Delayed Donor Lymphocyte Infusions Research Articles

Antibody-drug conjugates plus Janus kinase inhibitors enable MHC-mismatched allogeneic hematopoietic stem cell transplantation.

Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.

Murine allogeneic CD19 CAR T cells harbor potent antileukemic activity but have the potential to mediate lethal GVHD

AbstractAcute lymphoblastic leukemia (ALL) persisting or relapsing following bone marrow transplantation (BMT) has a dismal prognosis. Success with chimeric antigen receptor (CAR) T cells offers an opportunity to treat these patients with leukemia-redirected donor-derived T cells, which may be more functional than T cells derived from patients with leukemia but have the potential to mediate graft-versus-host disease (GVHD). We, together with others, have previously demonstrated tumor-specific T-cell dysfunction in the allogeneic environment. Here, we studied CAR T-cell function following BMT using an immunocompetent murine model of minor mismatched allogeneic transplantation followed by donor-derived CD19-CAR T cells. Allogeneic donor-derived CD19-CAR T cells eliminated residual ALL with equal potency to those administered after syngeneic BMT. Surprisingly, allogeneic CAR T cells mediated lethal acute GVHD with early mortality, which is atypical for this minor mismatch model. We demonstrated that both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells, with a higher peak but rapid deletion of allogeneic CAR T cells. Interestingly, CAR-mediated acute GVHD was only seen in the presence of leukemia, suggesting CAR-target interactions induced GVHD. Indeed, serum interleukin (IL)-6 was elevated only in the presence of both leukemia and CAR T cells, and IL-6 neutralization ameliorated the severity of GVHD in a delayed donor lymphocyte infusion model. Finally, allogeneic CD4+ CAR T cells were responsible for GVHD, which correlated with their ability to produce IL-6 upon CAR stimulation. Altogether, we demonstrate that donor-derived allogeneic CAR T cells are active but have the capacity to drive GVHD.

Long‐term graft‐versus‐tumor effect following reduced intensity hematopoietic stem cell transplantation in a child with metastatic renal cell carcinoma

Pediatric renal cell carcinoma (RCC) is rare and different from adult RCC. Although target agents have recently been introduced, allogeneic hematopoietic stem cell transplantation exploiting graft-versus-tumor effect still remains an important treatment option for metastatic RCC. A 2-year-old male with RCC developed hepatic metastases 6 months following radical nephrectomy and subsequent cytokine therapy. Allogeneic reduced-intensity stem cell transplantation (RIST) with early withdrawal of immunosuppression and delayed donor lymphocyte infusions was performed. A second transplantation was undertaken following marrow aplasia. Now he remains progression-free with regression of hepatic metastases 5.7 years after RIST, along with complete donor chimerism.

Alloreactivity Directed Against the Widely Distributed HY Antigen Impairs Antitumor Immunity and Results in T-Cell Dysfunction

Abstract 2964 INTRODUCTION:The curative potential of allogeneic transplant for high-risk malignancy is based on the observation that alloreactivity can result in a clinically significant graft-versus-tumor (GVT) effect. However, we have observed that alloreactivity directed against non-tumor restricted miHA's reduces quantitative responses to vaccines targeting tumor-specific antigens. The relative impact of the GVHD-mediating antigen on the potency of the GVT response when the antigen is shared has not been well studied. METHODS:A murine allotransplant system in which the clinically relevant GVHD antigen HY drives both graft-versus-host disease and the antitumor response was utilized. Following lethal irradiation, combinations of B6 male (HY-expressing) and female (HY-naïve) donors and recipients were used in T-cell depleted bone marrow transplants to control for HY expression in hematopoetic and non-hematopoetic compartments. Delayed donor lymphocyte infusion (DLI) with female HY-specific transgenic T-cells was then performed which allowed tracking of antigen-specific cells. Mice were subsequently challenged with an immunogenic HY-expressing tumor (MB49). In tumor protection studies, transplant recipients received a male dendritic cell vaccine at the time of DLI. Recipients were monitored for clinical GVHD scoring, weight loss, tumor-free and overall survival. Surface phenotyping of HY-specific CD8 T cells from recipient bone marrow, tumor-draining lymph node (LN) and spleen was performed serially by flow cytometry using congenic markers. Statistical analyses were performed using paired Student t-tests and Kaplan-Meier survival estimates. RESULTS:Transplantation of female marrow and HY-specific T cells into male recipients produces a mild HY-targeted GVHD, indicated by weight loss and skin GVHD scores. Female recipients of female marrow and HY-specific DLI had 100% survival following HY-expressing tumor challenge. In contrast, male recipients had only 20 +/− 4.7% tumor-free survival (p<0.0001), despite receiving HY-reactive female marrow and HY-specific DLI. Administration of an HY-expressing male dendritic cell vaccine did not improve either tumor growth velocity or tumor-free survival in male recipients. Despite a poor antitumor response in males, expression of HY on nonhematopoetic tissues produced a significant expansion of HY specific T-cells following DLI, regardless of tumor-bearing status (30.5 −77.4% total CD8 from spleen, draining LN and marrow, vs 0.01–1% from female recipient controls, p<0.0001). This suggested that impaired tumor control was due to dysfunction, rather than deletion, of HY-specific T cells. Indeed, nearly 100% of HY-specific CD8 isolated from the spleen, tumor-draining lymph node, and bone marrow of male recipients expressed high levels of PD-1, a phenomenon observed at all time points in tumor-bearing and non tumor-bearing male recipients with HY-directed GVHD. Non-HY specific CD8 cells did not express PD-1 (p<0.0001). Further, HY-specific CD8 from spleen and tumor-draining LN of male recipients display a significantly increased percentage of CD44+CD62L- effector memory (72.4 +/− 17.2%) vs. CD44+CD62L+ central memory (15.9 +/− 9.7%, p= 0.006) cells compared to non-HY specific CD8 cells (26.5% +/− 2.8 % vs. 28.2 +/− 12.7%, p= 0.52) from male and female recipient controls. CONCLUSIONS:In an experimental system where HY is expressed on both recipient nonhematopoetic tissue and tumor, HY-directed alloreactivity impairs the antitumor response despite antigen-specific DLI and effective vaccination. Characterization of alloreactive CD8 T cells in this setting reveals a persistence of effector memory and high levels of PD-1 expression, which suggest T-cell dysfunction as a possible mechanism. Further studies of T-cell dysfunction in this model may identify targets for therapeutic blockade following adoptive immunotherapy with particular relevance to those clinical situations where GVHD does not enhance GVT. Disclosures:No relevant conflicts of interest to declare.

