Current Concepts and Advances in Graft-Versus-Host Disease Immunology.

Worldwide, each year over 30,000 patients undergo an allogeneic hema-topoietic stem cell transplantation with the intent to cure high-risk hematologic malignancy, immunodeficiency, metabolic disease, or a life-threatening bone marrow failure syndrome. Despite substantial advances in donor selection and conditioning regimens and greater availability of allograft sources, transplant recipients still endure the morbidity and mortality of graft-versus-host disease (GVHD). Herein, we identify key aspects of acute and chronic GVHD pathophysiology, including host/donor cell effectors, gut dysbiosis, immune system and cytokine imbalance, and the interface between inflammation and tissue fibrosis. In particular, we also summarize the translational application of this heightened understanding of immune dysregulation in the design of novel therapies to prevent and treat GVHD.

Post-Transplantation Cyclophosphamide-Based Graft-versus-Host Disease Prophylaxis.

BACKGROUND: In patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT), a calcineurin inhibitor plus methotrexate has been a standard prophylaxis against graft-versus-host disease (GVHD). A phase 2 study indicated the potential superiority of a post-transplantation regimen of cyclophosphamide, tacrolimus, and mycophenolate mofetil. METHODS: In a phase 3 trial, we randomly assigned adults with hematologic cancers in a 1:1 ratio to receive cyclophosphamide-tacrolimus-mycophenolate mofetil (experimental prophylaxis) or tacrolimus-methotrexate (standard prophylaxis). The patients underwent HSCT from an HLA-matched related donor or a matched or 7/8 mismatched (i.e., mismatched at only one of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci) unrelated donor, after reduced-intensity conditioning. The primary end point was GVHD-free, relapse-free survival at 1 year, assessed in a time-to-event analysis, with events defined as grade III or IV acute GVHD, chronic GVHD warranting systemic immunosuppression, disease relapse or progression, and death from any cause. RESULTS: In a multivariate Cox regression analysis, GVHD-free, relapse-free survival was significantly more common among the 214 patients in the experimental-prophylaxis group than among the 217 patients in the standard-prophylaxis group (hazard ratio for grade III or IV acute GVHD, chronic GVHD, disease relapse or progression, or death, 0.64; 95% confidence interval [CI], 0.49 to 0.83; P = 0.001). At 1 year, the adjusted GVHD-free, relapse-free survival was 52.7% (95% CI, 45.8 to 59.2) with experimental prophylaxis and 34.9% (95% CI, 28.6 to 41.3) with standard prophylaxis. Patients in the experimental-prophylaxis group appeared to have less severe acute or chronic GVHD and a higher incidence of immunosuppression-free survival at 1 year. Overall and disease-free survival, relapse, transplantation-related death, and engraftment did not differ substantially between the groups. CONCLUSIONS: Among patients undergoing allogeneic HLA-matched HSCT with reduced-intensity conditioning, GVHD-free, relapse-free survival at 1 year was significantly more common among those who received cyclophosphamide-tacrolimus-mycophenolate mofetil than among those who received tacrolimus-methotrexate. (Funded by the National Heart, Lung, and Blood Institute and others; BMT CTN 1703 ClinicalTrials.gov number, NCT03959241.).

Alloengraftment without significant toxicity or GVHD in CD45 antibody-drug conjugate-conditioned Fanconi anemia mice.

ABSTRACT: Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage-Sca1+cKit+ cells within 48 hours. Fanca-/- recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc-/- and Fancg-/- recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca-/- recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.

B-cell-directed CAR-T cell therapy activates CD8+ cytotoxic CARneg bystander T-cells in non-human primates and patients.

