ALBERT

Description: DNA methylation is one of the major epigenetic mechanisms that controls cellular differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases mediates active DNA demethylation through the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates. Here we demonstrate that TET2 regulates CD8+ T cell differentiation in vivo following acute and chronic viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss enhances LCMV-specific CD8+ T cell memory differentiation in a cell-intrinsic manner without disrupting antigen-specific cell expansion or cytokine production. However, TET2-deficient memory CD8+ T cells exhibit altered recall responses with blunted re-expansion, retained expression of phenotypic memory markers and restricted re-expression of activation markers. During chronic viral infection with LCMV-clone 13, TET2 controls CD8+ T cell expansion and alters differentiation. Importantly, though mice with T-cell specific loss of TET2 developed similar levels of CD8+ T cell exhaustion as wild-type mice, TET2 loss specifically reduced PD-1 expression suggesting that TET2 may direct DNA demethylation of the PD-1 locus. Together, our data indicate that TET2 is an important regulator of CD8+ T cells following both acute and chronic viral infections and suggest targeting epigenetic regulators have potential for enhancing antiviral immunity. Disclosures No relevant conflicts of interest to declare.

Description: Regulation of DNA methylation is critical for proper T cell differentiation and function. Antigen-specific CD8+ T cells undergo global remodeling of DNA methylation following viral infection, suggesting that DNA methylation may direct antigen-specific T cell responses. TET2 is a member of the Ten-Eleven-Translocation (TET) family, which converts 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates ultimately leading to DNA demethylation. How TET2 regulates T cell differentiation and function is unknown. Here we demonstrate that TET2 expression is regulated by TCR signaling in primary murine T cells. Furthermore, using a novel flow cytometric assay to measure 5hmC levels on a single cell basis, we find that TCR signaling also regulates TET activity as evidenced by a rapid increase in global 5hmC levels after TCR stimulation that is blunted in TET2-deficient T cells. To determine the role of TET2 in T cell responses, we generated mice deficient in TET2 in the T cell compartment (TET2fl/flCD4Cre+) mice. These mice develop grossly normal thymic and peripheral T cell subsets. Given the regulation of TET2’s expression and activity by TCR stimulation, we used a murine model of acute viral infection, specifically LCMV-Armstrong, to test if TET2 regulates antigen-specific T cell responses in vivo. Following viral challenge, TET2fl/flCD4Cre+ mice have similar antigen-specific CD8+ T cell expansion and effector responses but exhibit significantly enhanced memory CD8+ T cell differentiation compared to control mice. These data demonstrate that TET2 plays a critical role in directing CD8+ T cell differentiation and function. Studies are ongoing to identify specific TET2 regulated genes important in the development of CD8+ T cell memory. Disclosures No relevant conflicts of interest to declare.

Description: Cytokines regulate T cell development and function. Interleukin (IL)-4 is a cytokine classically associated with CD4+ T helper (TH) differentiation, specifically TH2 differentiation. However, IL-4 has recently been shown to drive the development of CD8+ innate-like lymphocytes (ILLs). CD8+ ILLs are non-conventional thymocytes that develop with characteristics typically associated with innate and/or memory immune cells, including surface expression of the activation/memory markers CD44 and CD122, expression of the T-box transcription factor Eomesodermin (Eomes) and the rapid production of interferon (IFN)-γ after ex vivo stimulation. Here we show that IL-4 is sufficient to promote Eomes expression in CD8 single-positive (SP) thymocytes in short-term in vitro culture and direct CD8+ ILL development in fetal thymic organ culture. Using genetic deficiency and pharmacologic inhibitors, we demonstrate that IL-4 up-regulation of Eomes in CD8SP thymocytes requires STAT6 and Akt signaling pathways. Next, we investigated the possibility that in addition to directing the fate of developing thymocytes, IL-4 might impact the function of mature CD8+ cells undergoing activation. We found that that in naïve peripheral CD8+ T the combination of IL-4 and low dose T cell receptor (TCR) stimulation is a potent inducer of Eomes. Futhermore, when combined, these stimuli promote the persistence of CD8+ T cells in an adoptive transfer model. Understanding how IL-4 directs CD8+ T cell differentiation may provide a novel way to enhance CD8+ T cell function in adoptive T cell therapies. Disclosures: No relevant conflicts of interest to declare.

