ER-associated degradation adapter Sel1L is required for CD8+ T cell function and memory formation following acute viral infection.

The maintenance of antigen-specific CD8+ T cells underlies the efficacy of vaccines and immunotherapies. Pathways contributing to CD8+ T cell loss are not completely understood. Uncovering the pathways underlying the limited persistence of CD8+ T cells would be of significant benefit for developing novel strategies of promoting T cell persistence. Here, we demonstrate that murine CD8+ T cells experience endoplasmic reticulum (ER) stress following activation and that the ER-associated degradation (ERAD) adapter Sel1L is induced in activated CD8+ T cells. Sel1L loss limits CD8+ T cell function and memory formation following acute viral infection. Mechanistically, Sel1L is required for optimal bioenergetics and c-Myc expression. Finally, we demonstrate that human CD8+ T cells experience ER stress upon activation and that ER stress is negatively associated with improved T cell functionality in T cell-redirecting therapies. Together, these results demonstrate that ER stress and ERAD are important regulators of T cell function and persistence.

Biological insights into the role of TET2 in T cell lymphomas.

Peripheral T cell lymphomas (PTCL) are a heterogenous group of mature T cell lymphomas with an overall poor prognosis. Understanding the molecular heterogeneity in PTCL subtypes may lead to improved understanding of the underlying biological mechanisms driving these diseases. Mutations in the epigenetic regulator TET2 are among the most frequent mutations identified in PTCL, with the highest frequency in angioimmunoblastic T cell lymphomas and other nodal T follicular helper (TFH) lymphomas. This review dissects the role of TET2 in nodal TFH cell lymphomas with a focus on emerging biological insights into the molecular mechanism promoting lymphomagenesis and the potential for epigenetic therapies to improve clinical outcomes.

SOHO State of the Art Updates and Next Questions | New Pathways and New Targets in PTCL: Staying on Target.

While the peripheral T-cell lymphomas (PTCL) remain a therapeutic challenge, and increasingly account for a disproportionate number of lymphoma-related deaths, improved understanding of disease pathogenesis and classification, and the development of novel therapeutic agents over the past decade, all provide reasons for a more optimistic outlook in the next. Despite their genetic and molecular heterogeneity, many PTCL are dependent upon signaling input provided by antigen, costimulatory, and cytokine receptors. While gain-of-function alterations effecting these pathways are recurrently observed in many PTCL, more often than not, signaling remains ligand-and tumor microenvironment (TME)-dependent. Consequently, the TME and its constituents are increasingly recognized as "on target". Utilizing a "3 signal" model, we will review new-and old-therapeutic targets that are relevant for the more common nodal PTCL subtypes.

Adding venetoclax to lenalidomide and rituximab is safe and effective in patients with untreated mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a rare, incurable hematological malignancy with a heterogeneous presentation and clinical course. A wide variety of chemotherapy-based regimens are currently used in patients who are untreated. Over the last several years, several targeted or small-molecule therapies have shown efficacy in the relapsed/refractory setting and have since been explored in the frontline setting. Lenalidomide plus rituximab was explored in a phase 2 study of 38 patients with MCL who were untreated and ineligible to receive transplantation, in which the combination produced durable remissions. We looked to build upon this regimen by adding venetoclax to the combination. We conducted a multicenter, open-label, nonrandomized, single-arm study to evaluate this combination. We enrolled 28 unselected patients with untreated disease irrespective of age, fitness, or risk factors. Lenalidomide was dosed at 20 mg daily from days 1 to 21 of each 28-day cycle. The dose of venetoclax was determined using the time-to-event continual reassessment method. Rituximab was dosed at 375 mg/m2 weekly, starting on cycle 1, day 1 until cycle 2, day 1. No dose-limiting toxicities were noted. All patients were treated with venetoclax at the maximum tolerated dose of 400 mg daily. The most common adverse events were neutropenia and thrombocytopenia. The overall and complete response rates were 96% and 86%, respectively. In total, 86% of patients achieved minimal residual disease undetectability via next-generation sequencing. The median overall and progression-free survivals were not reached. The combination of lenalidomide, rituximab, and venetoclax is a safe and effective regimen in patients with untreated MCL. This trial was registered at www.clinicaltrials.gov as #NCT03523975.

Metabolomic Profiles of Human Glioma Inform Patient Survival.