IFN-γ promotes graft-versus-leukemia effects without directly interacting with leukemia cells in mice after allogeneic hematopoietic cell transplantation

AbstractThe ability of IFN-γ to enhance graft-versus-leukemia (GVL) activity without direct interaction with leukemia cells was examined by comparing GVL effects against IFN-γ receptor-deficient (GRKO) leukemia between wild-type (WT) and IFN-γ–deficient (GKO) allogeneic hematopoietic cell transplantation (allo-HCT). We established a primary IFN-γ–unresponsive T-cell leukemia model using virally-transduced GRKO B6 mouse bone marrow cells overexpressing Notch1. We first assessed GVL effects in lethally-irradiated B6 mice receiving CD4-depleted allo-HCT from WT or GKO BALB/c donors. Administration of CD4+ cell-depleted allo-HCT from WT, but not GKO, BALB/c donors mediated significant GVL effects against GRKO leukemia. Similar results were obtained in pre-established allogeneic chimeras receiving delayed donor lymphocyte infusion (DLI). Although both WT and GKO DLI achieved significant anti-tumor responses, the former was markedly stronger than the latter. These data indicate that IFN-γ is capable of promoting GVL effects via mechanisms independent of its interaction with leukemia cells.

Inhibition of STAT1 modulates GVHD after allogeneic BMT through induction of S100A8/A9 (169.38)

Abstract T cell depletion (TCD) of donor bone marrow (BM) reduces graft-versus-host-disease (GVHD) after allogeneic blood and marrow transplantation (alloBMT), but can result in poor T cell reconstitution and relapse. We have shown that transplantation of TCD BM deficient in the gamma interferon (IFNγ) receptor allows delayed donor lymphocyte infusions (DLI) without GVHD. We have replicated these results with TCD BM deficient in STAT1, which mediates IFNα and IFNγ signals, and demonstrated expanded plasmacytoid dendritic cells (pDCs). In this study, we further characterized the mechanism of GVHD resistance. After MHC-matched, minor histocompatibility antigen-mismatched alloBMT, TCD STAT1-deficient BM prevents DLI-induced GVHD. Treatment of wild type BM donors and recipients with the glucagon-like peptide-1 analog, Exenatide, which mediates off-target inhibition of STAT1, attenuates GVHD and increases splenic B220+ cell recovery. TCD IRF9-deficient BM, which lacks IFNα signaling, does not prevent GVHD, confirming that IFNγ signaling via STAT1 is the primary pathway of GVHD resistance. Selective deletion of STAT1 using CD11c cre/STAT1flox BM is sufficient to attenuate GVHD-associated weight loss, further implicating a DC subset. Although S100 proteins are not typically found in pDCs, pDCs from TCD STAT1-deficient BM recipients express increased S100A8. Finally, TCD S100A8/A9-deficient BM causes more severe weight loss after DLI, confirming the relevance of S100 proteins in GVHD.

Extracorporeal Photopheresis Attenuates Murine Graft-versus-Host Disease via Bone Marrow–Derived Interleukin-10 and Preserves Responses to Dendritic Cell Vaccination

Extracorporeal photopheresis (ECP) is emerging as a therapy for graft-versus-host-disease (GVHD), but the full mechanism of action and the impact on immunity have not been fully established. After murine minor histocompatibility antigen-mismatched bone marrow (BM) transplantation (allo-BMT), coinfusion of ECP-treated splenocytes with T cell-replete BM attenuated GVHD irrespective of the donor strain of the ECP-treated splenocytes, and was associated with increased numbers of regulatory T cells. Coculture of myeloid dendritic cells (DCs) with ECP-treated splenocytes resulted in increased interleukin (IL)-10 production after submaximal stimulation with lipopolysaccharide. Furthermore, male myeloid DCs exposed to ECP-treated splenocytes were less potent at inducing CD8(+) HY responses when used as a vaccine in vivo. The efficacy of ECP-treated splenocytes was enhanced when administered just before delayed donor lymphocyte infusion following T cell-depleted allo-BMT, allowing for the administration of sufficient numbers of T cells to respond to myeloid DC vaccination in the absence of a thymus. Finally, the therapeutic effect of ECP-treated splenocytes was lost in recipients of IL-10-deficient BM. We demonstrate that ECP-treated splenocytes attenuate GVHD irrespective of the source of ECP-treated cells via a mechanism that likely involves modulation of DCs and requires IL-10 produced by BM-derived cells. Importantly, the attenuation of GVHD by ECP-treated splenocytes permits donor lymphocyte infusion-dependent responses to DC vaccines after allo-BMT.

