Graft-versus-leukemia Responses Research Articles

Targeting PKCtheta Inhibits Alloreactivity and Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia and Anti-Infection Activities.

Abstract 2458Poster Board II-435 Introduction:When used as therapy for hematopoietic malignances, allogeneic hematopoietic stem cell transplantation (HCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host-disease (GVHD) is a potentially lethal complication of allogeneic BMT. Thus, inhibition of GVHD, while preserving GVL and protective responses against infectious agents, can enhance the therapeutic potential of BMT. GVHD is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues. Protein kinase C theta isoform (PKCθ) is crucial in TCR signaling and regulates the nature of lymphocyte-specific effector responses. The aim of this study was to investigate the significance of PKCθ in donor T-cell-mediated GVHD, anti-leukemia, and antiviral infection. Methods:Our results were validated using clinically relevant murine bone marrow transplantation (BMT) models. GVHD was measured by recipient survival and clinical signs such as body weight loss. Leukemia/lymphoma was induced by intravenous injection of a luciferase expressing A20 clone (a B lymphoma cell line derived from BALB/c mice). Tumor invasion was quantitated using the Xenogen IVIS-200 bioluminescence imaging system. Murine cytomegalovirus (MCMV) infection was induced by intraperitoneally injecting MCMV in the recipients after GVHD was established. Results:Using 3 distinct mouse models of allogeneic BMT, we found that T cells lacking PKCθ are unable to undergo robust expansion and cause damage to recipient hematopoietic or epithelial tissues, demonstrating that an essential requirement for PKCθ in alloreactivity and GVHD development. In contrast, PKCθ-/- T cells retain the ability to respond to virus infection post-BMT. Mechanistically, absence of PKCθ raises the threshold for T cell activation that selectively impacts alloresponses, but T cell responses triggered by infection or following administration of antigen plus microbial adjuvant are relatively well preserved in the absence of PKCθ Furthermore, we evaluated the role of PKCθ in GVHD and GVL responses, and found that PKCθ-/- T cells have at least a 10-fold reduction in the ability to induce GVHD but only a ∼2.5-fold reduction in GVL compared to WT T cells. Thus, absence of PKCθ impacts GVHD responses more significantly than GVL responses. Conclusion:These findings validate PKCθ as a potentially unique therapeutic target that is required for GVHD induction but not for GVL or protective responses to infectious agents in allogeneic BMT. Disclosures:No relevant conflicts of interest to declare.

Blockade of IL-23 Signaling Results in Targeted Protection of the Colon and Allows for Separation of Graft Versus Host and Graft Versus Leukemia Effects.

Abstract 231Separation of graft versus leukemia (GVL) and graft versus host (GVH) reactivity has been a longstanding but elusive goal in allogeneic bone marrow transplantation (BMT). The difficulty in dissociating these two responses stems in part from the fact that alloreactive donor T cells are equally capable of trafficking to sites of disease as well as GVHD target organs such as the colon, liver and skin. Recent studies from our laboratory have shown that donor APC-derived IL-23 secretion has a critical and selective role in mediating pathological damage in the colon during GVHD (Das et al, Blood, 2009). These results demonstrate the feasibility of regional GVHD protection and raise the question as to whether such localized protection might be a strategy to separate GVH and GVL responses. To directly address this question, we performed studies using a Balb/c-derived luciferase-transfected A20 leukemia cell line (A20-luc) in GVL experiments. Lethally irradiated Balb/c mice were administered 1 × 105 A20 cells and then transplanted with C57BL/6 (B6) BM alone or BM and spleen cells from B6 or IL-23−/− donors. Transplantation with BM and spleen cells from IL-23−/− donor mice resulted in prolonged survival when compared to mice reconstituted with similarly composed marrow grafts from B6 animals (63% versus 26% at day 70, p=0.03). A five-fold escalation of the A20 cell dose produced similar protective results in recipients of IL-23−/− marrow grafts indicating that the effects were not leukemia dose dependent. Studies were then conducted using an IL-23 specific (p19) antibody to block IL-23 signaling as a more clinically relevant strategy. To confirm that antibody blockade would also attenuate GVHD, lethally irradiated Balb/c mice were transplanted with B6 BM and spleen cells to induce GVHD. Mice that were administered p19 antibody (once/week for four weeks) had selective protection from colonic GVHD and a significant reduction in colonic proinflammatory cytokine production when compared to isotype control-treated mice, confirming results observed with use of transgenic IL-23−/− donors. Administration of p19 antibody on a similar schedule to A20-luc-bearing animals resulted in increased survival relative to isotype control antibody-treated mice demonstrating that antibody blockade also resulted in preservation of the GVL response. Serial bioluminescence imaging (BLI) studies and pathological examination confirmed that animals treated with p19 antibody had no evidence of leukemia recurrence. Since the colon is preferentially protected after blockade of IL-23 signaling, we considered that this site might serve as a sanctuary site for residual leukemia cells. BLI studies revealed that A20-luc cells migrated to the liver, lung and nodal sites but were not detectable in the colon. For that reason, we examined this question using a novel, clinically relevant murine model of chronic myelogenous leukemia (CML). In this model, FVB animals that have the bcr/abl oncogene under the control of a tetracycline-inducible repressor are used as donor animals (i.e. CML mice). Lethally irradiated normal FVB mice were transplanted with equivalent numbers of T cell depleted BM from B6 and FVB CML animals. Withdrawal of tetracycline from the drinking water induces expression of bcr/abl oncogene, the development of neutrophilia, and leukemic infiltration in the colon, liver and spleen. Real time PCR studies also demonstrated a 600-fold increase in bcr/abl mRNA levels in the colon of these mice, relative to controls, indicative of CML in this organ. To determine whether the colon would serve as a sanctuary site and limit the effectiveness of the GVL response, lethally irradiated FVB mice were transplanted with the same BM inoculum plus 3.5 × 106 T cells from wild type or IL-23−/− donors. Whereas mice that received adjunctive B6 T cells succumbed from GVHD, animals transplanted with T cells from IL-23−/− mice had significantly prolonged survival and no evidence of leukemia by either blood counts or pathological examination. Real time PCR studies also demonstrated absence of the bcr/abl oncogene in the colon of mice indicating that this organ did not serve as a reservoir for subsequent leukemic relapse. In conclusion, these studies demonstrate that targeting of IL-23 is able to reduce GVHD without loss of the GVL effect and suggest that this approach may be a viable clinical strategy for the dissociation of GVH and GVL responses. Disclosures:No relevant conflicts of interest to declare.

