T-Cell Receptor Stimulation Enhances the Expansion and Function of CD19 Chimeric Antigen Receptor-Expressing T Cells.

PURPOSE: Current protocols for CD19 chimeric antigen receptor-expressing T cells (CD19.CAR-T cells) require recipients to tolerate preinfusion cytoreductive chemotherapy, and the presence of sufficient target antigen on normal or malignant B cells. PATIENTS AND METHODS: We investigated whether additional stimulation of CD19.CAR-T cells through their native receptors can substitute for cytoreductive chemotherapy, inducing expansion and functional persistence of CD19.CAR-T even in patients in remission of B-cell acute lymphocytic leukemia. We infused a low dose of CD19.CAR-modified virus-specific T cells (CD19.CAR-VST) without prior cytoreductive chemotherapy into 8 patients after allogeneic stem cell transplant. RESULTS: Absent virus reactivation, we saw no CD19.CAR-VST expansion. In contrast, in patients with viral reactivation, up to 30,000-fold expansion of CD19.CAR-VSTs was observed, with depletion of CD19+ B cells. Five patients remain in remission at 42-60+ months. CONCLUSIONS: Dual T-cell receptor and CAR stimulation can thus potentiate effector cell expansion and CAR-target cell killing, even when infusing low numbers of effector cells without cytoreduction.

HIV-Specific, Ex Vivo Expanded T Cell Therapy: Feasibility, Safety, and Efficacy in ART-Suppressed HIV-Infected Individuals.

Adoptive T cell therapy has had dramatic successes in the treatment of virus-related malignancies and infections following hematopoietic stem cell transplantation. We adapted this method to produce ex vivo expanded HIV-specific T cells (HXTCs), with the long-term goal of using HXTCs as part of strategies to clear persistent HIV infection. In this phase 1 proof-of-concept study (NCT02208167), we administered HXTCs to antiretroviral therapy (ART)-suppressed, HIV-infected participants. Participants received two infusions of 2 × 107 cells/m2 HXTCs at a 2-week interval. Leukapheresis was performed at baseline and 12 weeks post-infusion to measure the frequency of resting cell infection by the quantitative viral outgrowth assay (QVOA). Overall, participants tolerated HXTCs, with only grade 1 adverse events (AEs) related to HXTCs. Two of six participants exhibited a detectable increase in CD8 T cell-mediated antiviral activity following the two infusions in some, but not all, assays. As expected, however, in the absence of a latency reversing agent, no meaningful decline in the frequency of resting CD4 T cell infection was detected. HXTC therapy in ART-suppressed, HIV-infected individuals appears safe and well tolerated, without any clinical signs of immune activation, likely due to the low residual HIV antigen burden present during ART.

Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma.

Purpose Transforming growth factor-ß (TGF-ß) production in the tumor microenvironment is a potent and ubiquitous tumor immune evasion mechanism that inhibits the expansion and function of tumor-directed responses; therefore, we conducted a clinical study to discover the effects of the forced expression of a dominant-negative TGF-ß receptor type 2 (DNRII) on the safety, survival, and activity of infused tumor-directed T cells. Materials and Methods In a dose escalation study, eight patients with Epstein Barr virus-positive Hodgkin lymphoma received two to 12 doses of between 2 × 107 and 1.5 × 108 cells/m2 of DNRII-expressing T cells with specificity for the Epstein Barr virus-derived tumor antigens, latent membrane protein (LMP)-1 and LMP-2 (DNRII-LSTs). Lymphodepleting chemotherapy was not used before infusion. Results DNRII-LSTs were resistant to otherwise inhibitory concentrations of TGF-ß in vitro and retained their tumor antigen-specific activity. After infusion, the signal from transgenic T cells in peripheral blood increased up to 100-fold as measured by quantitative polymerase chain reaction for the transgene, with a corresponding increase in the frequency of functional LMP-specific T cells. Expansion was not associated with any acute or long-term toxicity. DNRII-LSTs persisted for up to ≥ 4 years. Four of the seven evaluable patients with active disease achieved clinical responses that were complete and ongoing in two patients at > 4 years, including in one patient who achieved only a partial response to unmodified tumor-directed T cells. Conclusion TGF-ß-resistant tumor-specific T cells safely expand and persist in patients with Hodgkin lymphoma without lymphodepleting chemotherapy before infusion. DNRII-LSTs can induce complete responses even in patients with resistant disease. Expression of DNRII may be useful for the many other tumors that exploit this potent immune evasion mechanism.

Cytotoxic T lymphocytes simultaneously targeting multiple tumor-associated antigens to treat EBV negative lymphoma.

Although immunotherapy with Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can treat EBV-associated Hodgkin and non-Hodgkin lymphoma (HL/NHL), more than 50% of such tumors are EBV negative. We now describe an approach that allows us to consistently generate, in a single line, CTLs that recognize a wide spectrum of nonviral tumor-associated antigens (TAAs) expressed by human HL/NHL, including Survivin, MAGE-A4, Synovial sarcoma X (SSX2), preferentially expressed antigen in melanoma (PRAME) and NY-ESO-1. We could generate these CTLs from nine of nine healthy donors and five of eight lymphoma patients, irrespective of human leukocyte antigen (HLA) type. We reactivated TAA-directed T cells ex vivo, by stimulation with dendritic cells (DCs) pulsed with overlapping peptide libraries spanning the chosen antigens in the presence of an optimized Th1-polarizing, prosurvival/proliferative and Treg inhibitory cytokine combination. The resultant lines of CD4(+) and CD8(+), polycytokine-producing T cells are directed against a multiplicity of epitopes expressed on the selected TAAs, with cytolytic activity against autologous tumor cells. Infusion of such multispecific monocultures may extend the benefits of CTL therapy to treatment even of EBV negative HL and NHL.