Chronic Granulomatous Disease: Lessons from a Rare Disorder

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency with X-linked or autosomal recessive inheritance involving defects in genes encoding phox proteins, which are the subunits of the phagocyte NADPH oxidase. This results in failure to produce superoxide anion and downstream antimicrobial oxidant metabolites and to activate antimicrobial proteases. Affected patients are susceptible to severe, life-threatening bacterial and fungal infections and excessive inflammation characterized by granulomatous enteritis resembling Crohn's disease and genitourinary obstruction. Early diagnosis of CGD and rapid treatment of infections are critical. Prophylaxis with antibacterial and mold-active antifungal agents and the administration of interferon-γ has significantly improved the natural history of CGD. Currently, the only cure is allogeneic hematopoietic cell transplant (HCT), although there remains controversy as to which patients with CGD should get a transplant. Allele-based HLA typing of alternative donors, improved supportive care measures, and use of reduced toxicity conditioning have resulted in event-free survival (EFS) of at least 80% even with an unrelated donor and even better when the patient has no active infections/inflammation. Gene correction of CGD would eliminate the risks of graft-versus-host disease (GVHD) and the immunoablative chemotherapy required for allogeneic HCT. Based on gene therapy trials in patients with SCID-X1, ADA-SCID, and the early experience with CGD, it is clear that at least some degree of myeloablation will be necessary for CGD as there is no inherent selective growth advantage for gene-corrected cells. Current efforts for gene therapy focus on use of lentivector constructs, which are thought to be safer from the standpoint of insertional mutagenesis and more efficient in transducing hematopoietic stem cells (HSCs).

Adoptive Transfer of Natural Killer (NK) Cells to Prevent Gvhd and Enhance GVT Effects After Allogeneic Hematopoietic Cell Transplantation (HCT): The Timing of Donor NK Cell Infusions Critically Impacts Transplant Outcome.

Abstract 786Murine models have shown infusions of donor NK cells from hematopoietic stem cell transplant donors can prevent GVHD while simultaneously mediating a graft-vs-tumor (GVT) effect. Reduction of GVHD by alloreactive NK cells can be mediated indirectly through the eradication of host antigen presenting cells (APC) or directly by NK cell killing of alloreactive T cells. To assess how the timing of NK cell administration impacts these effects, donor NK cells from B10.d2 (H-2d) mice (1-2×106 cells) were infused into MHC-matched BALB/c (H-2d) recipients following lethal irradiation (950cGy) at one of the following time-points: 1) two days prior to a T cell replete (TR) HCT to target host APC or 2) at the time of a TR-HCT or 3) five days following a TR-HCT to target both host APC and in vivo primed alloreactive donor T cells. We also evaluated whether donor NK cells given on the day of a T cell deplete (TD) HCT could be used to prevent GVHD in mice that subsequently received a delayed donor lymphocyte infusion (DLI) given four days following HCT.Administration of donor NK cells two days prior to allogeneic TR-HCT did not result in a reduction of GVHD (figure). In contrast, the administration of NK cells given either at the time of a TR-HCT or five days following a TR-HCT reduced the incidence of GVHD (GVHD incidence 70% (p=0.2) and 40% (p=0.01) respectively) compared to controls that did not receive NK cells following a TR-HCT (GVHD incidence 100%). Similarly, mice that received a TD-HCT followed by a DLI on day four had a significantly lower incidence of GVHD when NK cells were infused on the day of transplantation compared to controls that did not receive donor NK cells (GVHD incidence 40% vs 100% respectively, p=0.01). Using bioluminescence imaging, we next investigated the impact of the timing of NK cell infusions on GVT effects in tumor bearing mice. Luciferase transduced RENCA tumors were injected intravenously into BALB/c mice ten days prior to allogeneic HCT. Tumor progression was significantly delayed in recipients of a TR-HCT when NK cells were infused on day five compared to when NK cells were infused two days prior to or at the same day of a TR-HCT (tumor doubling times 22.9 days, 7.6 days and 8.5 days respectively; p=0.03) (figure). This delay in tumor progression correlated with a significant improvement in overall survival; recipients of a TR-HCT given NK cells on day five had significantly longer survival compared to recipients of TR-HCT that did not receive NK cells (median survival 54±14 days vs 44±9; p=0.008), whereas infusion of NK cells prior to or concomitant with a TR-HCT did not significantly prolong survival (median survival 49±4 days and 50±12 days respectively; p=0.23). A comparable delay in tumor progression and longer survival was observed in mice that received a TD-HCT followed by a DLI on day four when NK cells were infused at the time of transplant compared to controls not receiving NK cells (tumor doubling time 19.7 days vs 7.2 days and survival 62±16 days vs. 50±9 days respectively; p=0.07). [Display omitted] In conclusion, these results show that the timing of adoptive donor NK cell transfer has a critical impact on the ability of NK cells to prevent GVHD and enhance GVT effects following both T-cell replete and T-cell depleted allogeneic HCT. Following a TR-HCT, a delayed add-back of NK cells maximizes GVHD reducing and anti-tumor effects of adoptively transferred donor NK cells. Disclosures:No relevant conflicts of interest to declare.