Human T Cells with Distinct Specificities Mediate Graft-Versus-Leukemia Reactivity and Xenogeneic Graft-Versus-Host Disease in a NOD/Scid Mouse Model for Human Acute Leukemia.

Abstract 1330Poster Board I-352Immunodeficient mice inoculated with human leukemia can be used as a model to investigate Graft-versus-Leukemia (GvL) effects of donor lymphocyte infusions (DLIs). In addition to GvL reactivity, treatment with DLI induces xenogeneic Graft-versus-Host Disease (GvHD) in mice, characterized by pancytopenia and weight loss. In patients treated with DLI for relapsed or residual leukemia after allogeneic stem cell transplantation, immune responses against non-leukemic cells may also cause GvHD. It has been suggested that GvL reactivity and GvHD, which co-develop in vivo, can be separated and that distinct T cells exist with the specific capacity to mediate GvL reactivity or GvHD. Since adoptive T cell transfer models that allow analysis of separation of GvL and GvHD are rare, we aimed to establish whether GvL reactivity and xenogeneic GvHD could be separated using our model of human leukemia-engrafted NOD/scid mouse after treatment with human donor T cells. In this study, non-conditioned NOD/scid mice engrafted with primary human acute lymphoblastic leukemic cells were treated with CD3+ DLI. Established tumors were effectively eliminated by emerging human T cells, but also induced xenogeneic GvHD. Flowcytometric analysis demonstrated that the majority of emerging CD8+ and CD4+ T cells were activated (HLA-DR+) and expressed an effector memory phenotype (CD45RA-CD45RO+CCR7-). To investigate whether GvL reactivity and xenogeneic GvHD were mediated by the same T cells showing reactivity against both human leukemic and murine cells, or displaying distinct reactivity against human leukemic and murine cells, we clonally isolated and characterized the T cells during the GvL response and xenogeneic GvHD. T cell clones were analyzed for reactivity against primary human leukemic cells and primary NOD/scid hematopoietic (BM and spleen cells) and non-hematopoietic (skin fibroblasts) cells in IFN-g ELISA. Isolated CD8+ and CD4+ T cell clones were shown to recognize either human leukemic or murine cells, indicating that GvL response and xenogeneic GvHD were mediated by different human T cells. Flowcytometric analysis demonstrated that all BM and spleen cells expressed MHC class I, whereas only 1-3 % of the cells were MHC class II +. Primary skin fibroblasts displayed low MHC class I and completely lacked MHC class II expression. Xeno-reactive CD8+ T cell clones were shown to recognize all MHC class I + target cells and xeno-reactive CD4+ T cells clones displayed reactivity only against MHC class II + target cells. To determine the MHC restriction of xeno-reactive T cell clones, NOD/scid bone marrow (BM) derived dendritic cells (DC) expressing high levels of murine MHC class I and class II were tested for T cell recognition in the presence or absence of murine MHC class I and class II monoclonal antibodies in IFN-g ELISA. Xeno-reactive CD8+ T cell clones were shown to be MHC class I (H-2Kd or H-2Db) restricted, whereas xeno-reactive CD4+ T cell clones were MHC class II (I-Ag7) restricted, indicating that xeno-reactivity reflects genuine human T cell response directed against allo-antigens present on murine cells. Despite production of high levels of IFN-gamma, xeno-reactive CD8+ and CD4+ T cell clones failed to exert cytolytic activity against murine DC, as determined in a 51Cr-release cytotoxicity assay. Absence of cytolysis by CD8+ T cell clones, which are generally considered as potent effector cells, may be explained by low avidity interaction between human T cells and murine DC, since flowcytometric analysis revealed sub-optimal activation of T cells as measured by CD137 expression and T cell receptor downregulation upon co-culture with murine DC, and therefore these results indicate that xenogeneic GvHD in this model is likely to be mediated by cytokines. In conclusion, in leukemia-engrafted NOD/scid mice treated with CD3+ DLI, we show that GvL reactivity and xenogeneic GvHD are mediated by separate human T cells with distinct specificities. All xeno-reactive T cell clones showed genuine recognition of MHC class I or class II associated allo-antigens on murine cells similar as GvHD-inducing human T cells. These data suggest that our NOD/scid mouse model of human acute leukemia may be valuable for studying the effectiveness and specificity of selectively enriched or depleted T cells for adoptive immunotherapy. Disclosures:No relevant conflicts of interest to declare.