Aims: Targeting tumor metabolism may improve the outcomes for patients with glioblastoma (GBM). To further preclinical efforts targeting metabolism in GBM, we tested the hypothesis that brain tumors can be stratified into distinct metabolic groups with different patient outcomes. Therefore, to determine if tumor metabolites relate to patient survival, we profiled the metabolomes of human gliomas and correlated metabolic information with clinical data. Results: We found that isocitrate dehydrogenase-wildtype (IDHwt) GBMs are metabolically distinguishable from IDH mutated (IDHmut) astrocytomas and oligodendrogliomas. Survival of patients with IDHmut gliomas was expectedly more favorable than those with IDHwt GBM, and metabolic signatures can stratify IDHwt GBMs subtypes with varying prognoses. Patients whose GBMs were enriched in amino acids had improved survival, while those whose tumors were enriched for nucleotides, redox molecules, and lipid metabolites fared more poorly. These findings were recapitulated in validation cohorts using both metabolomic and transcriptomic data. Innovation: Our results suggest the existence of metabolic subtypes of GBM with differing prognoses, and further support the concept that metabolism may drive the aggressiveness of human gliomas. Conclusions: Our data show that metabolic signatures of human gliomas can inform patient survival. These findings may be used clinically to tailor novel metabolically targeted agents for GBM patients with different metabolic phenotypes. Antioxid. Redox Signal. 39, 942-956.

The physical exam's role in determining dose-limiting toxicity prior to immunochemotherapy administration in lymphoma.

INTRODUCTION: The COVID-19 pandemic has introduced new challenges to healthcare access and delivery. It is critical to identify areas for innovation within oncologic clinical practice to maintain high quality care. We evaluated the potential utility of telemedicine initiatives for patients with lymphoma undergoing immunochemotherapy. We conducted a retrospective review of adult lymphoma patients receiving R-CHOP + /- R, R-ICE, R-GEMOX, and R-DHAP at our institution in the last three years (2017-2019) and identified cycles for which dose modifications were required. METHODS: We reviewed 1290 total treatment cycles in 301 unique patients, 1102 cycles (85.4%) were R-CHOP + /- R, 105 (8.1%) were R-ICE, 71 (5.5%) were R-GEMOX, and 12 (0.9%) were R-DHAP. We identified that 144 cycles (11.2%) were subject to dosing adjustments. We retrospectively reviewed laboratory results, patient history, and/or physical exam findings that informed dose modifications. RESULTS: Of the 144 dose adjustments, 11% of cycles contained dose increases due to a well-tolerated previous dose noted in the clinical assessment. The remaining 128 modified cycles were dose reductions. Notably, only 7/128 dose reductions were based on physical exam findings alone, due solely to a change in patient body weight. As patients are routinely weighed immediately prior to chemotherapy administration, effectively no dose modifications (0/144) were exclusively based on abnormal physical exam finding during a pre-infusion assessment. CONCLUSION: These findings suggest that pre-infusion assessments may be amenable to virtual visits for lymphoma patients undergoing immunochemotherapy.

Two cases of challenging cutaneous lymphoid infiltrates presenting in the context of COVID-19 vaccination: A reactive lymphomatoid papulosis-like eruption and a bona fide lymphoma.

COVID-19 infection and vaccination may be associated with a wide variety of cutaneous and immune manifestations. Here, we describe two patients who presented with monoclonal cutaneous T-cell infiltrates that showed cytologic and immunophenotypic features concerning for lymphoma shortly following COVID-19 vaccination. In one case, the eruption completely resolved. The second patient showed initial resolution, but her disease recurred and progressed following a breakthrough SARS-CoV-2 infection. These cases suggest that immune stimulation following exposure to SARS-Cov-2 protein(s) in vaccine or infection may facilitate the development of a lymphoma or lymphoproliferative disorder in susceptible individuals. Moreover, they show that separating these cases from pseudolymphomatous reactive conditions is often challenging and requires close clinical correlation.

OXPHOS promotes apoptotic resistance and cellular persistence in TH17 cells in the periphery and tumor microenvironment.

T cell proliferation and cytokine production are bioenergetically and biosynthetically costly. The inability to meet these metabolic demands results in altered differentiation, accompanied by impaired effector function, and attrition of the immune response. Interleukin-17-producing CD4 T cells (TH17s) are mediators of host defense, autoimmunity, and antitumor immunity in the setting of adoptive T cell therapy. TH17s are long-lived cells that require mitochondrial oxidative phosphorylation (OXPHOS) for effector function in vivo. Considering that TH17s polarized under standardized culture conditions are predominately glycolytic, little is known about how OXPHOS regulates TH17 processes, such as their ability to persist and thus contribute to protracted immune responses. Here, we modified standardized culture medium and identified a culture system that reliably induces OXPHOS dependence in TH17s. We found that TH17s cultured under OXPHOS conditions metabolically resembled their in vivo counterparts, whereas glycolytic cultures were dissimilar. OXPHOS TH17s exhibited increased mitochondrial fitness, glutamine anaplerosis, and an antiapoptotic phenotype marked by high BCL-XL and low BIM. Limited mitophagy, mediated by mitochondrial fusion regulator OPA-1, was critical to apoptotic resistance in OXPHOS TH17s. By contrast, glycolytic TH17s exhibited more mitophagy and an imbalance in BCL-XL to BIM, thereby priming them for apoptosis. In addition, through adoptive transfer experiments, we demonstrated that OXPHOS protected TH17s from apoptosis while enhancing their persistence in the periphery and tumor microenvironment in a murine model of melanoma. Together, our work demonstrates how metabolism regulates TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.

