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Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting T cell (Tc) immunoglobulin and mucin-containing molecule 3 (TIM-3) for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ Tc enhanced Tc activation, proliferation, and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion, and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3 treatment-mediated GVL effects are Tc induced. In contrast to anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We decipher the connections between oncogenic mutations found in AML and TIM-3 ligand expression and identify anti-TIM-3 treatment as a strategy for enhancing GVL effects via metabolic and transcriptional Tc reprogramming without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Ab in patients with AML relapse after allo-HCT.

STAT3 deficiency (STAT3-/-) in donor T cells prevents graft-versus-host disease (GVHD), but the impact on graft-versus-leukemia (GVL) activity and mechanisms of GVHD prevention remains unclear. Here, using murine models of GVHD, we show that STAT3-/- donor T cells induced only mild reversible acute GVHD while preserving GVL effects against nonsusceptible acute lymphoblastic leukemia (ALL) cells in a donor T cell dose-dependent manner. GVHD prevention depended on programmed death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) signaling. In GVHD target tissues, STAT3 deficiency amplified PD-L1/PD-1 inhibition of glutathione (GSH)/Myc pathways that regulate metabolic reprogramming in activated T cells, with decreased glycolytic and mitochondrial ATP production and increased mitochondrial ROS production and dysfunction, leading to tissue-specific deletion of host-reactive T cells and prevention of GVHD. Mitochondrial STAT3 deficiency alone did not reduce GSH expression or prevent GVHD. In lymphoid tissues, the lack of host-tissue PD-L1 interaction with PD-1 reduced the inhibition of the GSH/Myc pathway despite reduced GSH production caused by STAT3 deficiency and allowed donor T cell functions that mediate GVL activity. Therefore, STAT3 deficiency in donor T cells augments PD-1 signaling-mediated inhibition of GSH/Myc pathways and augments dysfunction of T cells in GVHD target tissues while sparing T cells in lymphoid tissues, leading to prevention of GVHD while preserving GVL effects.

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.

Transplantation of allogeneic hematopoietic cells faces two barriers: failure of engraftment due to a host versus graft reaction, and the attack of donor cells against the patient, the graft versus host (GVH) reaction. This reaction may lead to GVH disease (GVHD), but in patients transplanted due to leukemia or other malignant disorders, this may also convey the benefit of a graft versus leukemia (GVL) effect. The interplay of transplant conditioning with donor and host cells and the environment in the patient is complex. The microbiome, particularly in the intestinal tract, profoundly affects these interactions, directly and via soluble mediators, which also reach other host organs. The microenvironment is further altered by the modifying effect of malignant cells on marrow niches, favoring the propagation of the malignant cells. The development of stable mixed donor/host chimerism has the potential of GVHD prevention without necessarily increasing the risk of relapse. There has been remarkable progress with novel conditioning regimens and selective T-cell manipulation aimed at securing engraftment while preventing GVHD without ablating the GVL effect. Interventions to alter the microenvironment and change the composition of the microbiome and its metabolic products may modify graft/host interactions, thereby further reducing GVHD, while enhancing the GVL effect. The result should be improved transplant outcome., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Deeg.)

Autophagy is a vital cellular process whose role in T immune cells is poorly understood, specifically, in its regulation of allo-immunity. Stimulation of wild-type T cells in vitro and in vivo with allo-antigens enhances autophagy. To assess the relevance of autophagy to T-cell allo-immunity, we generated T-cell-specific Atg5 knock-out mice. Deficiency of ATG5-dependent autophagy reduced T-cell proliferation and increased apoptosis following in vitro and in vivo allo-stimulation. The absence of ATG5 in allo-stimulated T cells enhanced their ability to release effector cytokines and cytotoxic functions, uncoupling their proliferation and effector functions. Absence of autophagy reduced intracellular degradation of cytotoxic enzymes such as granzyme B, thus enhancing the cytotoxicity of T cells. In several in vivo models of allo-HSCT, ATG5-dependent dissociation of T-cell functions contributed to significant reduction in graft-versus-host disease (GVHD) but retained sufficient graft versus tumor (GVT) response. Our findings demonstrate that ATG5-dependent autophagy uncouples T-cell proliferation from its effector functions and offers a potential new strategy to enhance outcomes after allo-HSCT. SIGNIFICANCE: These findings demonstrate that induction of autophagy in donor T-cell promotes GVHD, while inhibition of T-cell autophagy mitigates GVHD without substantial loss of GVL responses., (©2020 American Association for Cancer Research.)

Acute graft-versus-host disease (aGVHD) is one major serious complication that is induced by alloreactive donor T cells recognizing host Ags and limits the success of allogeneic hematopoietic stem cell transplantation. In the current studies, we identified a critical role of Kras in regulating alloreactive T cell function during aGVHD. Kras deletion in donor T cells dramatically reduced aGVHD mortality and severity in an MHC-mismatched allogeneic hematopoietic stem cell transplantation mouse model but largely maintained the antitumor capacity. Kras-deficient CD4 and CD8 T cells exhibited impaired TCR-induced activation of the ERK pathway. Kras deficiency altered TCR-induced gene expression profiles, including the reduced expression of various inflammatory cytokines and chemokines. Moreover, Kras deficiency inhibited IL-6-mediated Th17 cell differentiation and impaired IL-6-induced ERK activation and gene expression in CD4 T cells. These findings support Kras as a novel and effective therapeutic target for aGVHD., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Allogeneic hematopoietic stem cell transplantation is a potentially curative procedure for many malignant diseases. Donor T cells prevent disease recurrence via graft-versus-leukemia (GVL) effect. Donor T cells also contribute to graft-versus-host disease (GVHD), a debilitating and potentially fatal complication. Novel treatment strategies are needed which allow preservation of GVL effects without causing GVHD. Using murine models, we show that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving GVL effects. Both CD8 + and CD4 + donor T cells from Itk -/- mice produce less inflammatory cytokines and show decrease migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). Itk -/- T cells exhibit reduced expression of IRF4 and decreased JAK/STAT signaling activity but upregulating expression of Eomesodermin (Eomes) and preserve cytotoxicity, necessary for GVL effect. Transcriptome analysis indicates that ITK signaling controls chemokine receptor expression during alloactivation, which in turn affects the ability of donor T cells to migrate to GVHD target organs. Our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment., Competing Interests: AA receives research support from 3M Corporation. WH receives research support from Mega Robo Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Mammadli, Huang, Harris, Sultana, Cheng, Tong, Pu, Gentile, Dsouza, Yang, Bah, August and Karimi.)