Host CD11c+ Dendritic Cells Are Required for Priming the Lympho-Haematopoietic Graft-Versus-Host Response but Not Graft-Versus-Host Disease.

Abstract 2450Poster Board II-427Following allogeneic bone marrow transplantation (BMT), delayed donor leukocyte infusion (DLI) at a point when conditioning-induced inflammation has resolved, is employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease (GVHD). We have previously shown that host bone marrow (BM)-derived antigen presenting cells (APC) are required to prime GVL responses following delayed donor T cell transfer into MHC-mismatched allogeneic chimeras. The APC population(s) required for induction of graft-versus-host reactivity are not known, although it has been demonstrated by others that certain host-derived dendritic cell (DC) populations (in situ or transferred) are capable of inducing GVHD in models of immediate T cell transfer to freshly conditioned recipients. We hypothesised that host CD11c+ conventional DC would be required for the induction of graft-versus-host reactivity in a model of delayed T cell transfer to established mixed chimeras (MC). To test this hypothesis, we reconstituted lethally irradiated B6 (H2b) mice with a mixture of BALB/c (H2d) and B6.CD11c-DTR T cell depleted bone marrow. In the resulting MC, host CD11chigh cells (predominantly conventional DC) are uniquely sensitive to killing by diphtheria toxin (DT) due to their selective expression of a high affinity DT receptor. 8-10 week old established MC received DLI in form of congenic Thy1.1+ BALB/c splenocytes. MC were injected with DT (or PBS) every 72 hours from day -1 to day 10 following DLI. Depletion of host conventional DC (>90%) abrogated accumulation of donor Thy1.1+ CD4 and CD8 T cells in recipient spleens and reduced in vivo cytotoxicity against host target B cells. These effects associated with a lack of conversion from mixed to full donor chimerism, demonstrating an absolute requirement for host CD11chigh DC in priming the lympho-haematopoietic graft-versus-host response in the steady state.We have shown previously that induction of systemic inflammation by injection of a synthetic TRL7 agonist (R-848) is sufficient to induce systemic GVHD in MC after delayed DLI (Chakraverty et al, JEM, 2006). We therefore considered the requirement for CD11chigh DC in priming graft-versus-host reactivity in the presence of inflammation. We generated BALB/c + B6.CD11c-DTR→ B6 MC as before and then used DT treatment to deplete host conventional DC in the presence of R-848 (given every 72h from day -1 to day 10 following DLI). In MC without depletion of CD11chigh DC, R-848 treatment enhanced accumulation of Thy1.1+ BALB/c CD4 and CD8 donor cells and increased in vivo cytotoxicity against host B cell targets as compared to non-R-848 treated MC. This was associated with histological evidence of GVHD in the skin, gut and liver. In MC with depletion of CD11chigh DC (>90% deletion), R-848 treatment was associated with equivalent donor T cell accumulation and in vivo killing of host targets as observed in non-DC depleted controls. Furthermore, DC-depleted MC treated with R-848 also developed histological GVHD. In vitro experiments using CD11c+ stimulator cells purified from the spleens and lymph nodes of R-848 or PBS-treated mice suggested that priming of the allo-response in the presence of inflammation was not due to a residual population of CD11chigh DC remaining after DT treatment.Taken together, these data demonstrate for the first time that conventional host CD11chigh DC are required to prime lympho-haematopoietic graft-versus-host responses in the steady state following delayed DLI to MC. In contrast, their role is redundant in the context of TLR-agonist induced GVHD. This suggests that other APC populations can prime GVHD in the presence of systemic inflammation. Disclosures:No relevant conflicts of interest to declare.

PKCθ is required for alloreactivity and GVHD but not for immune responses toward leukemia and infection in mice

When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform theta (PKCtheta), a key regulator of TCR signaling. In contrast, PKCtheta was required for alloreactivity and GVHD induction. Furthermore, absence of PKCtheta raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCtheta-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCtheta is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.

Targeted Protection of the Colon in the Absence of Donor Antigen-Presenting Cell -Derived Interleukin 23 Allows for Separation of Graft Versus Host and Graft Versus Leukemia Effects

Separation of graft versus leukemia (GVL) and graft versus host (GVH) reactivity has been a longstanding but elusive goal in allogeneic bone marrow transplantation (BMT). Recent studies from our laboratory have shown that donor APC-derived IL-23 secretion has a critical role in mediating pathological damage in the colon during GVHD, but has no protective effect in other GVHD target organs such as the lung and liver. These results demonstrate the feasibility of regional GVHD protection and raise the question as to whether such localized protection might be a strategy to separate GVH and GVL responses.