Immunology 101: fundamental immunology for the practicing hematologist.

From an evolutionary perspective, the immune system developed primarily to protect the host from pathogens. In the continuous balance between killing pathogens and protecting host tissues, selective pressures have shaped the discriminatory functions of the immune system. In addition to protection against microbial pathogens, the immune system also plays a critical role in antitumor immunity. Immune dysfunction, either under- or overactivity, is found in a wide range of hematologic disorders. Here we review the fundamental features of the immune system and the key concepts critical to understanding the impact of immune dysfunction on hematologic disorders.

Clinical application of next generation sequencing in lymphoma.

Successful treatment of relapsed/refractory and rare subtypes of lymphomas remains a therapeutic challenge. Though the use of tumor profiling is increasing, little is described about how providers ultimately utilize this information in clinical decision-making. We reviewed 92 adult lymphoma patients who underwent an IRB-approved tumor sequencing protocol at the University of Michigan, MI-ONCOSEQ. Of this cohort, 60 had a targeted treatment suggested by their test results, and 11 patients ultimately underwent the MI-ONCOSEQ recommended therapy. One obtained complete response based on precision treatment recommendations. The two main barriers for targeted treatment utilization included inopportune timing (the patient either was sequenced too early or too late in their disease course) and clinical trial availability. While this study demonstrates the success of sequencing lymphomas for the identification of novel therapies, it also underlines the clinical challenges, namely the optimal timing and availability of trials, inherent in the successful application of this technology.

DNA Methylation in T-Cell Development and Differentiation.

T lymphocytes undergo carefully orchestrated programming during development in the thymus and subsequently during differentiation in the periphery. This intricate specification allows for cell-type and context-specific transcriptional programs that regulate immune responses to infection and malignancy. Epigenetic changes, including histone modifications and covalent modification of DNA itself through DNA methylation, are now recognized to play a critical role in these cell-fate decisions. DNA methylation is mediated primarily by the actions of the DNA methyltransferase (DNMT) and ten-eleven-translocation (TET) families of epigenetic enzymes. In this review, we discuss the role of DNA methylation and its enzymatic regulators in directing the development and differentiation of CD4+ and CD8+ T-cells.

Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation.

T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild-type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions.

Interleukin-4 regulates eomesodermin in CD8+ T cell development and differentiation.

Interleukin (IL)-4 is a cytokine classically associated with CD4(+) T helper type 2 differentiation, but has been recently shown to also be required for the development of CD8(+) innate-like lymphocytes. CD8(+) innate-like lymphocytes are non-conventional lymphocytes that exhibit characteristics typically associated with memory CD8(+) T cells, including expression of the T-box transcription factor Eomesodermin (Eomes). Here we investigate the signaling pathways required for IL-4 induction of Eomes and CD8(+) innate-like lymphocyte markers in murine CD8SP thymocytes and peripheral CD8(+) T cells. We demonstrate that IL-4 is sufficient to drive Eomes expression and the CD8(+) innate-like lymphocyte phenotype through cooperation between STAT6- and Akt-dependent pathways. Furthermore, we show that while IL-4 has little effect on the induction of Eomes in the setting of robust T cell receptor (TCR) activation, this cytokine promotes Eomes in the setting of attenuated TCR stimulation in mature CD8(+) T cells suggesting that cytokine signaling pathways may direct cell fate when TCR signals are limiting.

Development of innate CD4+ and CD8+ T cells in Itk-deficient mice is regulated by distinct pathways.