Disease relapse and graft-versus-host disease (GVHD) are the major complications affecting the outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT). While the functions of αβT cells are extensively studied, the role of donor γδT cells in allo-HSCT is less well defined. Using TCRδ -/- donors lacking γδT cells, we demonstrated that donor γδT cells were critical in mediating graft-versus-leukemia (GVL) effect during allo-HSCT. In the absence of donor γδT cells, IFN-γ production by CD8 + T cells was severely impaired. Vγ4 subset was the major γδT cell subset mediating the GVL effect in vivo , which was partially dependent on IL-17A. Meanwhile, donor γδT cells could mitigate acute GVHD in a murine allo-HSCT model by suppressing CD4 + T cell activation and the major γδT cell subset that exerted this protective function was also Vγ4 γδT cells. Therefore, our findings provide evidence that donor γδT cells, especially Vγ4 subset, can enhance GVL effect and mitigate aGVHD during allo-HSCT., (Copyright © 2020 Song, Zhu, Hu, Liu, Lin, Jin, Yin, Dong, Wu and Liu.)

Although the prognosis of chronic myeloid leukemia (CML) in blastic crisis remains poor, some patients achieve long-term remission after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This may be attributable to graft-versus-leukemia (GVL) effects by donor lymphocytes, but their regulating mechanisms are unclear. Antitumor natural killer (NK) cell immunity is assumed to be important in CML, and we have previously shown that allelic polymorphisms of killer immunoglobulin-like receptors (KIRs) and histocompatibility leukocyte antigens (HLAs) are associated with the response of CML to tyrosine kinase inhibitors. Here, we report a case of CML in blastic phase who received HLA-matched but KIR3DL1 allelic-mismatched allo-HSCT. After transplant, decreased BCR-ABL transcript levels and enhanced NK cell activity were transiently observed. However, reconstitution of KIR3DL1-expressing NK cells occurred, which was associated with diminished NK cell activity and increased BCR-ABL. This case indicates the potential significance of KIR3DL1 in NK cell-mediated GVL activity following allo-HSCT. To the best of our knowledge, this is the first report to analyze the association between sequential KIR3DL1 expression and activity of NK cells after allo-HSCT. Selecting donors with KIR3DL1-null alleles may maintain competent GVL effects and provide improved outcomes in allo-HSCT for CML.

Patients undergoing allogeneic stem cell transplantation as treatment for hematological diseases face the risk of Graft-versus-Host Disease as well as relapse. Graft-versus-Host Disease and the favorable Graft-versus-Leukemia effect are mediated by donor T cells recognizing polymorphic peptides, which are presented on the cell surface by HLA molecules and result from single nucleotide polymorphism alleles that are disparate between patient and donor. Identification of polymorphic HLA-binding peptides, designated minor histocompatibility antigens, has been a laborious procedure, and the number and scope for broad clinical use of these antigens therefore remain limited. Here, we present an optimized whole genome association approach for discovery of HLA class I minor histocompatibility antigens. T cell clones isolated from patients who responded to donor lymphocyte infusions after HLA-matched allogeneic stem cell transplantation were tested against a panel of 191 EBV-transformed B cells, which have been sequenced by the 1000 Genomes Project and selected for expression of seven common HLA class I alleles (HLA-A ∗ 01:01, A ∗ 02:01, A ∗ 03:01, B ∗ 07:02, B ∗ 08:01, C ∗ 07:01, and C ∗ 07:02). By including all polymorphisms with minor allele frequencies above 0.01, we demonstrated that the new approach allows direct discovery of minor histocompatibility antigens as exemplified by seven new antigens in eight different HLA class I alleles including one antigen in HLA-A ∗ 24:02 and HLA-A ∗ 23:01, for which the method has not been originally designed. Our new whole genome association strategy is expected to rapidly augment the repertoire of HLA class I-restricted minor histocompatibility antigens that will become available for donor selection and clinical use to predict, follow or manipulate Graft-versus-Leukemia effect and Graft-versus-Host Disease after allogeneic stem cell transplantation., (Copyright © 2020 Fuchs, Honders, van der Meijden, Adriaans, van der Lee, Pont, Monajemi, Kielbasa, ’t Hoen, van Bergen, Falkenburg and Griffioen.)

Graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT) is characterized by interleukin-6 (IL-6) dysregulation. IL-6 can mediate effects via various pathways, including classical, trans, and cluster signaling. Given the recent availability of agents that differentially inhibit these discrete signaling cascades, understanding the source and signaling and cellular targets of this cytokine is paramount to inform the design of clinical studies. Here we demonstrate that IL-6 secretion from recipient dendritic cells (DCs) initiates the systemic dysregulation of this cytokine. Inhibition of DC-driven classical signaling after targeted IL-6 receptor (IL-6R) deletion in T cells eliminated pathogenic donor Th17/Th22 cell differentiation and resulted in long-term survival. After engraftment, donor DCs assume the same role, maintaining classical IL-6 signaling-dependent GVHD responses. Surprisingly, cluster signaling was not active after transplantation, whereas inhibition of trans signaling with soluble gp130Fc promoted severe, chronic cutaneous GVHD. The latter was a result of exaggerated polyfunctional Th22-cell expansion that was reversed by IL-22 deletion or IL-6R inhibition. Importantly, inhibition of IL-6 classical signaling did not impair the graft-versus-leukemia effect. Together, these data highlight IL-6 classical signaling and downstream Th17/Th22 differentiation as important therapeutic targets after alloSCT., (© 2019 by The American Society of Hematology.)

A subset of MHC-associated self-peptides presented by the recipient's cells and immunologically foreign to the donor can induce an allogeneic immune response after hematopoietic stem cell transplantation (HSCT). These immunogenic peptides originate from the genomic polymorphisms and are known as minor histocompatibility antigens (MiHA). MiHA mismatches trigger the post-transplant immune response, which could manifest in both the deleterious "graft-vs.-host" disease and the beneficial "graft-vs.-leukemia" effect. Importantly, some MiHAs are considered to be promising targets for posttransplant T-cell immunotherapy of hematopoietic malignancies. This creates a demand for a robust and fast approach to genotyping MiHA-encoding polymorphisms. We report a multiplex real-time PCR method for the genotyping of 20 polymorphisms that are encoding HLA-A * 02:01-restricted MiHAs. This method uses allele-specific primers and gene-specific hydrolysis probes. In 1 h it allows for the detection of MiHA mismatches in a donor-recipient pair without the need for electrophoresis, sequencing, or other time-consuming techniques. We validated the method with Sanger and NGS sequencing and demonstrated good performance over a wide range of DNA concentrations. We propose our protocol as a fast and accurate method of identifying mismatched MiHAs. The information on the MiHA mismatches is useful for studying the allogeneic immune response following HSCT and for selecting the targets for post-transplant T-cell therapy.