CAR-T cells hold promise as a therapy for B-cell-derived malignancies, yet despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. While recent studies have explored the mechanisms of in vivo CAR-T cell function, little is understood about the activation of surrounding CARneg bystander T-cells and their potential to enhance tumor responses. We performed single-cell RNA-Seq (scRNA-Seq) on non-human primate (NHP) and patient-derived T-cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T-cells following B-cell targeted CAR-T cell therapy. Utilizing a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T-cells emerging during CAR-T cell expansion. These bystander CD8+ CARneg T-cells exhibited a unique transcriptional signature with upregulation of NK-cell markers (KIR3DL2, CD160, KLRD1), chemokines and chemokine receptors (CCL5, XCL1, CCR9), and downregulation of naive T-cell-associated genes (SELL, CD28). A transcriptionally similar population was identified in patients following Tisagenlecleucel infusion. Mechanistic studies revealed that IL-2 and IL-15 exposure induced bystander-like CD8+ T-cells in a dose dependent manner. In vitro activated and patient-derived T-cells with the bystander phenotype efficiently killed leukemic cells through a TCR-independent mechanism. Collectively, this dataset provides the first comprehensive identification and profiling of CARneg bystander CD8+ T-cells following B-cell targeting CAR-T cell therapy and suggests a novel mechanism through which CAR-T cell infusion might trigger enhanced anti-leukemic responses.

Author Correction: Innovations present in the primate interneuron repertoire.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Engineering T cells to suppress acute GVHD and leukemia relapse after allogeneic hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) limits the therapeutic benefit of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and requires immunosuppressive prophylaxis that compromises antitumor and antipathogen immunity. OX40 is a costimulatory receptor upregulated on circulating T cells in aGVHD and plays a central role in driving the expansion of alloreactive T cells. Here, we show that OX40 is also upregulated on T cells infiltrating GVHD target organs in a rhesus macaque model, supporting the hypothesis that targeted ablation of OX40+ T cells will mitigate GVHD pathogenesis. We thus created an OX40-specific cytotoxic receptor that, when expressed on human T cells, enables selective elimination of OX40+ T cells. Because OX40 is primarily upregulated on CD4+ T cells upon activation, engineered OX40-specific T cells mediated potent cytotoxicity against activated CD4+ T cells and suppressed alloreactive T-cell expansion in a mixed lymphocyte reaction model. OX40 targeting did not inhibit antiviral activity of memory T cells specific to Epstein-Barr virus, cytomegalovirus, and adenoviral antigens. Systemic administration of OX40-targeting T cells fully protected mice from fatal xenogeneic GVHD mediated by human peripheral blood mononuclear cells. Furthermore, combining OX40 targeting with a leukemia-specific chimeric antigen receptor in a single T cell product provides simultaneous protection against leukemia and aGVHD in a mouse xenograft model of residual disease posttransplant. These results underscore the central role of OX40+ T cells in mediating aGVHD pathogenesis and support the feasibility of a bifunctional engineered T-cell product derived from the stem cell donor to suppress both disease relapse and aGVHD following allo-HSCT.

Higher abatacept exposure after transplant decreases acute GVHD risk without increasing adverse events.

In the ABA2 study, the T-cell costimulation blockade agent, abatacept, was safe and effective in preventing acute graft-versus-host disease (aGVHD) after unrelated-donor hematopoietic cell transplant (HCT), leading to US Food and Drug Administration approval. Here, we performed a determination of abatacept pharmacokinetics (PK), which enabled an examination of how abatacept exposure-response relationships affected clinical outcomes. We performed a population PK analysis of IV abatacept using nonlinear mixed-effect modeling and assessed the association between abatacept exposure and key transplant outcomes. We tested the association between the trough after dose 1 (Ctrough_1) and grade (GR) 2 or 4 aGVHD (GR2-4 aGVHD) through day +100. An optimal Ctrough_1 threshold was identified via recursive partitioning and classification tree analysis. This demonstrated that abatacept PK was characterized by a 2-compartment model with first-order elimination. The ABA2 dosing regimen was based on previous work targeting a steady-state abatacept trough of 10 µg/mL. However, a higher Ctrough_1 (≥39 µg/mL, attained in ∼60% of patients on ABA2) was associated with a favorable GR2-4 aGVHD risk (hazard ratio, 0.35; 95% confidence interval, 0.19-0.65; P < .001), with a Ctrough_1 <39 µg/mL associated with GR2-4 aGVHD risk indistinguishable from placebo (P = .37). Importantly, no significant association was found between Ctrough_1 and key safety indicators, including relapse, and cytomegalovirus or Epstein-Barr virus viremia. These data demonstrate that a higher abatacept Ctrough_1 (≥39 µg/mL) was associated with a favorable GR2-4 aGVHD risk, without any observed exposure-toxicity relationships. This trial was registered at www.clinicaltrials.gov as #NCT01743131.