T cell development in the thymus produces multiple lineages of cells, including innate T cells such as γδ TCR(+) cells, invariant NKT cells, mucosal-associated invariant T cells, and H2-M3-specific cells. Although innate cells are generally a minor subset of thymocytes, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional CD8(+) T cells develop as innate cells with characteristics of memory T cells. Thus, in Itk-deficient mice, mature CD4(-)CD8(+) (CD8 single-positive [SP]) thymocytes express high levels of the transcription factor eomesodermin (Eomes) and are dependent on IL-4 being produced in the thymic environment by a poorly characterized subset of CD4(+) thymocytes expressing the transcriptional regulator promyelocytic leukemia zinc finger. In this study, we show that a sizeable proportion of mature CD4(+)CD8(-) (CD4SP) thymocytes in itk(-/-) mice also develop as innate Eomes-expressing T cells. These cells are dependent on MHC class II and IL-4 signaling for their development, indicating that they are conventional CD4(+) T cells that have been converted to an innate phenotype. Surprisingly, neither CD4SP nor CD8SP innate Eomes(+) thymocytes in itk(-/-) or SLP-76(Y145F) mice are dependent on γδ T cells for their development. Instead, we find that the predominant population of Eomes(+) innate itk(-/-) CD4SP thymocytes is largely absent in mice lacking CD1d-specific invariant NKT cells, with no effect on innate itk(-/-) CD8SP thymocytes. In contrast, both subsets of innate Eomes(+)itk(-/-) T cells require the presence of a novel promyelocytic leukemia zinc finger-expressing, SLAM family receptor adapter protein-dependent thymocyte population that is essential for the conversion of conventional CD4(+) and CD8(+) T cells into innate T cells with a memory phenotype.

Requirements for eomesodermin and promyelocytic leukemia zinc finger in the development of innate-like CD8+ T cells.

Conventional and nonconventional T cell development occur in the thymus. Nonconventional thymocytes that bear characteristics typically associated with innate immune cells are termed innate-like lymphocytes (ILLs). Mice harboring a tyrosine to phenylalanine mutation in the adaptor protein Src homology 2 domain-containing leukocyte protein of 76 kDa at residue 145 (Y145F mice) develop an expanded population of CD8(+)CD122(+)CD44(+) ILLs, typified by expression of the T-box transcription factor eomesodermin. Y145F mice also have an expanded population of γδ T cells that produce copious amounts of IL-4 via a mechanism that is dependent on the BTB-ZF transcription factor promyelocytic leukemia zinc finger. Using mice with T cell-specific deletion of Eomes, we demonstrate that this transcription factor is required for CD8(+) ILL development in Y145F as well as wild-type mice. Moreover, we show that promyelocytic leukemia zinc finger and IL-4 are also required for the generation of this ILL population. Taken together, these data shed light on the cell-intrinsic and cell-extrinsic factors that drive CD8(+) ILL differentiation.

HES1 cooperates with pRb to activate RUNX2-dependent transcription.

UNLABELLED: The retinoblastoma protein, pRb, can activate the transcription factor RUNX2, an essential regulator of osteogenic differentiation, but the mechanism of this activation is unknown. Here we studied the interaction of pRb and RUNX2 with HES1, previously reported to augment RUNX2 activity. PRb can act to promote RUNX2/HES1 association with concomitant promoter occupancy and transcriptional activation in bone cells. INTRODUCTION: RUNX2 (also known as OSF2/CBFA1) is a transcription factor required for osteoblast differentiation and bone formation. We have reported that RUNX2 can associate with the retinoblastoma protein pRb, a common tumor suppressor in bone, and the resultant complex can bind and activate transcription from bone-specific promoters. This activity of the pRb/RUNX2 complex may thus link differentiation control with tumor suppressor activity. However, the mechanism through which pRb can activate RUNX2 is unknown. HES1 is a reported co-activator of RUNX2 that shares a binding site on RUNX2 with pRb. Thus, we have tested the cooperativity among these factors in activating transcription from bone specific promoters. MATERIALS AND METHODS: Coimmunoprecipitation, chromatin immunoprecipitation, and EMSA experiments were used to study the interaction of RUNX2, HES1, and pRb in cell lysates and on DNA. Transcriptional reporter assays were used to analyze the activity of RUNX2 in the presence and absence of HES1 and pRb. RESULTS: We showed that pRb can associate with HES1, a previously described RUNX2 interactor that can itself augment RUNX2-dependent transcription. The association of HES1 with RUNX2 is augmented by pRb. Furthermore, both pRb and HES1 increase the amount of RUNX2 bound to promoter sites in vivo, pRb and HES1 synergistically activate a RUNX2-dependent reporter gene, and depletion of HES1 reduces RUNX2/pRb activity. CONCLUSIONS: These data indicate that pRb acts as a RUNX2 co-activator at least in part by recruiting HES1 into the pRb/RUNX2 complex and further elucidate a novel role for pRb as a transcriptional co-activator in osteogenesis.

Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.