Despite recent advances in therapy, allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for a range of high-risk hematologic malignancies. However, acute graft-versus-host disease (aGVHD) continues to limit the long-term success of HSCT, and new therapies are still needed. We previously demonstrated that aGVHD depends on the ability of donor conventional T cells (T con s ) to express the lymph node trafficking receptor, CC-Chemokine Receptor 7 (CCR7). Consequently, we examined the ability of cosalane, a recently identified CCR7 small-molecule antagonist, to attenuate aGVHD in mouse HSCT model systems. Here we show that the systemic administration of cosalane to transplant recipients after allogeneic HSCT did not prevent aGVHD. However, we were able to significantly reduce aGVHD by briefly incubating donor T cons with cosalane ex vivo before transplantation. Cosalane did not result in T con toxicity and did not affect their activation or expansion. Instead, cosalane prevented donor T con trafficking into host secondary lymphoid tissues very early after transplantation and limited their subsequent accumulation within the liver and colon. Cosalane did not appear to impair the intrinsic ability of donor T con s to produce inflammatory cytokines. Furthermore, cosalane-treated T con s retained their graft-versus-leukemia (GVL) potential and rejected a murine P815 inoculum after transplantation. Collectively, our data indicate that a brief application of cosalane to donor T con s before HSCT significantly reduces aGVHD in relevant preclinical models while generally sparing beneficial GVL effects, and that cosalane might represent a viable new approach for aGVHD prophylaxis., (Copyright © 2019 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Despite the increasing use of humanized mouse models to study new approaches of graft-versus-host disease (GVHD) prevention, the pathogenesis of xenogeneic GVHD (xGVHD) in these models remains misunderstood. The aim of this study is to describe this pathogenesis in NOD/LtSz-Prkdc scid IL2rγ tm1Wjl (NSG) mice infused with human PBMCs and to assess the impact of the expression of HLA-A0201 by NSG mice cells (NSG-HLA-A2/HHD mice) on xGVHD and graft-versus-leukemia (GvL) effects, by taking advantage of next-generation technologies. We found that T cells recovered from NSG mice after transplantation had upregulated expression of genes involved in cell proliferation, as well as in TCR, co-stimulatory, IL-2/STAT5, mTOR and Aurora kinase A pathways. T cells had mainly an effector memory or an effector phenotype and exhibited a Th1/Tc1-skewed differentiation. TCRβ repertoire diversity was markedly lower both in the spleen and lungs (a xGVHD target organ) than at infusion. There was no correlation between the frequencies of specific clonotypes at baseline and in transplanted mice. Finally, expression of HLA-A0201 by NSG mice led to more severe xGVHD and enhanced GvL effects toward HLA-A2 + leukemic cells. Altogether our data demonstrate that the pathogenesis of xGVHD shares important features with human GVHD and that NSG-HLA-A2/HHD mice could serve as better model to study GVHD and GvL effects.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for several malignant and non-malignant disorders. The precise control of allogeneic T cells is critical for successful outcomes after BMT. The mechanisms governing desirable (graft-versus-leukemia) versus undesirable (graft-versus-host disease) allogeneic responses remain incompletely understood. Non-coding RNAs (ncRNA) are controllers of gene expression that fine-tune cellular responses. Multiple microRNAs (miRNAs), a type of ncRNA, have recently been shown to influence allogeneic T cell responses in both murine models and clinically. Here, we review the role of various miRNAs that regulate T cell responses, either positively or negatively, to allo-stimulation and highlight their potential relevance as biomarkers and as therapeutic targets for improving outcomes after allogeneic BMT.

Janus kinase 2 (JAK2) signal transduction is a critical mediator of the immune response. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Transfer of JAK2 -/- donor T cells to allogeneic recipients leads to attenuated GVHD yet maintains graft-versus-leukemia. Th1 differentiation among JAK2 -/- T cells is significantly decreased compared with wild-type controls. Conversely, iTreg and Th2 polarization is significantly increased among JAK2 -/- T cells. Pacritinib is a multikinase inhibitor with potent activity against JAK2. Pacritinib significantly reduces GVHD and xenogeneic skin graft rejection in distinct rodent models and maintains donor antitumor immunity. Moreover, pacritinib spares iTregs and polarizes Th2 responses as observed among JAK2 -/- T cells. Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after allo-HCT or solid organ transplantation. As such, a phase I/II acute GVHD prevention trial combining pacritinib with standard immune suppression after allo-HCT is actively being investigated (https://clinicaltrials.gov/ct2/show/NCT02891603)., Competing Interests: Conflict of interest statement: B.C.B. has participated in advisory boards related to pacritinib (CTI BioPharma) and ruxolitinib (Incyte) in GVHD. J.S. is employed by CTI BioPharma with related equity ownership. A.O. is an employee at BioSeek. All other authors have no competing financial interests to declare.

The graft-versus-leukemia (GVL) effect in allogeneic hematopoietic stem cell transplantation (alloSCT) is potent against chronic phase chronic myelogenous leukemia (CP-CML), but blast crisis CML (BC-CML) and acute myeloid leukemias (AML) are GVL resistant. To understand GVL resistance, we studied GVL against mouse models of CP-CML, BC-CML, and AML generated by the transduction of mouse BM with fusion cDNAs derived from human leukemias. Prior work has shown that CD4+ T cell-mediated GVL against CP-CML and BC-CML required intact leukemia MHCII; however, stem cells from both leukemias were MHCII negative. Here, we show that CP-CML, BC-CML, and AML stem cells upregulate MHCII in alloSCT recipients. Using gene-deficient leukemias, we determined that BC-CML and AML MHC upregulation required IFN-γ stimulation, whereas CP-CML MHC upregulation was independent of both the IFN-γ receptor (IFN-γR) and the IFN-α/β receptor IFNAR1. Importantly, IFN-γR-deficient BC-CML and AML were completely resistant to CD4- and CD8-mediated GVL, whereas IFN-γR/IFNAR1 double-deficient CP-CML was fully GVL sensitive. Mouse AML and BC-CML stem cells were MHCI+ without IFN-γ stimulation, suggesting that IFN-γ sensitizes these leukemias to T cell killing by mechanisms other than MHC upregulation. Our studies identify the requirement of IFN-γ stimulation as a mechanism for BC-CML and AML GVL resistance, whereas independence from IFN-γ renders CP-CML more GVL sensitive, even with a lower-level alloimmune response.

Programmed death ligand-1 (PD-L1) interacts with programmed death-1 (PD-1) and the immunostimulatory molecule CD80 and functions as a checkpoint to regulate immune responses. The interaction of PD-L1 with CD80 alone has been shown to exacerbate the severity of graft-versus-host disease (GVHD), whereas costimulation of CD80 and PD-1 ameliorates GVHD. Here we have demonstrated that temporary depletion of donor CD4+ T cells early after hematopoietic cell transplantation effectively prevents GVHD while preserving strong graft-versus-leukemia (GVL) effects in allogeneic and xenogeneic murine GVHD models. Depletion of donor CD4+ T cells increased serum IFN-γ but reduced IL-2 concentrations, leading to upregulation of PD-L1 expression by recipient tissues and donor CD8+ T cells. In GVHD target tissues, the interactions of PD-L1 with PD-1 on donor CD8+ T cells cause anergy, exhaustion, and apoptosis, thereby preventing GVHD. In lymphoid tissues, the interactions of PD-L1 with CD80 augment CD8+ T cell expansion without increasing anergy, exhaustion, or apoptosis, resulting in strong GVL effects. These results indicate that the outcome of PD-L1-mediated signaling in CD8+ T cells depends on the presence or absence of CD4+ T cells, the nature of the interacting receptor expressed by CD8+ T cells, and the tissue environment in which the signaling occurs.