Host Environment Determines the Development of Gvhd but Not the Extent of Activation and Expansion of Gvh-Reactive CD4 T Cells

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<0.001). FI mice died of GVHD by Day 9, whereas GVHD did not develop in MC. Similar studies were performed with directly class II MHC alloreactive CD4 T cells. Six weeks after establishing B6→B6D2F1 MC, a DLI consisting of 106 OTII CD4 T cells (non-specific T cells in this model because they do not recognize any recipient antigen), 106 4C TCR transgenic CD4 T cells (directly alloreactive for recipient IAd class-II antigen) and 107 polyclonal B6 spleen cells was injected into MC and FI B6D2F1 mice that also received B6 BMC. On Days 6 and 9, greater accumulation of 4C CD4 T cells compared to OTII CD4 T cells (p<0.05) was detected in the spleens of both groups. Expression of activation markers on 4C cells occurred in both FI mice and MC, with similar MFI for CD44 (mean 120 in FI mice and 198 in MC; NS), similar %CD69+ (average 54.9% positive in FI and 60.2% in MC; NS) and %CD25+ cells (average 19.6% positive in FI mice and 11.2% in MC; NS). Downregulation of CD62L was also similar but slightly delayed in MC. By day 16, FI mice developed significant weight loss and GVHD, whereas MC did not develop GVHD. These data confirm the influence of the host environment in the development of GVHD and show that, similar to GVH-reactive CD8 T cells, marked antigen-driven activation and proliferation of GVH-reactive CD4 T cells occurs in both FI mice and MC, although with different kinetics.

Disruption of Gamma Interferon Signaling through the STAT1 Pathway Enhances Alloreactivity While Abrogating GvHD

BACKGROUND: Strategies that allow for sufficient alloreactivity to mediate graft-versus-tumor (GVT) effects after allogeneic bone marrow transplant (BMT) while minimizing graft-versus-host-disease (GVHD) remain elusive. Historically, gamma interferon (IFNγ) has been implicated as a critical mediator of both GVT and GVHD pathophysiology, and high levels of IFNγ have been implicated in immunosuppression. Given that immunosuppression is also a hallmark of GVHD, we hypothesized that modulation of IFNγ signaling could have important effects on both GVHD and GVT following BMT. We therefore compared immunocompetence to tumor challenge and tumor vaccination, in the presence or absence of IFNγ receptor (R1) and STAT1 signaling (a primary downstream mediator of IFNγ signaling) on donor bone marrow.METHODS: We utilized a clinically relevant, minor histocompatibility antigen-mismatched, T cell-depleted BMT model, with delayed donor lymphocyte infusions (DLIs) into thymectomized mice to ensure that T cell recovery occurred exclusively from the DLI, as occurs in most clinical settings during the first 6 months post-BMT. Lethally irradiated female C57BL/6 (B6) × C3H.SW recipients were engrafted with normal B6, B6 IFNγR1-deficient, normal B6129, or B6129 STAT1-deficient bone marrow on day 0 and reconstituted with donor T cells on days 14 and 28 at a dose that induced sublethal GVHD, evidenced by weight loss, histology, and abrogated quantitative immune responses to an HY-directed tumor vaccine. Recipients were then challenged on day 42 with an HY-expressing tumor and followed for survival. Immune reconstitution was assessed by flow cytometry both prior to and after DLI administration.RESULTS: Mild GVHD prevents vaccine-mediated immune responses and HY-expressing tumor protection in thymectomized recipients. Both IFNγR1 and STAT1-deficient marrow abrogate GVHD. STAT1-deficient marrow increased absolute numbers of splenic plasmacytoid dendritic cells (CD11cint/Class II+/Gr-1+/B220+) prior to DLI administration. In addition, an increase in the percentage and absolute number of splenic regulatory T cells was observed at the time of tumor challenge. Surprisingly, despite the immunosuppressive milieu, recipients of IFNγR1-deficient marrow had enhanced vaccine-mediated tumor protection as measured by overall survival, similar to syngeneic BMT controls.CONCLUSION: IFNγ signaling is a critical mediator of GVHD pathophysiology, including the immunosuppression associated with this condition. Disrupting IFNγ signaling through the JAK1/STAT1 pathway is a novel strategy to selectively modulate alloreactivity, and results in enhanced anti-tumor immune responses following allogeneic BMT without exacerbating GVHD.