Description: DNA methylation is one of the major epigenetic mechanisms that control T cell differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines, intermediates in active DNA demethylation. Here we demonstrate that TET2 regulates CD8+ T cell differentiation in vivo following acute viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. However, following acute viral infection with LCMV-Armstrong, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting antigen-driven cell expansion or effector function. Integration of genome-wide methylation analysis and expression data suggest that TET2 loss leads to hypermethyation of the PRDM1 genomic locus (encoding Blimp-1) and alters the relative expression of Blimp-1 and Bcl-6, two antagonistic transcriptional repressors known to direct CD8+ T cell memory differentiation. Together, our data indicate that TET2 is an important regulator of CD8+ T cell fate decisions. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2161 The innate and adaptive arms of the immune system collaborate to protect the host against invading pathogens and perform immune surveillance against malignant transformation. As key effectors of the adaptive immune system, conventional T cells develop in the thymus and exit to the periphery as naïve cells, requiring antigenic stimulation and subsequent differentiation to gain effector functions, such as cytokine secretion or cytolytic activity. In contrast to conventional T cells, non-conventional T lymphocytes possess characteristics of innate immune cells, such as expression of surface markers associated with activation/memory and acquisition of effector function during thymic development, and thus are termed innate-like lymphocytes (ILLs). Recently, an expanded population of CD8+ ILLs was identified in mice with a mutation in the T cell receptor signaling protein SLP-76 (SLP-76 Y145F mice). These CD8+ ILLs are characterized by expression of activation/memory markers CD44 and CD122, the expression of the T-box transcription factor Eomesodermin (Eomes) and rapid production of IFN-γ after ex vivo stimulation. The development of these CD8+ ILLs occurs in a cell-extrinsic manner and requires IL-4. We demonstrate that IL-4 is sufficient to upregulate Eomes expression in wild-type CD8 single-positive (SP) thymocytes in short-term in vitro culture and potentiate CD8+ ILL development in fetal thymic organ culture. Using phospho-flow cytometry, we find that CD8+ ILLs from SLP-76 Y145F mice have increased STAT6 and Akt activation vs. CD8+ non-ILLs. In CD8SP thymocytes deficient in STAT6, Eomes expression is not upregulated in response to IL-4. In addition, we demonstrate that pharmacologic inhibition of Akt in SLP-76 Y145F fetal thymic organ culture blocks CD8+ ILL development and also prevents IL-4 induced Eomes upregulation in WT CD8SP thymocytes. Importantly, we have identified CD8+ ILLs in human fetal thymocytes and umbilical cord blood and found that IL-4 is sufficient to up-regulate Eomes expression in these cells. Taken together, our data suggest that IL-4 signaling via STAT6 and Akt pathways is required for IL-4 induction of Eomes expression and CD8+ ILL development. Understanding signal transduction pathways required for CD8+ ILL development will provide insight into the development of this unique lymphocyte subset that sits at the interface between innate and adaptive immunity and has been identified in human umbilical cord blood. Disclosures: No relevant conflicts of interest to declare.