Acute graft-versus-host disease (aGVHD) continues to be a frequent and devastating complication of allogeneic hematopoietic stem cell transplantation (HSCT), posing as a significant barrier against the widespread use of HSCTs as a curative modality. Recent studies suggested serum/plasma microRNAs (miRs) may predict aGVHD onset. However, little is known about the functional role of circulating miRs in aGVHD. In this article, we show in two independent cohorts that miR-29a expression is significantly upregulated in the serum of allogeneic HSCT patients at aGVHD onset compared with non-aGVHD patients. Serum miR-29a is also elevated as early as 2 wk before time of diagnosis of aGVHD compared with time-matched control subjects. We demonstrate novel functional significance of serum miR-29a by showing that miR-29a binds and activates dendritic cells via TLR7 and TLR8, resulting in the activation of the NF-κB pathway and secretion of proinflammatory cytokines TNF-α and IL-6. Treatment with locked nucleic acid anti-miR-29a significantly improved survival in a mouse model of aGVHD while retaining graft-versus-leukemia effects, unveiling a novel therapeutic target in aGVHD treatment or prevention., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Patients with leukemia who receive a T cell-depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD., Competing Interests: The authors have declared that no conflict of interest exists.

Beyond its critical role in T cells, T-bet regulates the functions of APCs including dendritic cells and B cells, as well as NK cells. Given that recipient APCs are essential for priming allogeneic T cells and recipient NK or T cells are able to reject allogeneic donor cells, we evaluated the role of T-bet on the host in acute graft-versus-host disease (GVHD) using murine models of allogeneic bone marrow transplantation. T-bet(-/-) recipients developed significantly milder GVHD than their wild type counterparts in MHC-mismatched or CD4-dependent minor histocompatibility Ag-mismatched models. Allogeneic donor T cells, in particular, CD4 subset, significantly reduced IFN-γ production, proliferation and migration, and caused less injury in liver and gut of T-bet(-/-) recipients. We further observed that T-bet on recipient hematopoietic cells was primarily responsible for the donor T cell response and pathogenicity in GVHD. T-bet(-/-) dendritic cells expressed higher levels of Trail, whereas they produced lower levels of IFN-γ and IL-12/23 p40, as well as chemokine CXCL9, resulting in significantly higher levels of apoptosis, less priming, and infiltration of donor T cells. Meanwhile, NK cells in T-bet(-/-) hosts partially contribute to the decreased donor T cell proliferation. Furthermore, although T-bet on hematopoietic cells was required for GVHD development, it was largely dispensable for the graft-versus-leukemia effect. Taken together with our previous findings, we propose that T-bet is a potential therapeutic target for the control of GVHD through regulating donor T cells and recipient hematopoietic cells., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The human leukocyte antigen (HLA) genomic region spanning about 4 Mb is the most gene dense and the polymorphic stretches in the human genome. A total of the 269 loci were identified, including 145 protein coding genes mostly important for immunity and 50 noncoding RNAs (ncRNAs). Biological function of these ncRNAs remains unknown, becoming hot spot in the studies of HLA-associated diseases. The genomic diversity analysis in the HLA region facilitated by next-generation sequencing will pave the way to molecular understanding of linkage disequilibrium structure, population diversity, histocompatibility in transplantation, and associations with autoimmune diseases. The 4-digit DNA genotyping of HLA for six HLA loci, HLA-A through DP, in the patients with Graves' disease (GD) and Hashimoto thyroiditis (HT) identified six susceptible and three resistant HLA alleles. Their epistatic interactions in controlling the development of these diseases are shown. Four susceptible and one resistant HLA alleles are shared by GD and HT. Two HLA alleles associated with GD or HT control the titers of autoantibodies to thyroid antigens. All these observations led us to propose a new model for the development of GD and HT. Hematopoietic stem cell transplantation from unrelated donor (UR-HSCT) provides a natural experiment to elucidate the role of allogenic HLA molecules in immune response. Large cohort studies using HLA allele and clinical outcome data have elucidated that (1) HLA locus, allele, and haplotype mismatches between donor and patient, (2) specific amino acid substitution at specific positions of HLA molecules, and (3) ethnic background are all responsible for the immunological events related to UR-HSCT including acute graft-versus-host disease (GVHD), chronic GVHD, graft-versus-leukemia (GvL) effect, and graft failure., (© 2016 Elsevier Inc. All rights reserved.)

MicroRNAs (miRs) play important roles in orchestrating many aspects of the immune response. The miR-17-92 cluster, which encodes 6 miRs including 17, 18a, 19a, 20a, 19b-1, and 92-1, is among the best characterized of these miRs. The miR-17-92 cluster has been shown to regulate a variety of immune responses including infection, tumor, and autoimmunity, but the role of this cluster in T-cell response to alloantigens has not been previously explored. By using major histocompatibility complex (MHC)-matched, -mismatched, and haploidentical murine models of allogeneic bone marrow transplantation (allo-BMT), we demonstrate that the expression of miR-17-92 on donor T cells is essential for the induction of graft-versus-host disease (GVHD), but dispensable for the graft-versus-leukemia (GVL) effect. The miR-17-92 plays a major role in promoting CD4 T-cell activation, proliferation, survival, and Th1 differentiation, while inhibiting Th2 and iTreg differentiation. Alternatively, miR-17-92 may promote migration of CD8 T cells to GVHD target organs, but has minimal impact on CD8 T-cell proliferation, survival, or cytolytic function, which could contribute to the preserved GVL effect mediated by T cells deficient for miR-17-92. Furthermore, we evaluated a translational approach and found that systemic administration of antagomir to block miR-17 or miR-19b in this cluster significantly inhibited alloreactive T-cell expansion and interferon-γ (IFNγ) production, and prolonged the survival in recipients afflicted with GVHD while preserving the GVL effect. Taken together, the current work provides a strong rationale and demonstrates the feasibility to target miR-17-92 for the control of GVHD while preserving GVL activity after allo-BMT., (© 2015 by The American Society of Hematology.)

Haplo-cord transplant has emerged as a feasible and reliable approach for haematopoietic stem cell transplant in patients who are unable to find matched-donor. This approach provides fast myeloid recovery, low incidence of graft vs host disease (GVHD) and favorable graft versus leukemia (GVL) effects. T cell recovery plays an important role in preventing infectious complications; it also mediates the GVHD and the GVL effects. Here, we utilized a novel RNA-based sequencing approach to quantitatively characterize the T cell receptor (TCRs) repertoire in patients underwent haplo-cord transplant in comparison with those underwent matched-donor transplant. Our study shows that higher percentage of cord cells early post transplant were associated with significantly higher TCR diversity. TCR diversity was significantly lower in patients with GVHD and in relapsed patients. A larger cohort study is needed to validate these data and to provide useful information on the specific TCR clones correlated with clinical outcome.