BET-bromodomain and EZH2 inhibitor-treated chronic GVHD mice have blunted germinal centers with distinct transcriptomes.

Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.

Enforced gut homing of murine regulatory T cells reduces early graft-versus-host disease severity.

Damage to the gastrointestinal tract following allogeneic hematopoietic stem cell transplantation is a significant contributor to the severity and perpetuation of graft-versus-host disease. In preclinical models and clinical trials, we showed that infusing high numbers of regulatory T cells reduces graft-versus-host disease incidence. Despite no change in in vitro suppressive function, transfer of ex vivo expanded regulatory T cells transduced to overexpress G protein-coupled receptor 15 or C-C motif chemokine receptor 9, specific homing receptors for colon or small intestine, respectively, lessened graft-versus-host disease severity in mice. Increased regulatory T cell frequency and retention within the gastrointestinal tissues of mice that received gut homing T cells correlated with lower inflammation and gut damage early post-transplant, decreased graft-versus-host disease severity, and prolonged survival compared with those receiving control transduced regulatory T cells. These data provide evidence that enforced targeting of ex vivo expanded regulatory T cells to the gastrointestinal tract diminishes gut injury and is associated with decreased graft-versus-host disease severity.

Repurposing a novel anti-cancer RXR agonist to attenuate murine acute GVHD and maintain graft-versus-leukemia responses.

The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic.

Robust expansion of HIV CAR T cells following antigen boosting in ART-suppressed nonhuman primates.

Chimeric antigen receptor (CAR) T cells targeting CD19+ hematologic malignancies have rapidly emerged as a promising, novel therapy. In contrast, results from the few CAR T-cell studies for infectious diseases such as HIV-1 have been less convincing. These challenges are likely due to the low level of antigen present in antiretroviral therapy (ART)-suppressed patients in contrast to those with hematologic malignancies. Using our well-established nonhuman primate model of ART-suppressed HIV-1 infection, we tested strategies to overcome these limitations and challenges. We first optimized CAR T-cell production to maintain central memory subsets, consistent with current clinical paradigms. We hypothesized that additional exogenous antigen might be required in an ART-suppressed setting to aid expansion and persistence of CAR T cells. Thus, we studied 4 simian/HIV-infected, ART-suppressed rhesus macaques infused with virus-specific CD4CAR T cells, followed by supplemental infusion of cell-associated HIV-1 envelope (Env). Env boosting led to significant and unprecedented expansion of virus-specific CAR+ T cells in vivo; after ART treatment interruption, viral rebound was significantly delayed compared with controls (P = .014). In 2 animals with declining CAR T cells, rhesusized anti-programmed cell death protein 1 (PD-1) antibody was administered to reverse PD-1-dependent immune exhaustion. Immune checkpoint blockade triggered expansion of exhausted CAR T cells and concordantly lowered viral loads to undetectable levels. These results show that supplemental cell-associated antigen enables robust expansion of CAR T cells in an antigen-sparse environment. To our knowledge, this is the first study to show expansion of virus-specific CAR T cells in infected, suppressed hosts, and delay/control of viral recrudescence.

Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion.

BACKGROUND AIMS: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of 'off the shelf' Treg. RESULTS: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. DISCUSSION: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile-desirable properties that support the possibility of off-the-shelf Treg therapeutics.

Advances in targeting co-inhibitory and co-stimulatory pathways in transplantation settings: the Yin to the Yang of cancer immunotherapy.

