Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.

Red cell exchange for rapid leukoreduction in adults with hyperleukocytosis and leukostasis.

ABSTRACT: We show that red cell exchange (RCE) treats hyperleukocytosis in acute leukemia. RCE provided similar leukoreduction to standard therapeutic leukoreduction and could be superior in patients with severe anemia or monocytic leukemias or when requiring rapid treatment.

On the road to eliminating long-lived plasma cells-"are we there yet?"

Central to protective humoral immunity is the activation of B cells and their terminal differentiation into antibody-secreting plasma cells. Long-lived plasma cells (LLPC) may survive for years to decades. Such long-lived plasma cells are also responsible for producing pathogenic antibodies that cause a variety of challenges such as autoimmunity, allograft rejection, and drug neutralization. Up to now, various therapeutic strategies aimed at durably eliminating pathogenic antibodies have failed, in large part due to their inability to efficiently target LLPCs. Several antibody-based therapies have recently gained regulatory approval or are in clinical phases of development for the treatment of multiple myeloma, a malignancy of plasma cells. We discuss the exciting potential of using these emerging cancer immunotherapies to solve the antibody problem.

Therapeutic Opportunities for Immunoreceptor-Engineered T Cell Therapy for Modulation of Alloimmunity.

Achieving immunosuppression-free immune tolerance to an allograft is one of the central goals of transplantation. In this article, we review recent developments in the fields of T cell-based therapies and T cell engineering using chimeric Ag receptors and their potential for effective and targeted immune modulation of T and B cell activity in an effort to eliminate pre-existing alloantibodies (desensitization) and achieve long-term tolerance. Approaches that span preclinical to early clinical studies in transplantation will be reviewed, with specific emphasis on advances in T cell immunotherapy that have shown promise. Lastly, we conclude with a forward-looking discussion of how T cell-based therapies in other fields of medicine can be potentially applied to solid organ transplantation.

Linking retroelements to autoimmunity.

In this issue, Stetson et al. (2008) report a mechanism by which host cells avert an autoimmune response to self-nucleic acids. They show that the nuclease Trex1 prevents the accumulation of DNA derived from endogenous retroelements that, if left unchecked, trigger elevated production of type I interferons leading to autoimmunity.

Conventional T cell therapies pave the way for novel Treg therapeutics.

Approaches to harness the immune system to alleviate disease have become remarkably sophisticated since the crude, yet impressively-effective, attempts using live bacteria in the late 1800s. Recent evidence that engineered T cell therapy can deliver durable results in patients with cancer has spurred frenzied development in the field of T cell therapy. The myriad approaches include an innumerable variety of synthetic transgenes, multiplex gene-editing, and broader application to diseases beyond cancer. In this article, we review the preclinical studies and over a decade of clinical experience with engineered conventional T cells that have paved the way for translating engineered regulatory T cell therapies.

Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy.

The mechanisms underlying the maintenance of long-lasting humoral immunity are not well understood. Studies in mice indicate that plasma cells (PCs) can survive up to a lifetime, even in the absence of regeneration by B cells, implying the presence of long-lived PCs as a mechanism for long-lasting immunity. Evidence from humans treated with anti-CD20, which depletes circulating B cells, also suggests B-cell-independent long-term survival of some PCs. On the other hand, antibody responses may be sustained solely by short-lived PCs with repopulation from clonally related memory B cells. To explore PC longevity and humoral immunity in humans, we investigated the fate of PCs and their antibodies in adult and pediatric patients who received chimeric antigen receptor-based adoptive T-cell immunotherapy targeting CD19 to treat B-cell lineage malignancies (CTL019). Treatment with CTL019 is frequently associated with B-cell aplasia that can persist for years. Serum antibody titers to vaccine-related antigens were measured, and quantitative assessment of B cells and PCs in blood and bone marrow was performed at various time points before and after CTL019 therapy. While total serum immunoglobulin concentrations decline following CTL019-induced B-cell aplasia, several vaccine/pathogen-specific serum immunoglobulin G and A (IgG and IgA) titers remain relatively stable for at least 6 and 12 months posttreatment, respectively. Analysis of bone marrow biopsies after CTL019 revealed 8 patients with persistence of antibody-secreting PCs at least 25 months post-CTL019 infusion despite absence of CD19(+)CD20(+) B cells. These results provide strong evidence for the existence of memory B-cell-independent, long-lived PCs in humans that contribute to long-lasting humoral immunity.