Killer cell Ig-like receptors (KIRs) bind cognate HLA class I ligands with distinct affinities, affecting NK cell licensing and inhibition. We hypothesized that differences in KIR and HLA class I genotypes predictive of varying degrees of receptor-ligand binding affinities influence clinical outcomes in autologous hematopoietic cell transplantation (AHCT) for acute myeloid leukemia (AML). Using genomic DNA from a homogeneous cohort of 125 AML patients treated with AHCT, we performed KIR and HLA class I genotyping and found that patients with a compound KIR3DL1(+) and HLA-Bw4-80Thr(+), HLA-Bw4-80Ile(-) genotype, predictive of low-affinity interactions, had a low incidence of relapse, compared with patients with a KIR3DL1(+) and HLA-Bw4-80Ile(+) genotype, predictive of high-affinity interactions (hazard ratio [HR], 0.22; 95% confidence interval [CI], 0.06-0.78; p = 0.02). This effect was influenced by HLA-Bw4 copy number, such that relapse progressively increased with one copy of HLA-Bw4-80Ile (HR, 1.6; 95% CI, 0.84-3.1; p = 0.15) to two to three copies (HR, 3.0; 95% CI, 1.4-6.5; p = 0.005) and progressively decreased with one to two copies of HLA-Bw4-80Thr (p = 0.13). Among KIR3DL1(+) and HLA-Bw4-80Ile(+) patients, a predicted low-affinity KIR2DL2/3(+) and HLA-C1/C1 genotype was associated with lower relapse than a predicted high-affinity KIR2DL1(+) and HLA-C2/C2 genotype (HR, 0.25; 95% CI, 0.09-0.73; p = 0.01). Similarly, a KIR3DL1(+) and HLA-Bw4-80Thr(+), HLA-Bw4-80Ile(-) genotype, or lack of KIR3DL1(+) and HLA-Bw4-80Ile(+) genotype, rescued KIR2DL1(+) and HLA-C2/C2 patients from high relapse (p = 0.007). These findings support a role for NK cell graft-versus-leukemia activity modulated by NK cell receptor-ligand affinities in AHCT for AML., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Obliterative bronchiolitis is a potentially life-threatening noninfectious pulmonary complication after allogeneic hematopoietic stem cell transplantation and the only pathognomonic manifestation of pulmonary chronic graft-versus-host disease (cGVHD). In the current study, we identified a novel effect of IL-26 on transplant-related obliterative bronchiolitis. Sublethally irradiated NOD/Shi-scidIL2rγ(null) mice transplanted with human umbilical cord blood (HuCB mice) gradually developed clinical signs of graft-versus-host disease (GVHD) such as loss of weight, ruffled fur, and alopecia. Histologically, lung of HuCB mice exhibited obliterative bronchiolitis with increased collagen deposition and predominant infiltration with human IL-26(+)CD26(+)CD4 T cells. Concomitantly, skin manifested fat loss and sclerosis of the reticular dermis in the presence of apoptosis of the basilar keratinocytes, whereas the liver exhibited portal fibrosis and cholestasis. Moreover, although IL-26 is absent from rodents, we showed that IL-26 increased collagen synthesis in fibroblasts and promoted lung fibrosis in a murine GVHD model using IL-26 transgenic mice. In vitro analysis demonstrated a significant increase in IL-26 production by HuCB CD4 T cells following CD26 costimulation, whereas Ig Fc domain fused with the N-terminal of caveolin-1 (Cav-Ig), the ligand for CD26, effectively inhibited production of IL-26. Administration of Cav-Ig before or after onset of GVHD impeded the development of clinical and histologic features of GVHD without interrupting engraftment of donor-derived human cells, with preservation of the graft-versus-leukemia effect. These results therefore provide proof of principle that cGVHD of the lungs is caused in part by IL-26(+)CD26(+)CD4 T cells, and that treatment with Cav-Ig could be beneficial for cGVHD prevention and therapy., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Hematopoietic-stem-cell transplantation (HSCT) is complicated by histocompatibility-dependent immune responses such as graft-versus-host disease, relapse, and graft rejection. The severity of these common adverse effects is directly related to the degree of human leukocyte antigen (HLA) incompatibility. In addition to the key role of classic HLA matching in influencing HSCT outcome, several lines of evidence suggest an important role for nonclassic major histocompatibility complex class I molecule, HLA-E. The interaction of HLA-E with NKG2A, its main receptor on natural killer cells, modulates cell-mediated cytotoxicity and cytokine production, an important role in innate immune responses. In addition, the HLA-E molecule can present peptides to different subtypes of T cells that may either support graft-versus-leukemia effects or be involved in bridging innate and acquired immunity. To date, the role of HLA-E and its polymorphisms in HSCT outcomes such as graft-versus-host disease, transplant-related mortality, and improved survival has been published by a number of groups. In addition, these data suggest an association between HLA-E polymorphisms and relapse. Whether the engagement of the HLA-E molecule in the modulation of donor T cells is involved in the graft-versus-leukemia effect, or whether a different mechanism of HLA-E dependent reduction of relapse is involved, requires further investigation., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Graft-versus-host disease (GvHD) is a life-threatening immunological complication after allogenic hematopoietic stem cell transplantation (allo-HCT). The intrinsic graft-versus-leukemia (GvL) effect, however, is the desirable curative benefit. Patients with acute GvHD are treated with cyclosporine A (CsA) or tacrolimus (FK506), which not only often causes severe adverse effects, but also interferes with the anticipated GvL. Both drugs inhibit calcineurin, thus at first suppressing activation of the nuclear factor of activated T cells (NFAT). Therefore, we explored the specific contribution of individual NFAT factors in donor T cells in animal models of GvHD and GvL. Ablation of NFAT1, NFAT2, or a combination of both resulted in ameliorated GvHD, due to reduced proliferation, target tissue homing, and impaired effector function of allogenic donor T cells. In contrast, the frequency of Foxp3(+) regulatory T (Treg) cells was increased and NFAT-deficient Tregs were fully protective in GvHD. CD8(+) T-cell recall response and, importantly, the beneficial antitumor activity were largely preserved in NFAT-deficient effector T cells. Thus, specific inhibition of NFAT opens an avenue for an advanced therapy of GvHD maintaining protective GvL.