T Cell Repertoire Complexity Is Conserved after LLME Treatment of Donor Lymphocyte Infusions

Slow reconstitution of the T cell repertoire after allogeneic blood or bone marrow stem cell transplantation is a major risk factor for patient mortality. The delivery of immunocompetent T cells as delayed donor lymphocyte infusions (DLIs) is a potential way of counteracting this problem. The development of graft-versus-host disease (GVHD) is a potential complication of this procedure, however. We previously found that in P→F1 haploidentical murine models, the ex-vivo treatment of donor lymphocytes with L-leucyl-L-leucine methyl ester (LLME) can prevent the onset of GVHD after DLI, likely by inducing cell death in most of the perforin-positive CD8 + T cells and in a fraction of CD4 + T cells. Our previous preclinical studies have formed the basis of an ongoing phase I clinical trial in which patients received LLME-treated DLI from their original donor in an attempt to accelerate T cell reconstitution. To understand how this treatment strategy might affect the complexity of the DLI T cell repertoire, we used T cell receptor Vβ spectratype analysis to evaluate the DLI product pre-LLME and post-LLME treatment. The results indicated that the LLME-treated DLI product exhibited CDR3-size distribution complexities similar to those of its untreated donor sample. In addition, comparisons of the CD4 + and CD8 + T cell repertoire from the donor before LLME treatment with that of the recipient post-DLI demonstrated equal complexity for most of the resolvable Vβ families. Finally, the in vitro proliferative capacity of LLME-treated DLI product in response to allo-stimulation in a one-way mixed lymphocyte reaction was comparable to that of the untreated product.

Loss of IFNγR1 Signaling on Donor Bone Marrow Abrogates GVHD but Maintains Immunocompetence.

BACKGROUND: Acute graft versus host disease (GVHD) remains as the major complication after allogeneic bone marrow transplant (BMT) resulting in organ toxicity and immune dysfunction. Indeed, we have previously demonstrated that GVHD impairs responses to dendritic cell vaccines. The pathophysiology of GVHD involves preparative regimen-induced inflammation of target organs, release of inflammatory mediators such as gamma interferon (IFNg), and subsequent activation of alloreactive T cells. Given that IFNg can both contribute to GVHD and provide beneficial immune responses, we explored the potential role of IFNg on GVHD and post-transplant immunocompetence.METHODS: We utilized a minor histocompatibility antigen mismatched, T cell-depleted BMT model, with delayed donor lymphocyte infusions (DLIs) as a means of controlling the induction of acute GVHD and to provide a source of immunocompetence in a thymectomized mouse. To study the role of IFNg on GVHD, we chose either IFNg receptor 1 (IFNgR1) −/− marrow or DLI to permit the normal production of IFNg in GVHD while influencing which cells that can respond to the cytokine. Normal C57BL/6 (B6) or IFNgR1 −/− B6 mice were used as bone marrow donors on day 0 into lethally irradiated, thymectomized B6 × C3H.SW (F1) mice. Normal B6 or IFNgR1 −/− DLIs given with or without a dendritic cell vaccine were introduced at days 14 and 28 post-BMT both to control the induction of GVHD and to provide a population of vaccine-responding cells. F1 recipients were observed for signs of GVHD. ELISPOT of the number of antigen-reactive IFNg-producing splenocytes were also performed to measure functional response to vaccine.RESULTS: The absence of IFNgR1 in the DLI abrogates GVHD as shown by % change in weight (B6 DLI = −6.3 +/− 4.7 vs. IFNgR1−/− DLI = 6.6 +/− 6.1, p=0.001) and allows for greater doses of DLI to be tolerated by the host, however, there is also decreased vaccine responses by ELISPOT (B6 DLI = 1631 vs. IFNgR1−/− DLI = 72, p=0.03). Surprisingly, using IFNgR1−/− bone marrow also abrogates GVHD as shown by % change in weight (B6 marrow = −6.3 +/− 4.7 vs. IFNgR1−/− marrow = 4.4 +/− 4.2, p less than 0.05) and splenocyte count (B6 marrow = 31.48 +/− 12.54 vs. IFNgR1−/− marrow = 63.54 +/− 15.92, p=0.008), but vaccine responses by ELISPOT can be restored to levels that are equivalent of syngeneic control mice, even in the presence of a normal B6 DLI (B6 marrow = 1631 vs. IFNgR1−/− marrow = 9283, p=0.0002). The abrogation of GVHD by IFNgR1−/− marrow does not appear to be a dominant effect since mixtures of IFNgR1 −/− and normal B6 bone marrow still cause GVHD.CONCLUSIONS: Recipients of allogeneic bone marrow and T cells developed GVHD and had decreased vaccine responses by ELISPOT. Loss of IFNgR1 on allogeneic donor lymphocytes abrogates their ability to cause GVHD, but also diminished their ability to respond to vaccine. Surprisingly, loss of IFNgR1 on a donor bone marrow-derived, non T cell results in equivalent abrogation of GVHD, while restoring immunocompetence through favorable responses to a vaccine. Further studies will attempt to identify the phenotype of the responsible bone marrow-derived cell. These results demonstrate a strategy of providing higher doses of DLIs to enhance anti-tumor activity without exacerbating GVHD, and thus, have implications for immune modulation post-allogeneic BMT.

Spectratype Analysis of In Vitro Donor Anti-Host T Cell Repertoire Responses Predict Those of In Vivo Post-BMT.