The thrombotic microangiopathy Registry of North America: A United States multi-institutional TMA network.

The thrombotic microangiopathy (TMA) Registry Network of North America (TRNA) is a collaborative network organized for the purpose of developing a multi-institutional registry and network to conduct clinical studies in a rare patient population. The TRNA was founded in 2013 by four academic medical centers (Columbia University Medical Center, Duke University Medical Center, University of Alabama at Birmingham, and University of Pennsylvania) to develop a national and demographically diverse dataset of patients with TMA. A clinical database was developed by network members using REDCap (Research Electronic Data Capture), a web-based database developed for clinical research. To facilitate rapid Institutional Review Board (IRB) approval at multiple sites, the TRNA utilized IRBshare, a streamlined IRB process to allow patient recruitment and enrollment into the TMA registry. This article reviews the process used to establish the TRNA network and discusses the significance of the first multi-institutional clinical apheresis network developed in the United States. J. Clin. Apheresis 31:448-453, 2016. © 2015 Wiley Periodicals, Inc.

Lipoprotein apheresis.

Lipoprotein apheresis is an extracorporeal therapy whereby apo B-containing lipoproteins (LDL, vLDL, and Lp(a)) are selectively removed from the plasma of a patient. It is a variation of plasma exchange, which non-selectively removes all plasma proteins. Lipoprotein apheresis is most commonly used to treat patients with familial hypercholesterolemia, but is also often used for other hyperlipidemic patients, including those with elevated Lp(a), to limit and/or reverse the atherosclerotic complications of high cholesterol. Herein, we provide an overview of lipoprotein apheresis including indications, techniques, efficacy, and adverse events, as well as guidelines and other considerations important for selection of patients for this therapy.

Ubiquitylation in innate and adaptive immunity.

Protein ubiquitylation has emerged as a key mechanism that regulates immune responses. Much like phosphorylation, ubiquitylation is a reversible covalent modification that regulates the stability, activity and localization of target proteins. As such, ubiquitylation regulates the development of the immune system and many phases of the immune response, including its initiation, propagation and termination. Recent work has shown that several ubiquitin ligases help to prevent the immune system from attacking self tissues. The dysfunction of several ubiquitin ligases has been linked to autoimmune diseases.

Making inroads to precision medicine for the treatment of autoimmune diseases: Harnessing genomic studies to better diagnose and treat complex disorders.

Precision Medicine is an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle. Autoimmune diseases are those in which the body's natural defense system loses discriminating power between its own cells and foreign cells, causing the body to mistakenly attack healthy tissues. These conditions are very heterogeneous in their presentation and therefore difficult to diagnose and treat. Achieving precision medicine in autoimmune diseases has been challenging due to the complex etiologies of these conditions, involving an interplay between genetic, epigenetic, and environmental factors. However, recent technological and computational advances in molecular profiling have helped identify patient subtypes and molecular pathways which can be used to improve diagnostics and therapeutics. This review discusses the current understanding of the disease mechanisms, heterogeneity, and pathogenic autoantigens in autoimmune diseases gained from genomic and transcriptomic studies and highlights how these findings can be applied to better understand disease heterogeneity in the context of disease diagnostics and therapeutics.

Immunotherapy targeting B cells and long-lived plasma cells effectively eliminates pre-existing donor-specific allo-antibodies.

Pre-existing anti-human leukocyte antigen (HLA) allo-antibodies constitute a major barrier to transplantation. Current desensitization approaches fail due to ineffective depletion of allo-specific memory B cells (Bmems) and long-lived plasma cells (LLPCs). We evaluate the efficacy of chimeric antigen receptor (CAR) T cells targeting CD19 and B cell maturation antigen (BCMA) to eliminate allo-antibodies in a skin pre-sensitized murine model of islet allo-transplantation. We find that treatment of allo-sensitized hosts with CAR T cells targeting Bmems and LLPCs eliminates donor-specific allo-antibodies (DSAs) and mitigates hyperacute rejection of subsequent islet allografts. We then assess the clinical efficacy of the CAR T therapy for desensitization in patients with multiple myeloma (MM) with pre-existing HLA allo-antibodies who were treated with the combination of CART-BCMA and CART-19 (ClinicalTrials.gov: NCT03549442) and observe clinically meaningful allo-antibody reduction. These findings provide logical rationale for clinical evaluation of CAR T-based immunotherapy in highly sensitized candidates to promote successful transplantation.