PR1 vaccination in myeloid malignancies

The graft-versus-leukemia (GVL) effect following allogeneic stem cell transplantation is testament to the effectiveness of the immune system in recognizing and eliminating leukemia cells. The successful identification of a range of leukemia-associated antigens (LAAs) that drive the GVL response in recent years has stimulated research in the development of vaccines to treat hematological malignancies. Here, we review the current experience with the PR1 vaccine. PR1 is a nine amino acid, HLA-A*0201-restricted peptide, shared by two myeloid LAAs, proteinase (PR)3 and neutrophil elastase (NE). PR3 and NE are found in the primary (azurophil) granule proteins of normal granulocytes and are overexpressed in myeloid leukemia cells. PR1 induces powerful HLA-A*0201-restricted CD8+ T-cell responses that selectively kill myeloid leukemia cells in vitro. The detection of low frequencies of PR1-specific CD8+ T cells in patients with chronic myeloid leukemia and at higher frequencies in patients entering molecular remission after allogeneic stem cell transplantation supports the concept that there is natural immunity to PR1, which can be boosted further by vaccination to enhance immunity to leukemia. Preliminary reports indicate that PR1 peptide vaccination induces significant increases in PR1-specific CD8+ T cells, with rapid and durable remissions in some patients with myeloid leukemia. These promising early results point the way to optimizing the administration of peptide vaccines to improve the treatment of otherwise refractory myeloid leukemias.

Quantitative MRD monitoring identifies distinct GVL response patterns after allogeneic stem cell transplantation for chronic lymphocytic leukemia: results from the GCLLSG CLL3X trial

The purpose of this study was to prospectively analyze minimal residual disease(MRD) kinetics after reduced-intensity allogeneic stem cell transplantation (allo-SCT) in high-risk chronic lymphocytic leukemia (CLL). Subjects were the first 30 consecutive patients from a prospective clinical trial, and seven pilot patients treated identically. Using real-time quantitative-PCR (RQ-PCR) and/or flow-based MRD monitoring (sensitivity >or=10(-4)), five distinct patterns of MRD kinetics could be identified: patients who promptly achieved durable MRD negativity without direct evidence of graft-versus-leukemia (GVL) effects (Group 1) (n=4; no clinical relapse); patients with complete and sustained MRD response after GVL induced by immunosuppression tapering (Group 2) or donor lymphocyte infusions (Group 3) (n=18; one relapse); patients without MRD response due to lack of GVL (Group 4) (n=2; two relapses); patients with incomplete and transient MRD response to GVL (Group 5) (n=4; three relapses). In summary, this study provides a comprehensive map of possible MRD courses and their prognostic implications after T-replete allo-SCT in high-risk CLL, indicating that effective GVL activity is induced virtually in all patients who develop chronic GVHD. However, in a significant proportion of cases, this does not translate into sustained disease control due to development of secondary GVL resistance.

Vigorous Priming of Minor Histocompatibility Antigen Specific T Cells by Frequent High Dose Peptide Vaccination May Lead to Prolonged T Cell Receptor Downregulation, Impaired T Cell Functionality, and Promotion of Tumor Escape.

Allogeneic stem cell transplantation can be successfully applied in the treatment of hematological malignancies and relies on the graft versus leukemia (GVL) effect mediated by donor T cells directed against minor histocompatibility antigens (mHag) selectively expressed on malignant hematopoietic cells of the patient. However, due to insufficient in-vivo priming of donor T cells the GVL response may not be adequately initiated or amplified. Vaccination strategies using immunogenic peptides derived from hematopoeisis specific mHag like HA-1 may form a strategy to initiate or boost the in-vivo GVL response. However, it has been reported that repetitive vaccination with HLA-class I binding 9-mer peptides can lead to the induction of T cell anergy. We hypothesized that repetitive strong priming of the mHag specific T cells may also lead to prolonged downregulation of the T cell receptor (TCR) resulting in inability of the T cells to subsequently attack tumor cells expressing the mHag, allowing tumor escape despite the presence of potentially effective T cells. We tested this hypothesis in an in-vitro model using CFSE-labeled HA-1+ CD34+ chronic myeloid leukemia (CML) cells as target/stimulator cells, and HA-1 specific T cells as effector cells. In previous studies we have demonstrated the resistance of a small population of quiescent CML stem cells to all high avidity T cells, allowing the subsequent outgrowth of malignant progeny from this population. To mimick a peptide vaccination strategy, we loaded CD34+ CML cells from an HA-1+ patient with various concentrations (E-12-E-6M) of the 9-mer HA-1 peptide, and investigated the direct and residual functional cytotoxic capacity of an HA-1 specific CD8+ T cell clone. In accordance with our previous results, we observed complete deletion of all proliferating CML precursor cells after 24–48 hours of exposure to the CTLs, whereas a small subpopulation of quiescent CD34+ cells was resistant to T cell attack. The exogenous peptide loading resulted in more rapid lysis and also attack of part of the quiescent stem cell population. However, in the next days malignant progeny was formed from the quiescent stem cell population in the conditions of high peptide stimulation despite the continuous presence of the T cells, suggesting impaired residual cytotoxic function of these T cells. Therefore, we analyzed the level of TCR downregulation after exposure to the HA-1 positive CML CD34+ cells in the absence or presence of E-12-E-6 M HA-1 peptide loaded to the target cells. We observed strong dose-dependent TCR downregulation as measured by specific tetramer staining (20%–78% decrease in fluorescence intensity after 24 hours of exposure to targets loaded with 0-E-6M HA-1 peptide). At high peptide concentrations it took 6–9 days before proper functional TCR expression could be again demonstrated. In conclusion, we here demonstrate that high affinity T cells show a prolonged TCR downregulation after vigorous stimulation by peptide loaded target cells. In this period the T cells showed a dramatic loss of function and allowed the outgrowth of a leukemic subpopulation expressing the HA-1 antigen. Milder vaccination strategies using longer peptides requiring uptake and processing by the target cells may lead to expression of more physiological levels of the mHag and less vigorous priming of the mHag specific T cells, thereby preserving their functional capacity and responsiveness.