Immunotherapeutic strategies have gained recognition as viable alternatives to more conventional modalities for the treatment of cancer. In this regard, adoptive T cell therapy through allogeneic blood and marrow transplantation (BMT) has provided the strongest evidence that anti-tumor effects could be achieved against hematological malignancies. However, the major complications of BMT still include graft failure, opportunistic infections, disease relapse and graft-versus-host disease (GVHD). The presence of mature donor T cells in the transplant inoculum reduces the incidence of the first three complications, while unfortunately increasing the risk of GVHD, which can be directed against either HLA or minor histocompatibilty antigen (miHA) disparities. Thus, a major objective in the field has been to develop tactics that could facilitate the separation of graft-versus-tumor (GVT) effects from the deleterious effects of GVHD. One such approach would be to selectively deplete donor alloreactive T cells in the donor inoculum while allowing residual T cells to provide some protection against infection and to support a tumor-specific GVT response. For a more targeted approach, delayed donor lymphocyte infusion (DLI) of positively-selected donor GVT-reactive T cells could be used weeks to months post-transplant, if these elements were identifiable. In this regard, TCR Vβ repertoire analysis by CDR3-size spectratyping can be a powerful tool for the characterization of alloreactive T cell responses. Theoretically, molecular analysis of T cell responses in vitro, given the high sensitivity of the PCR-based spectratyping technique, should identify the most potentially critical Vβ families involved in the later development of GVHD and GVT effects in patients. To this end, we tested the hypothesis that T cell repertoire analysis of HLA-matched sibling (SIB) or matched unrelated donors (URD) from in vitro, host-stimulated, mixed lymphocyte cultures (MLC) would be predictive of the TCR-Vβ spectratype analysis of the T cell repertoire in the patient following BMT. In this study, we examined 17 patient pairs and report that for the resolvable Vβ families, we observed overall 71.2 ± 11.9% (mean ± SD.; range 40%–85%) of the in vitro anti-host T cell responses were predictive of those in the patient post-transplant. Of the 28.8% non-predictive Vβ families, 6.9 ± 6.3% (range 0%–27%) exhibited skewing in the MLC but no skewing in the patient post-transplant repertoire, 9.3 ± 6.3% (range 0%–18.8%) exhibited skewing in different peaks within the same Vβ family, and 12.5 ± 10.8% (range 0%–40%) showed skewing in the patient post-transplant and none in the MLC. Taken together, these results suggest that the in vitro MLC T cell responses show good consistency with post-transplant patient responses. Thus, in vitro spectratyping may be useful for predicting the alloreactive T cell responses involved in GVHD and could be used to guide custom-designed select Vβ family T cell-depleted transplants to improve patient outcomes. The additional advantage of this approach is that minimization of GVHD risk can be obtained without any direct knowledge of the specific miHA involved in the individual donor-patient pair.

Ex Vivo Generated Cytotoxic T Cells (CTL) for Adoptive Immunotherapy across MHC Barriers Retain Anti-Leukemic Capacity (GVL) without Eliciting Graft Versus Host Disease (GVHD): Crucial Role of Recipient-Derived Antigen Presenting Cells (APC).

BACKGROUND: While recipient-derived APC have proven to be crucial in mediating GVL after allogeneic hematopoietic stem cell transplantation (HCT), this desired effect seemed to be inevitably linked to the risk of developing severe GVHD. It has been elegantly shown in murine models that delayed donor lymphocyte infusions (DLI) into mixed rather than full hematopoietic chimeras produce dramatically improved GVL effects and virtually no GVHD. However, avoidance of GVHD has been more difficult when this intelligent concept was transferred into the clinical setting. Here we demonstrate that donor-derived T cells of MHC-mismatched origin retain strong anti-leukemic activity and lose nonspecific alloreactivity upon specific in vitro priming and consecutive short term expansion.METHODS: We chose a fully MHC-mismatched allogeneic bone marrow transplantation model using B10.A mice (H-2a) as donors and C57BL/6 (H-2b) mice as recipients to produce either full (controls) or stable mixed hematopoietic chimeras. After full hematopoietic recovery mice were challenged with a lethal dose of a C57BL/6-derived leukemic cell line (C1498) and consecutively injected with either naive DLI, ex vivo generated CTL or PBS only. CTL were generated by specifically priming lymphocytes of MHC-mismatched donor origin on bone marrow-derived dendritic cells (DC) (either donor- or recipient-derived) that had been pulsed with recipient-derived leukemia cell lysates. This was followed by expansion using αCD3/αCD28 coated microspheres.RESULTS: We show thatex vivo generated CTL of allogeneic origin can mediate anti-leukemic effects only when primed on recipient-derived DC that had been pulsed with leukemia cell lysates (CTL versus controls, p < 0.05 ). In contrast, loading recipient-derived leukemia lysates on donor-derived DC (T cells and DC syngeneic to each other) abrogates the anti-leukemia effect of adoptively transferred CTL in vitro and in vivo.CTL exhibit a strong proliferative response in vitro when stimulated with irradiated leukemic cells of allogeneic origin (C1498), however, this response is significantly weaker (3-fold) when compared to naive DLI. Remarkably, the nonspecific proliferative response (alloreactivity) of CTL is completely abolished when irradiated C57BL/6 splenocytes are used as stimulators.CTL maintain their responsiveness to leukemia in vivo as demonstrated by cure rates from leukemia in up to 40% of mice. Importantly, upon adoptive transfer into leukemia bearing mice after MHC-mismatched HCT, CTL lose their unspecific alloreactivity in vivo as well as proven on microscopic sections of GVH target organs.Anti-leukemia effects of CTL across MHC-barriers are comparable to naive DLI in leukemia bearing mice (40% versus 50% cure rate).CTL are able to convert mixed hematopoietic chimerism into full hematopoietic chimerism (30% donor →100% donor). In contrast, mixed chimerism remains unchanged when CTL are transferred that had been primed ex vivo on donor-derived DC. This data is encouraging in that it provides further evidence that strong GVL effects can be obtained across MHC barriers without eliciting severe GVHD.