MAVS and MyD88 are essential for innate immunity but not cytotoxic T lymphocyte response against respiratory syncytial virus.

Infection by RNA viruses is detected by the host through Toll-like receptors or RIG-I-like receptors. Toll-like receptors and RIG-I-like receptors signal through the adaptors MyD88 and MAVS, respectively, to induce type I IFNs (IFN-I) and other antiviral molecules, which are thought to be essential for activating the adaptive immune system. We investigated the role of these adaptors in innate and adaptive immune responses against respiratory syncytial virus (RSV), a common human pathogen. Deletion of Mavs abolished the induction of IFN-I and other proinflammatory cytokines by RSV. Genome-wide expression profiling in the lung showed that the vast majority of RSV-induced genes depended on MAVS. Although Myd88 deficiency did not affect most RSV-induced genes, mice lacking both adaptors harbored a higher and more prolonged viral load and exhibited more severe pulmonary disease than those lacking either adaptor alone. Surprisingly, Myd88(-/-)Mavs(-/-) mice were able to activate a subset of pulmonary dendritic cells that traffic to the draining lymph node in response to RSV. These mice subsequently mounted a normal cytotoxic T-lymphocyte response and demonstrated delayed but effective viral clearance. These results provide an example of a normal and effective adaptive immune response in the absence of innate immunity mediated by MAVS and MyD88.

Absolute lymphocyte count proliferation kinetics after CAR T-cell infusion impact response and relapse.

CD19-directed chimeric antigen receptor (CAR) T cells show characteristic proliferation kinetics after infusion that correlate with response. Clearance of circulating disease, B-cell aplasia (BCA), and cytokine release syndrome (CRS) are used to observe CAR T-cell function, given the lack of commercial CAR T-cell measurement assays. We investigated the utility of common hematology laboratory parameters in 166 patients with B-cell acute lymphoblastic leukemia (B-ALL) who were treated with CAR T-cell therapy targeting CD19. CAR T-cell infusion was followed by disappearance of circulating blasts in 86% of patients at a median of 6 days. After a lag phase, there was a rapid expansion in absolute lymphocyte count (ALC) in the second week that coincided with the appearance of atypical lymphocytes. The expansion phase was followed by a contraction phase with a concomitant decrease in atypical lymphocytes. In vitro CAR T-cell studies showed similar kinetics and morphological changes. Peak ALC and overall expansion was greater in sustained responders compared with that in nonresponders. Patients with early loss of BCA and those with eventual CD19+ minimal residual disease/relapse showed lower overall lymphocyte expansion compared with the controls. Pleomorphic lymphocytosis was noted in the cerebrospinal fluid at post-CAR time points. We conclude that lymphocyte counts and differential can also be used to evaluate CAR T-cell expansion after infusion, along with BCA and CRS. This is the first report to characterize the morphology of CAR T cells and determine the utility of lymphocyte kinetics.

Adoptive T cell immunotherapy for medullary thyroid carcinoma targeting GDNF family receptor alpha 4.

Metastatic medullary thyroid cancer (MTC) is a rare but often aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single-chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC.

BET bromodomain protein inhibition reverses chimeric antigen receptor extinction and reinvigorates exhausted T cells in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.

Respiratory syncytial virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice.

BACKGROUND: Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity. METHODS: Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood. RESULTS: RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes. CONCLUSIONS: RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.

CAR T-cell therapy is effective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy.

Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19- subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)- deep remission, whereas 67 patients had a recurrence after achieving a MRD- deep remission: 28 patients with CD19+ leukemia and 39 patients with CD19- leukemia. Return of CD19+ leukemia was associated with loss of CAR T-cell function, whereas CD19- leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19- events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD- remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.

The Pharmacology of T Cell Therapies.

Adoptive cellular therapy using T cells with tumor specificity derived from either natural T cell receptors (TCRs) or an artificial chimeric antigen receptor (CAR) has reached late phase clinical testing, with two CAR T cell therapies achieving regulatory approval within the United States in 2017. The effective use of these therapies depends upon an understanding of their pharmacology, which is quite divergent from traditional small molecule or biologic drugs. We review the different types of T cell therapy under clinical development, the factors affecting cellular kinetics following infusion, and the relationship between these cellular kinetics and anti-cancer activity. We also discuss the toxicity associated with T cell therapies, with an emphasis on cytokine release syndrome and neurotoxicity, and the gaps in knowledge regarding these frequent and unique adverse effects.

Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19- leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.