Minor histocompatibility (H) antigens are key molecules driving allo-immune responses in both graft-versus-host-disease (GvHD) and in graft-versus-leukemia (GvL) reactivity in human leukocyte antigen (HLA)-matched hematopoietic stem-cell transplantation (HSCT). Dissection of the dual function of minor H antigens became evident through their different modes of tissue and cell expression, i.e. hematopoietic system-restricted or broad. Broadly expressed minor H antigens can cause both GvHD and GvL effects, while hematopoietic system-restricted minor H antigens are more prone to induce GvL responses. This phenomenon renders the latter group of minor H antigens as curative tools for HSCT-based immunotherapy of hematological malignancies and disorders, in which minor H antigen-specific responses are enhanced in order to eradicate the malignant cells. This article describes the immunogenetics of minor H antigens and methods that have been developed to identify them. Moreover, it summarizes the clinical relevance of minor H antigens in transplantation, with special regards to allogeneic HSCT and solid-organ transplantation., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Immunoregulation following allogeneic hematopoietic stem cell transplantation (allo-HSCT) involves the delicate balance between the desirable graft-versus-leukemia (GvL) effect and the prevention of the undesirable graft-versus-host disease (GvHD). Emerging evidence has shown that microRNAs (miRNAs) play a role in the pathogenesis of different inflammatory and malignant diseases. Especially in autoimmune diseases, allergy and GvHD numerous dysregulated miRNAs have been identified. In this review, we provide an overview of current knowledge about the role of miRNAs in the immunoregulation after allo-HSCT. Moreover, we give an outlook on potential new diagnostic and therapeutic approaches, including the use of miRNAs as clinical biomarkers and the manipulation of immune responses using miRNA mimetics., (© Georg Thieme Verlag KG Stuttgart · New York.)

Mixed-lineage leukemia (MLL)/AF4-positive ALL is associated with a poor prognosis even after allogeneic hematopoietic SCT (allo-HSCT). We reported previously that MLL/AF4-positive ALL shows resistance to TNF-α, which is the main factor in the GVL effect, by upregulation of S100A6 expression followed by interference with the p53-caspase 8-caspase 3 pathway in vitro. We examined whether inhibition of S100A6 can induce an effective GVL effect on MLL/AF4-positive ALL in a mouse model. MLL/AF4-positive ALL cell lines (SEM) transduced with lentiviral vectors expressing both S100A6 siRNA and luciferase (SEM-Luc-S100A6 siRNA) were produced. SEM-Luc-S100A6 siRNA cells and SEM-Luc-control siRNA cells were injected into groups of five SCID mice (1 × 10(7)/body). After confirmation of engraftment of SEM cells by in vivo imaging, the mice in each group were injected with 4.8 × 10(7) human PBMCs. SEM-Luc-S100A6 siRNA-injected mice showed significantly longer survival periods than SEM-Luc-control siRNA-injected mice (P=0.002). SEM-Luc-S100A6 siRNA-injected mice showed significantly slower tumor growth than those injected with SEM-Luc-control siRNA (P<0.0001). These results suggested that inhibition of S100A6 may be a promising therapeutic target for MLL/AF4-positive ALL in combination with allo-HSCT.

In allogeneic stem cell transplantation (SCT), natural killer (NK) cells lacking their cognate inhibitory ligand can induce graft-versus-leukemia responses, without the induction of severe graft-versus-host disease (GVHD). This feature can be exploited for cellular immunotherapy. In this study, we examined selective expansion of NK cell subsets expressing distinct killer immunoglobulin-like receptors (KIRs) within the whole human peripheral blood NK cell population, in the presence of HLA-Cw3 (C1) or Cw4 (C2) transfected K562 stimulator cells. Coculture of KIR(+) NK cells with C1 or C2 positive K562 cells, in the presence of IL-2+IL-15, triggered the outgrowth of NK cells that missed their cognate ligand. This resulted in an increased frequency of alloreactive KIR(+) NK cells within the whole NK cell population. Also, after preculture with K562 cells lacking their cognate ligand, we observed that this alloreactive NK population revealed higher numbers of CD107(+) cells when cocultured with the relevant K562 HLA-C transfected target cells, as compared to coculture with untransfected K562 cells. This enhanced reactivity was confirmed using primary leukemic cells as target. This study demonstrates that HLA class I expression can mediate the skewing of the NK cell repertoire and enrich the population for cells with enhanced alloreactivity towards leukemic target cells. This feature may support future clinical applications of NK cell-based immunotherapy.

Since relapse following allogeneic hematopoietic stem cell transplantation (HSCT) can be due to the escape of the residual malignant cells from the graft-versus-leukemia (GvL) effect and given the role of NK cells in GvL and the importance of HLA-E in the modulation of NK cell function, we investigated whether polymorphisms of HLA-E molecule could impact on the incidence of relapse and the improvement of Disease-free Survival (DFS) after allogeneic HSCT. The study group included 56 pairs of donors and patients with malignant hematological disorders undergoing HLA-E matched allogeneic HSCT. The median follow-up was 43.6 (range 20.5-113.1) months. They were genotyped for HLA-E locus using a sequence-specific primer (SSP)-PCR. We found a lower incidence of relapse (p=0.02) in the patients with HLA-E*0103/0103 genotype compared to those with other genotypes of HLA-E. We also showed an association between HLA-E*0103/0103 genotype and a better DFS (p=0.001). Our results suggest a protective role for HLA-E*0103/0103 genotype against relapse and an association between this genotype and an improved DFS following HLA-E matched allogeneic HSCT., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T(CM)) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC-expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC-expanded T cells modified with a suicide gene in the setting of haplo-HSCT.

CD73 functions as an ecto-5'-nucleotidase to produce extracellular adenosine that has anti-inflammatory and immunosuppressive activity. We here demonstrate that CD73 helps control graft-versus-host disease (GVHD) in mouse models. Survival of wild-type (WT) recipients of either allogeneic donor naïve CD73 knock-out (KO) or WT T cells was similar suggesting that donor naïve T cell CD73 did not contribute to GVHD. By contrast, donor CD73 KO CD4(+)CD25(+) regulatory T cells (Treg) had significantly impaired ability to mitigate GVHD mortality compared to WT Treg, suggesting that CD73 on Treg is critical for GVHD protection. However, compared to donor CD73, recipient CD73 is more effective in limiting GVHD. Pharmacological blockade of A2A receptor exacerbated GVHD in WT recipients, but not in CD73 KO recipients, suggesting that A2 receptor signaling is primarily implicated in CD73-mediated GVHD protection. Moreover, pharmacological blockade of CD73 enzymatic activity induced stronger alloreactive T cell activity, worsened GVHD and enhanced the graft-versus-leukemia (GVL) effect. These findings suggest that both donor and recipient CD73 protects against GVHD but also limits GVL effects. Thus, either enhancing or blocking CD73 activity has great potential clinical application in allogeneic bone marrow transplants.