In the past decade, the power of harnessing T-cell co-signaling pathways has become increasingly understood to have significant clinical importance. In cancer immunotherapy, the field has concentrated on two related modalities: First, targeting cancer antigens through highly activated chimeric antigen T cells (CAR-Ts) and second, re-animating endogenous quiescent T cells through checkpoint blockade. In each of these strategies, the therapeutic goal is to re-ignite T-cell immunity, in order to eradicate tumors. In transplantation, there is also great interest in targeting T-cell co-signaling, but with the opposite goal: in this field, we seek the Yin to cancer immunotherapy's Yang, and focus on manipulating T-cell co-signaling to induce tolerance rather than activation. In this review, we discuss the major T-cell signaling pathways that are being investigated for tolerance induction, detailing preclinical studies and the path to the clinic for many of these molecules. These include blockade of co-stimulation pathways and agonism of coinhibitory pathways, in order to achieve the delicate state of balance that is transplant tolerance: a state which guarantees lifelong transplant acceptance without ongoing immunosuppression, and with preservation of protective immune responses. In the context of the clinical translation of immune tolerance strategies, we discuss the significant challenge that is embodied by the fact that targeted pathway modulators may have opposing effects on tolerance based on their impact on effector vs regulatory T-cell biology. Achieving this delicate balance holds the key to the major challenge of transplantation: lifelong control of alloreactivity while maintaining an otherwise intact immune system.

Defining success with cellular therapeutics: the current landscape for clinical end point and toxicity analysis.

Cellular therapies play a major and expanding role in the treatment of hematologic diseases. For each of these therapies, a narrow therapeutic window exists, where efficacy is maximized and toxicities minimized. This review focuses on one of the most established cellular therapies, hematopoietic stem cell transplant, and one of the newest cellular therapies, chimeric antigen receptor-T cells. In this review, I will discuss the current state of the field for clinical end point analysis with each of these therapeutics, including their critical toxicities, and focus on the major elements of success for each of these complex treatments for hematologic disease.

Busulfan Combined with Immunosuppression Allows Efficient Engraftment of Gene-Modified Cells in a Rhesus Macaque Model.

Busulfan conditioning is utilized for hematopoietic stem cell (HSC) depletion in the context of HSC gene-therapy conditioning but may result in insufficient immunosuppression. In this study, we evaluated whether additional immunosuppression is required for efficient engraftment of gene-modified cells using a rhesus HSC lentiviral gene-therapy model. We transduced half of rhesus CD34+ cells with an enhanced green fluorescent protein (GFP)-encoding vector (immunogenic) and the other half with a γ-globin-encoding vector (no predicted immunogenicity). After autologous transplantation of both transduced cell populations following myeloablative busulfan conditioning (5.5 mg/kg/day for 4 days), we observed immunological rejection of GFP-transduced cells up to 3 months post-transplant and stable engraftment of γ-globin-transduced cells in two animals, demonstrating that ablative busulfan conditioning is sufficient for engraftment of gene-modified cells producing non-immunogenic proteins but insufficient to permit engraftment of immunogenic proteins. We then added immunosuppression with abatacept and sirolimus to busulfan conditioning and observed engraftment of both GFP- and γ-globin-transduced cells in two animals, demonstrating that additional immunosuppression allows for engraftment of gene-modified cells expressing immunogenic proteins. In conclusion, myeloablative busulfan conditioning should permit engraftment of gene-modified cells producing non-immunogenic proteins, while additional immunosuppression is required to prevent immunological rejection of a neoantigen.

Human CD19-Targeted Mouse T Cells Induce B Cell Aplasia and Toxicity in Human CD19 Transgenic Mice.

The clinical success of chimeric antigen receptor (CAR) T cell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR T cells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR T cells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T cells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have ∼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR T cells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR T cell safety.

Building a Safer and Faster CAR: Seatbelts, Airbags, and CRISPR.

Therapeutic T cell engineering has recently garnered widespread interest because of the success of CD19 chimeric antigen receptor (CAR) therapy. CARs are synthetic receptors for antigen that redirect the specificity and reprogram the function of the T cells in which they are genetically introduced. CARs targeting CD19, a cell surface molecule found in most leukemias and lymphomas, have yielded high remission rates in patients with chemorefractory, relapsed disease, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. The toxicities of this treatment include B cell aplasia, cytokine release syndrome (CRS), and neurotoxicity. Although reversible in most instances, these toxicities may require specific medical interventions, including transfer to intensive care to treat severe CRS. Guidelines for managing these toxicities are emerging. The recent report of a nonhuman primate model for CRS is poised to help advance the management of this syndrome. Finally, new engineering modalities, based on the use of targeted nucleases like CRISPR, may further enhance the efficacy and safety of CAR T cells.

