +3) in freshly irradiated DLI recipients; 2) 2C CD8+ T cells showed greater reductions in expression of IL-7Rαfollowing delayed DLI; and 3) high rates of 2C CD8+ T cell apoptosis, as indicated by annexin V staining, were observed following delayed DLI with absolute numbers up to 6-fold greater than in TBI recipients. Thus, in contrast to responses in freshly irradiated mice that develop GVHD, GVH reactions induced by delayed DLI are characterized by delayed kinetics, a distinct distribution, marked apoptosis and reduced expression of cytokine receptors important for CD8+ T-cell survival.

Donor lymphocyte infusion (DLI) may lead to lymphohematopoietic graft-versus-host (GVH) reactions without graft-versus-host-disease (GVHD). The graft-versus-leukemia (GVL) effect of DLI is greater in mixed chimeras than in full chimeras following fully MHC-mismatched bone marrow transplantation (BMT). Some studies showed, following MHC-mismatched BMT, that only CD8+ T cells are involved in GVL effects of DLI against MHC class I+ class II− tumors, but others showed that the GVL effect of DLI is dependent on both CD8+ and CD4+ T cells. Previously, we have observed that GVL against EL4 is mediated by CD8+ T cells but not CD4+ T cells following DLI in freshly irradiated mice treated with cytokines to reduce GVHD mortality in fully MHC-mismatched BMT. The exact contribution of CD4+ and CD8+ T cells to the GVL effects of DLI in established mixed chimeras is not known. We hypothesized that lethal conditioning leads to the production of many proinflammatory cytokines which may help to generate a CD4 cell-independent, CD8 cell-mediated anti-tumor effect. However, in delayed DLI recipients, CD4+ helper T cells might be needed to induce CD8+ anti-tumor effector cells. We evaluated the role of T-cell subsets in GVL effects of DLI following fully major histocompatibility complex (MHC)-mismatched BMT in freshly irradiated mice and mixed chimeric delayed DLI recipients. While GVL effects of delayed DLI given to established mixed chimeras were dependent on both CD4+ and CD8+ T cells, CD4+ T cells did not contribute to GVL effects in freshly irradiated mice. Consistent with this, MHC class II expression on host antigen-presenting cells (APCs) was needed to optimize GVL effects of DLI in established mixed chimeras. To address whether cross-presentation of tumor antigens (Ag) via class I would permit GVL responses in full chimeras, we generated full chimeras in which donor and recipient shared a class I locus. However, there were no GVL effects of delayed DLI in full chimeras, providing no evidence that cross-presentation of tumor Ag through class I molecules shared by donor and the tumor led to anti-tumor effects in this model. We conclude that following delayed DLI to established mixed chimeras, the GVL response requires extensive MHC disparity, MHC class II expression on host APCs and the presence of CD4+ T cells. In contrast, the requirement for CD4+ T cells can be bypassed in freshly irradiated recipients, but this occurs at the expense of a greater risk of GVHD. Administration of delayed DLI to mixed chimeras permits achievement of optimal GVL effects because large numbers of GVH-reactive T cells can be administered without inducing GVHD.