Challenges of T cell therapies for virus-associated diseases after hematopoietic stem cell transplantation.

IMPORTANCE OF THE FIELD: Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for many hematological malignancies and genetic disorders. The majority of patients do not have a human leukocyte antigen (HLA) identical sibling donor, and alternative stem cell sources include HLA-matched or mismatched unrelated donors and haploidentical related donors. However, alternative donor HSCT are associated with three major complications i) graft rejection; ii) graft-versus-host disease (GvHD); and iii) delayed immune reconstitution leading to viral infections and relapse. AREAS COVERED IN THIS REVIEW: Graft rejection and the risk of GvHD can be significantly reduced by using intensive conditioning regimens, including in vivo T cell depletion as well as ex vivo T cell depletion of the graft. However, the benefits of removing alloreactive T cells from the graft are offset by the concomitant removal of T cells with anti-viral or anti-tumor activity as well as the profound delay in endogenous T cell recovery post-transplant. Thus, opportunistic infections, many of which are not amenable to conventional small-molecule therapeutics, are frequent in these patients and are associated with significant morbidity and high mortality rates. This review discusses current cell therapies to prevent or treat viral infections/reactivations post-transplant. WHAT THE READER WILL GAIN: The reader will gain an understanding of the current state of cell therapy to prevent and treat viral infections post-HSCT, and will be introduced to preclinical studies designed to develop and validate new manufacturing procedures intended to improve therapeutic efficacy and reduce associated toxicities. TAKE HOME MESSAGE: Reconstitution of HSCT recipients with antigen-specific T cells, produced either by allodepletion or in vitro reactivation, can offer an effective strategy to provide both immediate and long-term protection without harmful alloreactivity.

Erythroid GATA1 function revealed by genome-wide analysis of transcription factor occupancy, histone modifications, and mRNA expression.

The transcription factor GATA1 regulates an extensive program of gene activation and repression during erythroid development. However, the associated mechanisms, including the contributions of distal versus proximal cis-regulatory modules, co-occupancy with other transcription factors, and the effects of histone modifications, are poorly understood. We studied these problems genome-wide in a Gata1 knockout erythroblast cell line that undergoes GATA1-dependent terminal maturation, identifying 2616 GATA1-responsive genes and 15,360 GATA1-occupied DNA segments after restoration of GATA1. Virtually all occupied DNA segments have high levels of H3K4 monomethylation and low levels of H3K27me3 around the canonical GATA binding motif, regardless of whether the nearby gene is induced or repressed. Induced genes tend to be bound by GATA1 close to the transcription start site (most frequently in the first intron), have multiple GATA1-occupied segments that are also bound by TAL1, and show evolutionary constraint on the GATA1-binding site motif. In contrast, repressed genes are further away from GATA1-occupied segments, and a subset shows reduced TAL1 occupancy and increased H3K27me3 at the transcription start site. Our data expand the repertoire of GATA1 action in erythropoiesis by defining a new cohort of target genes and determining the spatial distribution of cis-regulatory modules throughout the genome. In addition, we begin to establish functional criteria and mechanisms that distinguish GATA1 activation from repression at specific target genes. More broadly, these studies illustrate how a "master regulator" transcription factor coordinates tissue differentiation through a panoply of DNA and protein interactions.

SCL and associated proteins distinguish active from repressive GATA transcription factor complexes.

GATA-1 controls hematopoietic development by activating and repressing gene transcription, yet the in vivo mechanisms that specify these opposite activities are unknown. By examining the composition of GATA-1-associated protein complexes in a conditional erythroid rescue system as well as through the use of tiling arrays we detected the SCL/TAL1, LMO2, Ldb1, E2A complex at all positively acting GATA-1-bound elements examined. Similarly, the SCL complex is present at all activating GATA elements in megakaryocytes and mast cells. In striking contrast, at sites where GATA-1 functions as a repressor, the SCL complex is depleted. A DNA-binding defective form of SCL maintains association with a subset of active GATA elements indicating that GATA-1 is a key determinant for SCL recruitment. Knockdown of LMO2 selectively impairs activation but not repression by GATA-1. ETO-2, an SCL-associated protein with the potential for transcription repression, is also absent from GATA-1-repressed genes but, unlike SCL, fails to accumulate at GATA-1-activated genes. Together, these studies identify the SCL complex as a critical and consistent determinant of positive GATA-1 activity in multiple GATA-1-regulated hematopoietic cell lineages.

The Set2 methyltransferase associates with Ssn6 yet Tup1-Ssn6 repression is independent of histone methylation.

The Tup1-Ssn6 corepressor regulates the expression of diverse classes of genes in Saccharomyces cerevisiae. Chromatin is an important component of Tup1-Ssn6-mediated repression. Tup1 binds to underacetylated tails of histones H3 and H4, and requires multiple histone deacetylases for the repression. Here we examine if histone methylation, in addition to histone deacetylation, plays a role in Tup1-Ssn6 repression. We found that like other genes, Tup1-Ssn6 target genes exhibit increased levels of histone H3 lysine 4 trimethylation upon activation. However, deletion of individual or multiple histone methyltransferases and other SET-domain containing genes has no apparent effect on Tup1-Ssn6-mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Although deletion of SET2 does not affect Tup1-Ssn6 repression of a number of target genes, Ssn6 may utilize Set2 in specific contexts to regulate gene repression.