Recent developments in cell processing and immunosuppressive strategies has allowed the safe infusion of high numbers of donor T cells in the context of clinical haploidentical hematopoietic stem cell transplantation (HSCT). Haploidentical T cells display an intrinsic ability to recognize and eliminate residual patient leukemic cells, largely due to alloreactivity against the patient-specific human leukocyte antigen (HLA) molecules encoded on the mismatched haplotype. However, recent evidence has shown that leukemia, like many other tumors displaying pronounced genomic instability, is frequently able to evade this potent graft-versus-leukemia effect by undergoing de novo genomic mutations, which result in the permanent loss of only those HLA molecules targeted by haploidentical donor T-cell alloreactivity. This review summarizes the recent clinical and experimental evidence regarding this phenomenon, and its therapeutic and clinical consequences., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Relapse of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT) leaves few therapeutic options, and mechanisms of immune escape of recurring leukemic cells remain poorly understood. Recently, acquired loss of mismatched human leukocyte antigen (HLA) was demonstrated in patients with AML undergoing haploidentical allogeneic HSCT and was suggested not to occur in HLA-matched HSCT. We hypothesized that this mechanism applies to extramedullary AML relapse which occurs frequently after allogeneic HSCT and might also not be restricted to haploidentical HSCT., Methods: DNA from extramedullary AML relapse after HSCT was compared with bone marrow at diagnosis with array comparative genomic hybridization to investigate relapse-specific genomic aberrations in relapsing AML after allogeneic HSCT. Formalin-fixed, paraffin-embedded tissues from the same points of time were assessed for HLA, major histocompatibility complex class I chain-related gene A, and TAP2 immunohistochemistry staining to assess cell surface expression of deleted loci encoded on chromosome 6p., Results: Array comparative genomic hybridization revealed a partial loss of chromosome 6p in extramedullary myeloid sarcoma relapse of AML after sustained complete remission was achieved through matched related allogeneic HSCT. Among others, a deleted region 6p21.32-p21.33, which included several HLA class I genes, was detected., Conclusions: These results suggest that the loss of HLA class I haplotype also occurs in AML relapse after HLA-matched related HSCT. Partial loss of several HLA class I genes and subsequent reduced presentation of minor histocompatibility antigens and reduced ligation of activating natural killer-cell receptors may explain the loss of graft-versus-leukemia response and extramedullary AML relapse in tissue with reduced immunologic surveillance.

During pregnancy women can develop B- and T-cell immunity against the inherited paternal antigens (IPAs) of the fetus, such as HLA, peptides of minor histocompatibilty antigens, and possibly onco-fetal antigens. The biological and pathological role of these pregnancy-induced immunological events is only understood in part. However, anti-IPA immunity in the mother persists for many decades after delivery and may reduce relapse in offspring with leukemia after HLA-haploidentical transplantation of maternal hematopoietic stem cells (HSC). We hypothesized that maternal anti-IPA immune elements cross the placenta and might confer a potent graft-versus-leukemia effect when cord blood (CB) is used in unrelated HSC transplantation. In a retrospective study of single-unit CB recipients with all grafts provided by the New York Blood Center, we show that patients with acute myeloid or lymphoblastic leukemia (n = 845) who shared one or more HLA-A, -B, or -DRB1 antigens with their CB donor's IPAs had a significant decrease in leukemic relapse posttransplantation [hazard ratio (HR) = 0.38, P < 0.001] compared with those that did not. Remarkably, relapse reduction in patients receiving CB with one HLA mismatch (HR = 0.15, P < 0.001) was not associated with an increased risk of severe acute graft-versus-host disease (HR = 1.43, P = 0.730). Our findings may explain the unexpected low relapse rate after CB transplantation, open new avenues in the study of leukemic relapse after HSC transplantation (possibly of malignancies in general), and have practical implications for CB unit selection.

Accumulating experimental and clinical evidence has been obtained over recent years in support of the notion that the immune system has the potential to cure cancer. The most convincing example is the graft versus leukaemia effect observed after allogeneic haematopoietic stem cell transplantation. In the autologous setting, however, the isolation and expansion of naturally occurring tumour-specific T cells is a challenging task. Cancer antigens are often self-antigens and cancer-specific T cells are thus subject to selective mechanisms of central and peripheral tolerance. The significant advances in gene-transfer technologies developed over the last decade have offered new tools to overcome these limitations. Natural T cells can be genetically modified to generate high numbers of 'supernatural' tumour-reactive T cells from virtually every cancer patient. Supernatural T cells may express clonal receptors providing new specificities, factors increasing T-cell performance or safety factors enabling their elimination in case of toxicity. Technological improvements applied to novel concepts of T-cell biology and oncogenesis will allow to simultaneously equip T cells with different transgenes and expand a real 'army' of lymphocytes trained to selectively eradicate cancer cells., (© 2011 John Wiley & Sons A/S.)

Since the advent of next-generation sequencing (NGS) in 2005, there has been an explosion of published studies employing the technology to tackle previously intractable questions in many disparate biological fields. This has been coupled with technology development that has occurred at a remarkable pace. This review discusses the potential impact of this new technology on the field of blood and marrow stem cell transplantation. Hematologic malignancies have been among the forefront of those cancers whose genomes have been the subject of NGS. Hence, these studies have opened novel areas of biology that can be exploited for prognostic, diagnostic, and therapeutic means. Because of the unprecedented depth, resolution and accuracy achievable by NGS, this technology is well-suited for providing detailed information on the diversity of receptors that govern antigen recognition; this approach has the potential to contribute important insights into understanding the biologic effects of transplantation. Finally, the ability to perform comprehensive tumor sequencing provides a systematic approach to the discovery of genetic alterations that can encode peptides with restricted tumor expression, and hence serve as potential target antigens of graft-versus-leukemia responses. Altogether, this increasingly affordable technology will undoubtedly impact the future practice and care of patients with hematologic malignancies., (Copyright © 2012 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Previously, we have shown that IL-21R(-/-) splenocytes ameliorate GVHD as compared with wild-type splenocytes. Here, we investigated whether or not IL-21R(-/-) splenocytes diminish the graft-versus-leukemia (GVL) effect. Surprisingly, IL-21R(-/-) splenocytes efficiently eliminate leukemic cells as well as wild-type splenocytes, suggesting the retention of GVL effects in the absence of IL-21 signaling. To compare the GVL effect between IL-21R(-/-) and wild-type cells, we titrated the number of splenocytes required for the elimination of leukemic cells and found that the threshold of GVL effect was obtained between 5 × 10(5) and 5 × 10(6) with both types of splenocytes. Cotransplantation with CD8-depleted splenocytes but not with purified CD8 T-cells resulted in a significant reduction in anti-leukemic effect of IL-21R(-/-) cells compared with wild-type cells, suggesting that the lack of IL-21 signaling primarily impairs CD4 T-cell rather than CD8 T-cell function and the comparable GVL effect with IL-21R(-/-) bulk splenocytes results from cooperative compensation by CD8 T-cells.

Gene-engineering of T cells offers the possibility of uniformly changing the characteristics of their immune responses. The ability to direct polyclonal T cells to a single antigenic specificity is a powerful tool with which to probe anti-tumour immune responses, and in turn ask which tumour antigens represent the best targets for immune therapy. The intracellular components of TCR signalling pathways can also be manipulated to optimise anti-tumour responses. Such manipulated T cells may in the future represent an important adjunctive therapy alongside allogeneic haematopoietic stem cell transplantation (ASCT), in order to specifically boost a graft versus leukaemia (GVL) effect. In addition, the ability to confer a suppressive phenotype to CD4 cells or to engineer a susceptibility gene into effector cells provides new therapeutic avenues for graft versus host disease (GVHD), the most challenging adverse effect arising post ASCT. Within this review, mechanisms of GVL and GVHD are discussed and we consider how they may be separated through T cell gene engineering. In addition, we highlight recently investigated safety issues which will impact the future clinical application of gene-manipulated immune cells., (Copyright © 2011. Published by Elsevier Ltd.)