Can graft-versus-leukemia reactivity be dissociated from graft-versus-host disease?

Dissociation of graft-versus-leukemia (GvL). effects from graft-versus-host disease (GvHD) is the ultimate goal of allogeneic hematopoietic stem cell transplantation (alloHSCT) in the treatment of hematological malignancies. The pivotal role of donor T cells in both anti-leukemic and anti-host reactivity of allogeneic stem cell grafts has been known since the first transplants for fatal leukemia were performed over 25 years ago. Growing understanding of the T cell-mediated GvL response has revealed the importance of host-type antigen-presenting cells and the capacity of adoptively transferred donor T cells in inducing anti-leukemic responses, and has led to a re-evaluation of the relative roles of the pre-transplant conditioning regimen and the allogeneic stem cell graft. Key advances in clinical practice such as reduced-intensity stem cell transplantation and donor lymphocyte infusions are now routinely applied and allow for the induction of potent antileukemic effects, while GvHD can to some extent be controlled. Other strategies to separate T cell-mediated antileukemic effects from GvHD are antigen-specific adoptive T cell-therapy and recipient lymphocyte infusion (RLI) and these are in an experimental stage. Importantly, a role for alloreactive natural killer cells in mediating GvL without GvHD has emerged in patient studies of MHC haplotype-mismatched alloHSCT. Finally, experimental studies indicate that naturally occurring regulatory T cells may differentially affect GvHD and GvL.

WT1-Specific CD8+ T Lymphocytes May Participate in the Elimination of Acute Lymphoblastic Leukemia Following Allogeneic Stem Cell Transplantation.

There is clinical evidence that a graft-versus-leukemia (GVL) effect occurs following allogeneic stem cell transplantation for acute lymphoblastic leukemia (ALL). However, the potency of this GVL effect is often associated with unwanted graft-versus-host-disease (GVHD) and disease relapse remains a major contributor to treatment failure. Wilms' tumor gene 1 (WT1) is overexpressed in 70–90% of cases of ALL and has been identified as a convenient minimal residual disease (MRD) marker. WT1 is an attractive immunotherapeutic target in ALL because peptides derived from WT1 can induce CD8+ T-cell responses, and being non-allelic, WT1 would be unlikely to provoke GVHD. We investigated whether CD8+ T-cells directed against an HLA-A*0201 restricted epitope of WT1 (WT126) occur in ALL patients during the early phase of immune reconstitution post-SCT (days 30–180). We analyzed CD8+ T-cell responses against WT1 in 10 HLA-A*0201+ ALL SCT recipients and their respective donors using WT1/HLA-A*0201 tetrameric complexes and flow cytometry for intracellular IFN-gamma. We studied the kinetics WT1-specific CD8+ T-cell responses in consecutive samples obtained post-SCT. CD8+ T-cells recognizing WT1 were detected ex vivo in samples from 5 of 10 ALL patients post-SCT but not in patients pre-SCT. WT1-tetramer+ CD8+ T cells had a predominantly effector memory phenotype (CD45RO+CD27−CD57+). WT1 gene expression in pre-SCT and donor samples was assayed by quantitative real-time PCR (RQ-PCR). WT1 expression in PBMC from healthy donors was significantly lower than in patients (median 0, range 0–66 ×10−4 WT1/ABL compared to patients, median 12, range 0–2275 ×10−4 WT1/ABL) (P < 0.01). There was a strong correlation between the emergence of WT1-specific CD8+ T cells and a reduction in WT1 gene expression (P < 0.001) (as depicted below) suggesting direct anti-ALL activity post-SCT. Disappearance of WT1-specific CD8+ T-cells from the blood coincided with reappearance of WT1 gene transcripts, consistent with a molecular relapse, further supporting the direct involvement of WT1-specific CD8+ T-cells in the GVL response. These results provide evidence for the first time of spontaneous T-cell reactivity against a leukemia antigen in ALL patients. Our results support the immunogenicity of WT1 in ALL patients post-SCT and a potential application for WT1 peptides in post-transplant immunotherapy of ALL. [Display omitted]

Vaccination with Host Dendritic Cells Induces Graft-Versus-Leukemia Responses without Severe Graft-Versus-Host Disease in a Preclinical Mouse Model for Allogeneic Stem Cell Transplantation.