Reprint of: Building a Safer and Faster CAR: Seatbelts, Airbags, and CRISPR.

Therapeutic T cell engineering has recently garnered widespread interest because of the success of CD19 chimeric antigen receptor (CAR) therapy. CARs are synthetic receptors for antigen that redirect the specificity and reprogram the function of the T cells in which they are genetically introduced. CARs targeting CD19, a cell surface molecule found in most leukemias and lymphomas, have yielded high remission rates in patients with chemorefractory, relapsed disease, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. The toxicities of this treatment include B cell aplasia, cytokine release syndrome (CRS), and neurotoxicity. Although reversible in most instances, these toxicities may require specific medical interventions, including transfer to intensive care to treat severe CRS. Guidelines for managing these toxicities are emerging. The recent report of a nonhuman primate model for CRS is poised to help advance the management of this syndrome. Finally, new engineering modalities, based on the use of targeted nucleases like CRISPR, may further enhance the efficacy and safety of CAR T cells.

Systems analysis uncovers inflammatory Th/Tc17-driven modules during acute GVHD in monkey and human T cells.

One of the central challenges of transplantation is the development of alloreactivity despite the use of multiagent immunoprophylaxis. Effective control of this immune suppression-resistant T-cell activation represents one of the key unmet needs in the fields of both solid-organ and hematopoietic stem cell transplant (HCT). To address this unmet need, we have used a highly translational nonhuman primate (NHP) model to interrogate the transcriptional signature of T cells during breakthrough acute graft-versus-host disease (GVHD) that occurs in the setting of clinically relevant immune suppression and compared this to the hyperacute GVHD, which develops in unprophylaxed or suboptimally prophylaxed transplant recipients. Our results demonstrate the complex character of the alloreactivity that develops during ongoing immunoprophylaxis and identify 3 key transcriptional hallmarks of breakthrough acute GVHD that are not observed in hyperacute GVHD: (1) T-cell persistence rather than proliferation, (2) evidence for highly inflammatory transcriptional programming, and (3) skewing toward a T helper (Th)/T cytotoxic (Tc)17 transcriptional program. Importantly, the gene coexpression profiles from human HCT recipients who developed GVHD while on immunosuppressive prophylactic agents recapitulated the patterns observed in NHP, and demonstrated an evolution toward a more inflammatory signature as time posttransplant progressed. These results strongly implicate the evolution of both inflammatory and interleukin 17-based immune pathogenesis in GVHD, and provide the first map of this evolving process in primates in the setting of clinically relevant immunomodulation. This map represents a novel transcriptomic resource for further systems-based efforts to study the breakthrough alloresponse that occurs posttransplant despite immunoprophylaxis and to develop evidence-based strategies for effective treatment of this disease.

CMV reactivation drives posttransplant T-cell reconstitution and results in defects in the underlying TCRß repertoire.

Although cytomegalovirus (CMV) reactivation has long been implicated in posttransplant immune dysfunction, the molecular mechanisms that drive this phenomenon remain undetermined. To address this, we combined multiparameter flow cytometric analysis and T-cell subpopulation sorting with high-throughput sequencing of the T-cell repertoire, to produce a thorough evaluation of the impact of CMV reactivation on T-cell reconstitution after unrelated-donor hematopoietic stem cell transplant. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme B(high)/CD28(low)/CD57(high)/CD8(+) effector memory T cells (Tem) and resulted in a linked contraction of all naive T cells, including CD31(+)/CD4(+) putative thymic emigrants. T-cell receptor ß (TCRß) deep sequencing revealed a striking contraction of CD8(+) Tem diversity due to CMV-specific clonal expansions in reactivating patients. In addition to querying the topography of the expanding CMV-specific T-cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the underlying TCR repertoire. Our results reveal new evidence for significant defects in the underlying CD8 Tem TCR repertoire in patients who reactivate CMV, providing the first molecular evidence that, in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T-cell repertoire. This trial was registered at www.clinicaltrials.gov as #NCT01012492.