Graft-versus-leukemia (GVL) against chronic-phase chronic myelogenous leukemia (CP-CML) is potent, but it is less efficacious against acute leukemias and blast-crisis chronic myelogenous leukemia (BC-CML). The mechanisms underlying GVL resistance are unknown. Previously, we found that alloreactive T cell targeting of GVL-sensitive bcr-abl-induced mouse CP-CML (mCP-CML) required TCR-MHC interactions and that multiple and redundant killing mechanisms were in play. To better understand why BC-CML is resistant to GVL, we performed a comprehensive analysis of GVL against mouse BC-CML (mBC-CML) induced by the retroviral transfer of the bcr-abl and NUP98/HOXA9 fusion cDNAs. Like human BC-CML, mBC-CML was GVL resistant, and this was not due to accelerated kinetics or a greater leukemia burden. To study T cell recognition and killing mechanisms, we generated a panel of gene-deficient leukemias by transducing bone marrow from gene-deficient mice. T cell target recognition absolutely required that mBC-CML cells express MHC molecules. GVL against both mCP-CML and mBC-CML required leukemia expression of ICAM-1. We hypothesized that mBC-CML would be resistant to some of the killing mechanisms sufficient to eliminate mCP-CML, but we found instead that the same mechanisms were effective against both types of leukemia, because GVL was similar against wild-type or mBC-CML genetically lacking Fas, TRAIL-R, Fas/TRAIL-R, or TNFR1/R2 or when donor T cells were perforin(-/-). However, mCP-CML, but not mBC-CML, relied on expression of programmed death-1 ligands 1 and 2 (PD-L1/L2) to resist T cell killing, because only GVL against mCP-CML was augmented when leukemias lacked PD-L1/L2. Thus, mBC-CML cells have cell-intrinsic mechanisms, distinct from mCP-CML cells, which protect them from T cell killing.

IL-21 is a proinflammatory cytokine produced by Th17 cells. Abrogation of IL-21 signaling has recently been shown to reduce GVHD while retaining graft-versus-leukemia/lymphoma (GVL) responses. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL remain incompletely understood. In a murine MHC-mismatched BM transplantation model, we observed that IL-21 receptor knockout (IL-21R KO) donor T cells mediate decreased systemic and gastrointestinal GVHD in recipients of a transplant. This reduction in GVHD was associated with expansion of transplanted donor regulatory T cells and with tissue-specific modulation of Th-cell function. IL-21R KO and wild-type donor T cells showed equivalent alloactivation, but IL-21R KO T cells showed decreased infiltration and inflammatory cytokine production within the mesenteric lymph nodes. However, Th-cell cytokine production was maintained peripherally, and IL-21R KO T cells mediated equivalent immunity against A20 and P815 hematopoietic tumors. In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T-cell function and GVL capacity are retained. IL-21 is thus an exciting target for therapeutic intervention and improvement of clinical transplantation outcomes.

Background: Minor histocompatibility antigens (mHA) mediate much of the graft vs. leukemia (GvL) effect and graft vs. host disease (GvHD) in patients who undergo allogeneic stem cell transplantation (SCT). Therapeutic decision making and treatments based upon mHAs will require the evaluation of multiple candidate mHAs and the selection of those with the potential to have the greatest impact on clinical outcomes. We hypothesized that common, immunodominant mHAs, which are presented by HLA-A, B, and C molecules, can mediate clinically significant GvL and/or GvHD, and that these mHAs can be identified through association of genomic data with clinical outcomes., Methodology/principal Findings: Because most mHAs result from donor/recipient cSNP disparities, we genotyped 57 myeloid leukemia patients and their donors at 13,917 cSNPs. We correlated the frequency of genetically predicted mHA disparities with clinical evidence of an immune response and then computationally screened all peptides mapping to the highly associated cSNPs for their ability to bind to HLA molecules. As proof-of-concept, we analyzed one predicted antigen, T4A, whose mHA mismatch trended towards improved overall and disease free survival in our cohort. T4A mHA mismatches occurred at the maximum theoretical frequency for any given SCT. T4A-specific CD8+ T lymphocytes (CTLs) were detected in 3 of 4 evaluable post-transplant patients predicted to have a T4A mismatch., Conclusions/significance: Our method is the first to combine clinical outcomes data with genomics and bioinformatics methods to predict and confirm a mHA. Refinement of this method should enable the discovery of clinically relevant mHAs in the majority of transplant patients and possibly lead to novel immunotherapeutics.

The effectiveness of allogeneic graft-versus-leukemia (GVL) activity in control of acute lymphoblastic leukemia is generally regarded as poor. One possible factor is dynamic adaptation of the leukemia cell to the allogeneic environment. This work tested the hypothesis that the pattern of gene expression in acute lymphoblastic leukemia cells in an allogeneic environment would differ from that in a non-allogeneic environment. Expression microarray studies were performed in murine B lineage acute lymphoblastic leukemia cells recovered from mice that had undergone allogeneic MHC-matched but minor histocompatibility antigen mismatched transplants. A limited number of genes were found to be differentially expressed in ALL cells surviving in the allogeneic environment. Functional analysis demonstrated that genes related to immune processes, antigen presentation, ubiquitination and GTPase function were significantly enriched. Several genes with known immune activities potentially relevant to leukemia survival (Ly6a/Sca-1, TRAIL and H2-T23) were examined in independent validation experiments. Increased expression in vivo in allogeneic hosts was observed, and could be mimicked in vitro with soluble supernatants of mixed lymphocyte reactions or interferon-gamma. The changes in gene expression were reversible when the leukemia cells were removed from the allogeneic environment. These findings suggest that acute lymphoblastic leukemia cells respond to cytokines present after allogeneic transplantation and that these changes may reduce the effectiveness of GVL activity.

Allogeneic stem cell transplantation is the most potent form of effective adoptive immunotherapy. The graft-versus-leukemia (GVL) effect mediated by the allogeneic graft, however, is typically coexpressed with graft-versus-host disease (GVHD), which is the major complication of allogeneic stem cell transplantation. In this study, we used genetic and antibody-based strategies to examine the effect that blockade of interleukin 23 (IL-23) signaling had on GVH and GVL reactivity in murine transplantation recipients. These studies demonstrate that the selective protection of the colon that occurs as a consequence of inhibition of IL-23 signaling reduces GVHD without loss of the GVL effect. The separation of GVH and GVL reactivity was noted in both acute and chronic hematologic malignancy models, indicating that this approach was not restricted by the kinetic profile of the underlying leukemia. Furthermore, a potent GVL response could be mounted in the colon under conditions where tumor cells migrated to this site, indicating that this organ did not serve as a sanctuary site for subsequent systemic relapse in GVHD-protected animals. These studies demonstrate that blockade of IL-23 signaling is an effective strategy for separating GVH and GVL responses and identify IL-23 as a therapeutic target for the regulation of alloresponses in humans.