Allogeneic stem cell transplantation is a potent treatment for patients with leukemia. This therapeutic effectiveness is largely attributed to the graft-versus-leukemia (GVL) response during which alloreactive donor CD8+ T cells eradicate minor histocompatibility antigens (MiHA)-expressing leukemic cells. Unfortunately, recognition of MiHA expressed in healthy tissues, including the liver, skin and gut, often results in morbidity and even mortality due to the development of graft-versus-host disease (GVHD). Antigen presenting cells (APC) play an essential role in the activation of alloreactive T cells and thus in the induction of GVL responses and GVHD. Using several in vivo models it has been suggested that host APC, which present endogenous MiHA to donor CD8+ T cells, are more potent inducers of alloreactive responses than donor APC. Here, we compared the ability of host APC versus donor APC to activate alloreactive T cells in vitro and to induce alloreactions in vivo. For this purpose, we isolated T cells and cultured bone marrow-derived dendritic cells (DC), the most potent APC, from B6.SJL ‘host' mice and MHC (H-2b)-matched but MiHA-mismatched C3.SW ‘donor' mice. Following their activation by the TLR4 ligand lipopolysaccharide for no longer than 5 hours, both host and donor DC showed a fully mature phenotype (MHCIhigh MHCIIhigh CD40+ CD80high CD86high), and comparable migratory behavior towards lymph node-specific chemokines. In primary mixed leukocyte reactions host DC were 2–3 times more potent than donor DC in inducing donor CD8+ T cell proliferation. To determine the role of host and donor DC in the development of GVL and GVHD, we established a preclinical mouse model (C3>B6) for allogeneic stem cell transplantation. Repopulation analyses demonstrated that T cells, B cells and myeloid cells from blood and spleen of transplanted mice were for more than 95% of donor origin. Transplanted mice were subsequently vaccinated intravenously with 3 weekly doses of 3 × 105 host or donor DC. DC vaccination, which did not lead to significant GVHD, was followed by an intravenous challenge with 106 GFP-expressing mouse AML (C1498) cells. While sham-vaccinated mice died quickly due to leukemia progression, vaccination with both host and donor DC resulted in diminished leukemia growth. However, only vaccination with host DC, and not donor DC, resulted in a (partial) rescue from challenge. These results suggest that host DC are the most potent inducers of GVL reactions without causing significant GVHD. We believe that these data will be instrumental in designing appropriate DC-based immunotherapies in transplanted leukemia patients.

Host MHC class II+ antigen-presenting cells and CD4 cells are required for CD8-mediated graft-versus-leukemia responses following delayed donor leukocyte infusions

AbstractFollowing bone marrow transplantation, delayed donor leukocyte infusions (DLIs) can induce graft-versus-leukemia (GVL) effects without graft-versus-host disease (GVHD). These antitumor responses are maximized by the presence of host hematopoietic antigen-presenting cells (APCs) at the time of DLI. Using a tumor-protection model, we demonstrate here that GVL activity following administration of DLIs to established mixed chimeras is dependent primarily on reactivity to allogeneic MHC antigens rather than minor histocompatibility or tumor-associated antigens. CD8+ T-cell–dependent GVL responses against an MHC class II–negative tumor following delayed DLI require CD4+ T-cell help and are reduced significantly when host APCs lack MHC class II expression. CD4+ T cells primed by host APCs were required for maximal expansion of graft-versus-host reactive CD8+ T cells but not their synthesis of IFN-γ. In contrast, the GVL requirement for CD4+ T-cell help was bypassed almost completely when DLI was administered to freshly irradiated recipients, indicating that the host environment is a major factor influencing the cellular mechanisms of GVL.

Antibody Responses Associated with the Graft-versus-Leukemia Effect in Adult T-Cell Leukemia

Adult T-cell leukemia (ATL) is a peripheral T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). The prognosis of ATL, especially the acute and lymphoma subtypes, is poor with conventional and high-dose chemotherapy. The effectiveness of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for ATL has been reported, suggesting the presence of a graft-versus-leukemia (GVL) effect against this malignancy. To identify the target antigens associated with tumor rejection, we used SEREX (serological identification of antigens by recombinant cDNA expression cloning) to screen ATL complementary DNA expression libraries with sera from an ATL patient who had a GVL response after allo-HSCT. Among the isolated clones, autocrine motility factor receptor (AMFR), which encodes a glycosylated transmembrane protein, was found to have selective reactivity with the sera obtained during tumor regression. Real-time reverse transcription polymerase chain reaction analysis for AMFR showed highest expression in the testis among normal tissues. Furthermore, aberrant AMFR expression was found in at least some ATL patients. Taken together, these findings suggest that AMFR may be one of the GVL antigens that provoke effective antitumor immunity against ATL in allogeneic settings.

Successful Generation of Mature Professional Acute Myeloid Leukemia (AML) – Antigen Presenting Cells (APC) Does Not Predict Successful Induction of AML Reactive T Cells for Adoptive Immunotherapy.

