Kras-Deficient T Cells Attenuate Graft-versus-Host Disease but Retain Graft-versus-Leukemia Activity.

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.

IL-6 dysregulation originates in dendritic cells and mediates graft-versus-host disease via classical signaling.

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.

Targeting JAK2 reduces GVHD and xenograft rejection through regulation of T cell differentiation.

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).

The Ex Vivo Treatment of Donor T Cells with Cosalane, an HIV Therapeutic and Small-Molecule Antagonist of CC-Chemokine Receptor 7, Separates Acute Graft-versus-Host Disease from Graft-versus-Leukemia Responses in Murine Hematopoietic Stem Cell Transplantation Models.

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 (Tcons) 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 Tcons with cosalane ex vivo before transplantation. Cosalane did not result in Tcon toxicity and did not affect their activation or expansion. Instead, cosalane prevented donor Tcon 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 Tcons to produce inflammatory cytokines. Furthermore, cosalane-treated Tcons 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 Tcons 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.

Serum miR-29a Is Upregulated in Acute Graft-versus-Host Disease and Activates Dendritic Cells through TLR Binding.

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.

T-bet Promotes Acute Graft-versus-Host Disease by Regulating Recipient Hematopoietic Cells in Mice.

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.

Selective NFAT targeting in T cells ameliorates GvHD while maintaining antitumor activity.

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.

MicroRNA-17-92 controls T-cell responses in graft-versus-host disease and leukemia relapse in mice.

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.

KIR and HLA genotypes predictive of low-affinity interactions are associated with lower relapse in autologous hematopoietic cell transplantation for acute myeloid leukemia.

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.

Regulation of pulmonary graft-versus-host disease by IL-26+CD26+CD4 T lymphocytes.

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.

Indirect evidence that maternal microchimerism in cord blood mediates a graft-versus-leukemia effect in cord blood transplantation.

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.

Alloreactive killer cells: hindrance and help for haematopoietic transplants.

Haematopoietic-cell transplantation is a treatment for leukaemia and lymphoma. To reduce the incidence of graft-versus-host disease (GVHD) caused by transplanted T cells, donors and recipients are HLA matched. For patients for whom a matched donor is not available, one option is transplantation from an HLA-mismatched relative who shares one HLA haplotype. This procedure is distinguished by the use of a stronger conditioning regimen for the patient and of a T-cell-depleted graft containing numerous stem cells. After transplantation, natural killer cells are prevalent, and they can include alloreactive cells that kill tumour cells and prevent GVHD. The alloreactions seem to be determined by the mismatched HLA class I ligands and their killer-cell immunoglobulin-like receptors.

Minor histocompatibility antigens: past, present, and future.

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.

Graft-versus-leukemia effect of HLA-haploidentical central-memory T-cells expanded with leukemic APCs and modified with a suicide gene.

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.

Oncogene-induced TIM-3 ligand expression dictates susceptibility to anti-TIM-3 therapy in mice.

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.

Abrogation of donor T-cell IL-21 signaling leads to tissue-specific modulation of immunity and separation of GVHD from GVL.

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.

Graft-versus-leukemia (GVL) against mouse blast-crisis chronic myelogenous leukemia (BC-CML) and chronic-phase chronic myelogenous leukemia (CP-CML): shared mechanisms of T cell killing, but programmed death ligands render CP-CML and not BC-CML GVL resistant.

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.

Donor T cell STAT3 deficiency enables tissue PD-L1-dependent prevention of graft-versus-host disease while preserving graft-versus-leukemia activity.

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.

Applications of next-generation sequencing to blood and marrow transplantation.

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.

Loss of mismatched HLA in leukemia after stem-cell transplantation.

BACKGROUND: Transplantation of hematopoietic stem cells from partially matched family donors is a promising therapy for patients who have a hematologic cancer and are at high risk for relapse. The donor T-cell infusions associated with such transplantation can promote post-transplantation immune reconstitution and control residual disease. METHODS: We identified 43 patients who underwent haploidentical transplantation and infusion of donor T cells for acute myeloid leukemia or myelodysplastic syndrome and conducted post-transplantation studies that included morphologic examination of bone marrow, assessment of hematopoietic chimerism with the use of short-tandem-repeat amplification, and HLA typing. The genomic rearrangements in mutant variants of leukemia were studied with the use of genomic HLA typing, microsatellite mapping, and single-nucleotide-polymorphism arrays. The post-transplantation immune responses against the original cells and the mutated leukemic cells were analyzed with the use of mixed lymphocyte cultures. RESULTS: In 5 of 17 patients with leukemia relapse after haploidentical transplantation and infusion of donor T cells, we identified mutant variants of the original leukemic cells. In the mutant leukemic cells, the HLA haplotype that differed from the donor's haplotype had been lost because of acquired uniparental disomy of chromosome 6p. T cells from the donor and the patient after transplantation did not recognize the mutant leukemic cells, whereas the original leukemic cells taken at the time of diagnosis were efficiently recognized and killed. CONCLUSIONS: After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donor's antileukemic T cells through the loss of the mismatched HLA haplotype. This event leads to relapse.