Donor CD8+ T Cells Mediate GVL without GVHD in Recipients Conditioned with Anti-CD3 mAb.

Donor CD8+ T cells play a critical role in mediating graft versus leukemia (GVL), but also induce graft versus host disease (GVHD) in recipients conditioned with total body irradiation (TBI). Here, we report that injections of donor C57BL/6 (H-2b) or FVB/N (H-2q) CD8+ T with bone marrow cells induced chimerism and eliminated BCL1 leukemia/lymphoma cells without GVHD in anti-CD3-conditioned BALB/c (H-2d) recipients. In contrast, the same dose of donor CD8+ T and marrow cells induced lethal GVHD in TBI-conditioned recipients. In addition, the anti-CD3-conditioned long-term complete chimeras without prior exposure to host-type BCL1 cells also eliminated the tumors when being challenged with BCL1 cells 120 days after HCT. This is in contrast to the report that long-term complete chimeras induced with delayed donor lymphocyte infusion lost GVL activity. Using in vivo and ex vivo bioluminescent imaging, we observed that donor CD8+ T cells expanded rapidly and infiltrated GVHD target tissues in TBI-conditioned recipients, but donor CD8+ T cell expansion in anti-CD3-conditioned recipients was confined to lympho-hematological tissues. This confinement was associated with lack of up-regulated expression of α4β7 integrin and chemokine receptors (i.e. CXCR3) on donor CD8+ T cells. In addition, host-reactive donor CD8+ T cells in anti-CD3-conditioned recipients were only partially deleted, and the residual cells were rendered heterogeneous: some unresponsive/anergic, some Tc2, some Foxp3+ suppressive cells, and some effector cells. The whole population of residual donor CD8+ T cells from anti-CD3-conditioned recipients mediated GVL without GVHD in TBI-conditioned secondary recipients. These results indicate that anti-CD3-conditioning separates GVL from GVHD via confining donor CD8+ T cell expansion to host lympho-hematological tissues as well as tolerization of the residual donor CD8+ T cells, in which the residual host-reactive effector cells mediate persistent GVL, and the regulatory CD8+ T cells prevent them from damaging host tissues.

Minimal HLA Disparity and KIR Ligand Compatibility in Host Versus Graft Direction May Facilitate Donor Engraftment Following In Vivo and Ex Vivo T Cell Depleted (TCD) Nonmyeloablative Haploidentical Stem Cell Transplantation for Hematologic Malignancies.

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.

In Vivo and Ex Vivo T-Cell Depleted (TCD) NonmyeloablativeHaploidentical Stem Cell Transplantation (NSCT) for Hematologic Malignancy (HM).

Based on our nonmyeloablative MHC-mismatched murine bone marrow transplant (BMT) models, in which mixed lymphohematopoietic chimerism (MLC) is reliably induced as a platform for adoptive cellular immunotherapy via delayed donor lymphocyte infusions (DLI), we developed a similar clinical strategy for haploidentical NSCT. Conditioning for NSCT has consisted of high dose cyclophosphamide, in vivo T-cell depletion (TCD) with anti-T-cell antibody therapy, pre-transplant thymic irradiation and cyclosporine (CYA) for GVHD prophylaxis (with taper and discontinuation by day 35 for patients with MLC and no evidence of GVHD). Because of a high incidence of ≥ grade II GVHD (10 of 14 patients) using equine anti-thymocyte globulin for in vivo TCD, we substituted MEDI-507 (an anti-CD2 monoclonal antibody) for ATG. Of 8 patients who underwent BMT with MEDI-507 for in vivo TCD, 5 experienced graft loss. The protocol was then modified to include ex vivo T-cell depleted high dose peripheral blood stem cell transplantation (PBSCT). Graft loss occurred in 2 of 4 patients. 2 pts who received DLI for recurrent disease converted to full donor chimerism (FDC) with no (n=1) or grade II (n=1) GVHD. Fludarabine was then added to the conditioning regimen. Ten patients with advanced HM (NHL, n=4; HD, n=3; MDS, n=1; CLL, n=1 CML, n=1) have received NSCT from HLA 5/6 (n=5) or 4/6 (n=5) matched haploidentical donors on a protocol using cyclophosphamide 60 mg/kg x 2, MEDI-507 0.1 mg/kg on day - 8 and 0.6 mg/kg on day 7 and - 6, fludarabine 25 mg/m2 on days - 5 through -1, thymic irradiation on day - 1, and ex vivo TCD (via CD34+ cell selection using the Isolex® device) PBSCT on day 0. Median #/kg (range) of infused CD34+ and CD3+ cells were 6.6X10E6 (3.2X10E6-1.5X10E7) and 3.5X10E4 (6.3X10E2-1.18X10E5), respectively. Of 9 evaluable patients, all initially achieved MLC without GVHD, but 2 patients subsequently lost detectable chimerism. 2 patients with MLC have received DLI, one in conjunction with chemotherapy, for disease progression. 5 pts (n=3 following rapid tapering of immunosuppression to induce a GVT effect, n=2 after DLI) have developed ≥ grade II acute GVHD (3 grade II, 2 grade III-IV). 5 patients have experienced disease progression, of whom one achieved a CR following DLI. Two pts have died due to transplant related complications (GVHD, n=1;late idiopathic pneumonitis, n=1) Split lineage MLC has occurred in each case, with a predominance of early donor granulocyte chimerism (mean 64%,77%,61%,>99% at 2,4,12,26 weeks) and a lower percentage of donor T-cell chimerism (mean 14%,37%,53%,>99% at 2,4,12,26 weeks). Conversion to FDC or near FDC occurred spontaneously in 4 pts and in 2 pts following DLI. Of 22 patients treated with MEDI-507 based haploidentical NSCT regimens, 8 are alive from < 1 to 70 months post-transplant. In conclusion, in vivo and ex vivo TCD haploidentical NSCT reliably leads to split lineage MLC with the potential for spontaneous chimerism conversion or conversion following DLI. Sustained anti-tumor responses in patients with advanced, chemorefractory HM have been achieved.