Treatment of relapsed acute myeloid leukemia (AML) after allogeneic stem cell transplantation (alloSCT) with donor lymphocyte infusion (DLI) can induce a graft-versus-leukemia (GVL) reaction but also graft-versus-host disease (GVHD). GVHD may be avoided and the likelihood of a GVL response increased by infusion of in-vitro generated donor-derived cytotoxic T cell lines reactive with the AML cells. Treatment of patients with relapsed AML after alloSCT with DLI often fails, possibly due to a limited antigen presenting function of the primary AML cells. Therefore, to improve this antigen presenting function, we studied the ability to modify AML cells into maturated professional APC and the consequences for the subsequent generation and isolation of AML-reactive T cells using these AML-APC as stimulator cells. AML-APC were generated as follows:primary AML cells were cultured in GM-CSF, SCF, TNFα and IL-4, and after 3–6 days maturation was induced by DKTP (diphtheria, whooping cough, tetanus and polio) vaccine, by addition of monocyte conditioned medium (MCM) mimic (containing IL-1β, IL-6 and TNFα) with PGE2, or by CD40-cross-linking, orAML cells were cultured for 1–4 days with IL-4, GM-CSF and calciumionophore (CI).The highest numbers of mature AML-APC could be generated after maturation by CD40 cross-linking or MCM mimic/PGE2. Induction or upregulation of CD80, CD86 and CD83 expression was determined in 5 -100% of the cells, IL-12 production was induced after CD40 cross-linking and the AML-APC did not produce IL-10. Cell-populations of leukemic cells containing more than 25% fully maturated AML-APC could be obtained in 8 out of 15 cases. AML-reactive T cell lines were generated in 9 HLA-identical donor-patient combinations. In 4 of these 9 combinations cell suspensions containing > 25% fully maturated professional AML-APC could be generated. Donor T cells were stimulated with AML-APC in a ratio of 10:1, and at day 7 and 11 of the culture, 10 IU/ml IL-2 was added. At day 14 responder cells were restimulated with AML-APC, and 16 hours after restimulation IFNγ producing AML-reactive T cells were stained using the IFNγ secretion assay (Miltenyi Biotech) and isolated by FACS sorting. Using a flowcytometry-based cytotoxicity assay in which the AML cells were labeled with CFSE we analyzed the isolated IFNγ positive T cells for their ability to kill the leukemic cells. In 2 of the 4 combinations in which optimal AML-APC could be induced, IFNγ producing T cells were capable of killing the leukemia. However, when we compared the capability of unmodified primary AML cells and professional AML-APC to induce AML-reactive T cells in these 2 responses, we found in both cases that the unmodified AML cells and AML-APC were equally capable to generate AML-reactive T cells, suggesting that the generation of professional AML-APC is not crucial for the induction of AML-reactive T cells. This is further supported by the fact that also in 2 of the 5 donor-patient combinations where no fully maturated professional AML-APC could be generated AML-reactive T cells could be induced. The expansion rate of responding T cells was higher after stimulation with fully maturated AML-APC. In conclusion, mature professional AML-APC could be generated in the majority of cases, but this modification of the malignant cells did not correlate with their ability to successfully induce AML-reactive T cell lines for adoptive immunotherapy.

A crucial role for antigen-presenting cells and alloantigen expression in graft-versus-leukemia responses

Graft-versus-leukemia (GVL) response after allogeneic bone marrow transplantation (BMT) represents one of the most potent forms of immunotherapy against malignant diseases. Antigen-presenting cells (APCs) are crucial for the induction of graft-versus-host disease (GVHD), the most serious complication of allogeneic BMT, but their role in GVL responses is unclear. Using a series of clinically relevant mouse GVL tumor models, we found that APCs and alloantigen expression on tumors are crucial for GVL. Moreover, APCs of host origin predominated in GVL responses although donor APCs contributed as the acuity of tumor burden decreased.

Identification of a 40S Ribosomal Protein S4–Derived H-Y Epitope Able to Elicit a Lymphoblast-Specific Cytotoxic T Lymphocyte Response

The superior graft-versus-leukemia (GVL) effect of the female-to-male stem cell transplantation is partially independent from the concomitant graft-versus-host reactivity. However, the antigenic basis of this selective GVL response remains enigmatic, because no H-Y antigens with hematopoietic-restricted expression were identified. In this study, we report a novel H-Y epitope that is preferentially recognized on activated proliferating lymphocytes. We generated a CTL clone YKIII.8 that showed reactivity toward male B*5201+ CD40-activated B cells, EBV-lymphoblastoid cell lines, and phytohemagglutinin-activated T-cell blasts but little or no reactivity toward fibroblasts, CD14+ cells, or unstimulated B and T cells. The antigen recognized by YKIII.8 was identified by screening of a cDNA expression library, and its pattern of expression was investigated. cDNA of the male isoform of 40S ribosomal protein S4 was found to encode the antigenic peptide TIRYPDPVI, which was recognized by YKIII.8. Western blot analysis showed that rapidly proliferating cells overexpress the RPS4 protein in comparison with nonrecognized cell subsets. Retroviral transfer of YKIII.8 T-cell receptor resulted in preservation of the lymphoblast-specific reactivity pattern. Our findings suggest that CTL specific to certain epitopes of ubiquitously expressed H-Y antigens may specifically target lymphoblasts, contributing to the selective GVL effect of female-to-male stem cell transplantation.

Survival Difference of AML Patients Receiving HLA Matched Sibling Allogeneic Bone Marrow Transplants (ABMT) Correlates with HLA-Cw Ligand Groups for Killer Immunoglobulin-Like Receptors (KIRs).

The reactivity of NK cells and some T cell populations is regulated by the interaction of KIRs with target cell HLA class I molecules. KIR interactions have been suggested to influence outcomes of haploidentical and HLA-identical allogeneic hematopoietic stem cell transplants, particularly for AML patients. We analyzed the KIR ligand phenotypes of 60 AML patients who received HLA-identical sibling myeloablative ABMT from 4/9/97-11/5/03. The median age was 45 yrs (range 8–62). At the time of transplant 24 patients (40%) were in CR. All patients received a busulfan/cyclophosphamide-based preparative regimen and all received bone marrow (T-cell replete) as their stem cell source. Patient HLA KIR ligands were categorized as: 1) HLA-Cw groups C1/C1, C2/C2, C1/C2; 2) HLA-Bw4 (+ or −); 3) HLA-A3 or A11 (+ or −) (as reviewed in Farag et al Blood 100:1935, 2002). Recursive partitioning analysis for post-transplant time-related outcomes (acute GVHD, grade 3/4 acute GVHD, chronic GVHD, extensive chronic GVHD, freedom from relapse and survival) was performed for each KIR ligand group. Patients with C1/C1 or C2/C2 (n=26) had improved survival compared to the C1/C2 group (n=34) (median survival 43.5 months vs. 5.8 months, respectively, p=0.018), as shown below: [Display omitted] This survival difference was associated with more relapse in the C1/C2 group (p=0.048), but not with incidence or severity of acute or chronic GVHD, age, infection or pretransplant disease status. There were 26/34 (76%) deaths in the C1/C2 group with 15 (58%) due to relapse as compared to 13/26 (50%) deaths in the C1/C1 + C2/C2 group where 4/13 (31%) were due to relapse. The median follow up of survivors was 36.3 mos (range 7.8–72.4 mos). No significant differences in outcomes were observed when patients were analyzed for the presence or absence of HLA-Bw4 or A3/11. The majority of patients had KIR genotyping performed for those KIRs with established HLA ligands. Among those tested there were no cases in which the donor did not have at least one inhibitory KIR gene specific for a Cw ligand present in the patient or donor. This may suggest that KIR expression at the cellular level rather than KIR genotype alone should be investigated. In conclusion, AML patients undergoing matched sibling donor ABMT who were heterozygous for HLA-Cw KIR ligand groups (C1/C2) had reduced survival compared to patients homozygous for these groups. The higher relapse rate observed in the heterozygous ligand group may suggest a less effective graft-versus-leukemia (GVL) response. Since C1/C2 heterozygotes have a greater opportunity to engage inhibitory KIRs than do C1 or C2 homozygotes, they may more effectively inhibit KIR-positive NK and T cell populations involved in GVL responses.

Requirement for CD4 Help for Optimal GVL Effects of Alloreactive T Cells Given to Established Mixed Chimeras but Not Freshly Irradiated Mice.

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.

Sequential Blockade and Engagement of Co-Stimulatory Pathways: A Potential Strategy for Amplifying Graft-Versus-Leukemia Responses without GVHD.

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 <0.1% of splenocytes by day 60. The decline in GVH-reactive T cells was associated with marked apoptosis and a sustained reduction in the expression of IL-7Rα. By day 60, no CTL activity against host cells was detectable. We reasoned that strategies that augment the survival of GVH TE/M might enhance the durability of the GVL response and, in the absence of tissue inflammation induced by conditioning, might not lead to GVHD. Co-stimulation through the tumor necrosis family receptor, OX40, which is expressed on activated T cells, is anti-apoptotic and enhances recruitment of TE/M to the memory pool. Following DLI, OX40 expression on 2C CD8+ T cells peaked on day 7 with somewhat earlier and sustained expression on DLI-derived CD4+ T cells. Since OX40 expression was specific for GVH-reactive T cells, we examined the effect of giving agonistic anti-OX40 antibody on day +5 following DLI. This was associated with rapid and complete conversion to full donor chimerism by day +14, whereas DLI + control antibody recipients had only partially converted by day +28. By day 60 post-DLI, anti-host CTL activity was clearly detectable in anti-OX40 recipients but not in controls. No clinical evidence of GVHD was observed, although histological examination revealed transient mild lymphocytic infiltration of the lamina propria on day +13, which resolved completely by day +18. In further experiments, anti-OX40 administration was associated with marked increases in the numbers of 2C CD8+ T cells in spleen, lymph node and bone marrow following DLI. Furthermore, effector differentiation, as assayed by intracellular expression of interferon-γ by 2C CD8+ T cells, was increased in recipients of anti-OX40 antibody. Of note, we observed a complete inhibition IL-7Rα down-regulation that is normally observed on activated CD8+ T cells following DLI. We conclude that OX40 co-stimulation following delayed DLI to established MC represents a potential means to enhance the magnitude and duration of a GVH reaction without the induction of significant GVHD.