Haploidentical Stem Cell Transplantation: The Always Present but Overlooked Donor

Haploidentical stem cell transplantation is a treatment option for the approximately 70% of patients who do not have an HLA-identical sibling donor. The availability of a haploidentical donor in most families is a potential advantage, both for avoiding the need to find an alternative unrelated donor, and for the potentially more potent graft-versus-tumor effect that can be induced. The early complications of severe graft-versus-host disease (GVHD) following T-cell replete stem cell transplantation (SCT), and graft failure and recurrent malignancy (after T-cell depleted SCT) have limited the applications of this approach. Newer strategies employing T-cell depletion of the graft, using either very high-dose peripheral blood stem cells and/or more intensive conditioning therapy have overcome some of the problems of conventional transplantation. Nonmyeloablative SCT approaches have overcome some of the morbidity and mortality associated with the early complications of SCT and have been associated with favorable engraftment and GVHD profiles. Induction of mixed lymphohematopoietic chimerism as a platform for adoptive cellular immunotherapy (via delayed donor lymphocyte infusions) may have important application in avoiding early GVHD, while ultimately capturing a very potent graft-versus-tumor effect. Current strategies are focusing on improvement of immune reconstitution and prevention of recurrence of the underlying malignancy.

ICSBP Gene Deficient Mice Are Highly Susceptible to the Development of Graft-versus-Host Disease (GVHD) Following Administration of Delayed Donor Lymphocyte Infusions (DLI).

Delayed administration of DLI to mixed hematopoietic chimeras following allogeneic bone marrow transplantation (alloBMT) leads to conversion from mixed to full donor chimerism without GVHD. However, in the clinical setting development of GVHD is still the most common complication. Donor T cells are the main mediators of GVHD in alloBMT. However, the precise role of host dendritic cells (DC) and their different subsets needs still to be elucidated. CD8a+ DCs seem to have a regulatory function and to reduce experimental GVHD. Recently, it has been shown that the ICSBP (Interferon Consensus Sequence Binding Protein) gene is essential for the development and activation of CD8a+ DCs. ICSBP-gene deficient mice (ICSBP KO) are characterized by several immune defects and systemic expansion of myeloid cells mimicking human CML and may be therefore an attractive in vivo model to evaluate the role of DCs in alloBMT. The aim of the study was to evaluate the 1. feasibility of inducing mixed hematopoietic chimerism following nonmyeloablative conditioning in ICSBP KO mice using fully MHC-mismatched BMT (Balb/c [H2Dd] to B6 x 129 [H2b]), and 2. susceptibility of ICSBP KO mice to the development of GVHD following administration of DLI. Nonmyeloablative conditioning consisted of in-vivo T cell depletion (anti-CD4-[GK1.5], anti-CD8-[2,43] mAb, d-5), fludarabine (30 mg/kg, d-4 to d-2), cyclophosphamid (200 mg/kg, d-1) and 3 Gy TBI (d0) followed by i.v. injection of 1,5 x 10E7 Balb/c bone marrow cells. Stable mixed chimerism was observed in ICSBP wildtype (wt) mice through week 35 post-BMT without signs of clinical GVHD. In contrast, we observed increasing donor CD4 and CD8 T cell chimerism over time in ICSBP KO mice suggesting a lack of donor T cell tolerance. Furthermore, chimerism was higher in granulocyte, B cells and monocyte compared to ICSBP wt mice. In a second experiment we were able to observe spontaneous conversion to full donor hematopoietic chimerism in 2 of 9 mice after nonmyeloablative BMT. In two separate experiments ICSBP wt or KO mice with mixed chimerism received (DLI) on day 35 post BMT. After injection of DLI all wt mice (n = 10) showed a rapid conversion to full donor chimerism and remained healthy until the end of the observation period without signs of GVHD. Despite myeloid hyperproliferation all ICSBP KO mice showed conversion to full donor chimerism. Furthermore, in ICSBP KO mice conversion to full donor chimerism was associated with development of severe GVHD (n = 9). In vitro studies using mixed lymphocyte reactions showed that splenic stimulator cells from ICSBP KO mice had a higher stimulatory capacity compared to wt stimulators (SI 1,5 versus 2,3). Using the same mixed lymphocyte culture (MLC) conditions, supernatants collected from BALB/c responders with ICSBP KO stimulators had higher levels of TNF-alpha and IFN-g compared to MLC using ICSBP wt stimulators. In conclusion, following alloBMT a defective development of host-specific tolerance and high susceptibility for GVHD in ICSBP KO mice is suggested. Further studies are warranted to delineate the precise underlying mechanism by addressing the potential contribution of defective regulatory host T cells and the lack of host CD8a+ DCs in ICSBP KO mice.