Description: Germline mutations in the shelterin component protection of telomeres 1 (POT1) were recently found to be associated with familial chronic lymphocytic leukemia (CLL), melanoma, glioma, and several other familial cancer syndromes. The role of POT1 mutations in myeloid neoplasms and other hematologic malignancies, however, remains unknown. To explore the role of POT1 variants in hematologic neoplasms, we analyzed POT1 variants in 3323 consecutive patients who underwent next-generation sequencing (NGS) of a panel of hematologic malignancy-associated genes at our institution and characterized the clinical and pathological characteristics of patients with germline and somatic POT1 mutations. Of 3323 consecutive patients who underwent NGS, 2770 patients had a hematologic malignancy (lymphoid n = 1299, myeloid n = 934, and both lymphoid and myeloid n = 537), while 553 patients were evaluated for non-malignant cytopenias. All 57 patients (2.06%) carrying either a POT1 disease-associated variant or variant of uncertain significance had a hematologic malignancy compared to no identified POT1 variants in 553 patients with benign cytopenias (OR = 23.5, p 〈 0.001), suggesting that the presence of POT1 variants was predictive of a hematologic malignancy. Of 57 patients, 33 had lymphoid malignancies, 23 had myeloid neoplasms, and 2 had a lymphoid and myeloid neoplasm (Fig 1). Patient variants were classified as germline or somatic using constitutional DNA sequencing, POT1 emergence/disappearance over time, or POT1 maintenance in remission. In the absence of these data, likely germline or likely somatic designations were made by assessing variant allele frequencies against clinical/pathologic characteristics. 18 patients (33%) were found to have germline or likely germline POT1 variants (29% and 42% in the lymphoid and myeloid malignancy groups, respectively). Another 6 patients (11%) had variants whose germline status could not be determined. Of the 17 unique germline POT1 variants, 10 were missense and located within mapped functional protein domains, while 7 were classified as predicted loss-of-function (pLOF) due to a disruption of start, premature stop, frameshift, or spice site alteration. Patients with hematological malignancies had a ~5-8x increased odds of having a germline pLOF POT1 variant compared to cancer-free individuals in the Genome Aggregation Database (gnomAD, n = 113,108 exomes, OR = 7.5, p 〈 0.001) or in the Penn Medicine BioBank (PMBB, n = 7877, OR = 5.0, p = 0.010), with a prevalence of 0.25% compared to 0.03% and 0.05%, respectively. Germline pLOF POT1 variants were significantly more enriched in patients with myeloid (gnomAD: OR = 6.1, p = 0.02) and lymphoid (gnomAD: OR = 9.8, p 〈 0.001; PMBB: OR = 6.5, p = 0.004) malignancies. In 33 patients with lymphoid malignancies and POT1 variants, the most common diagnoses were CLL/SLL (n = 21, germline n = 6, somatic n = 12), CD5- CD10- indolent B cell neoplasms (n = 4, germline n = 1, somatic n = 3), and multiple myeloma (n = 3, all somatic) (Table 1). Lymphoid malignancies with a germline POT1 variant had a relative paucity of additional mutations; in contrast, somatic POT1 variants frequently co-occurred with other mutations, most commonly with TP53 (Fig 2, n = 5, 23%). Among 23 patients with myeloid malignancies, patients with germline POT1 variants developed malignancies at a significantly younger age compared to those whose POT1 variants were somatic (median age 59.5 vs 70.5 years, p = 0.04). The most common diagnosis in patients with myeloid neoplasms carrying germline POT1 variants was MPN (germline n = 5, somatic n = 1). AML, MDS/MPN, and MDS occurred in 4, 3, and 1 patients respectively. All patients with myeloid neoplasms had additional disease-associated mutations, with the most common co-occurring variants in TET2 (n = 7), JAK2 (n = 6, co-occurring with 50% of germline POT1 myeloid variants), and NRAS (n = 6). In conclusion, this is the first comprehensive analysis of POT1 variants in an unselected hospital-based population undergoing molecular testing for variants associated with hematologic malignancies. Our results show that the presence of POT1 variants is predictive of having a hematologic neoplasm and that over 30% of POT1 variants in hematologic malignancy patients are germline. Our study expands the spectrum of POT1-associated familial neoplasms and highlights the needs for better recognition of familial hematologic cancer syndromes. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Bendamustine-associated lymphopenia is well-established. However, the risk of opportunistic infections (OI) and the utility of routine antimicrobial prophylaxis in this setting is unclear. In this retrospective analysis, we analyzed the incidence of lymphopenia, the use of antimicrobial prophylaxis, the incidence, type and severity of infections, and risk factors for developing infections in patients that received bendamustine-based therapies. Methods: We conducted a retrospective review of patients with lymphoma who were treated with bendamustine between January 2015 and January 2019 at University of Michigan Rogel Cancer Center. Baseline demographics and clinical characteristics such as age, sex, lymphoma diagnosis, absolute lymphocyte count (ALC) and absolute neutrophil count (ANC) at baseline, during treatment and post-treatment, details of infectious events and use of antimicrobial prophylaxis were collected. Results: 230 patients who received bendamustine were identified. Lymphoma diagnoses included 34% follicular lymphoma, 21% chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), 18% mantle cell lymphoma, 10% marginal zone lymphoma, 9% Hodgkin's lymphoma, 2% diffuse large B cell lymphoma, and 5% Waldenstrom's macroglobulinemia. Median age at the time of treatment was 64.5 years. After excluding CLL and leukemic phase of mantle cell lymphoma, the median ALC count at baseline, during treatment and following the last treatment were 1.4 K/mm3 (range: 0.1-16 K/mm3), 0.6 K/mm3 (range: 0.1-7.2 K/mm3) and 0.4 K/mm3 (range: 0.1-4.4 K/mm3), respectively. At the end of therapy, lymphopenia (ALC 〈 1 K/mm3) and severe lymphopenia (ALC 〈 0.5 K/mm3) were noted in 83.5% (n=192, median ALC: 0.3 K/mm3) and 46.5% (n=107, median ALC: 0.4 K/mm3) of patients, respectively. The median time to achieving ALC 〉 0.5 K/mm3 was 4 months from the end of therapy (range: 2-8 months) and the median time to complete count recovery was 9.5 months from the end of therapy (range: 4-14 months). Of 230 patients, 44.7% did not receive any antimicrobial prophylaxis, 38.3% received antiviral and pneumocystis pneumonia (PJP) prophylaxis, 13.9% received only antiviral prophylaxis, 2.1% received only PJP prophylaxis, 0.4% received antifungal prophylaxis and 0.4% received antibacterial prophylaxis. Fifty-six patients (24%) developed severe infection, defined as any infection requiring hospitalization. OI were relatively rare and were observed in 15 patients (6.5%). Among those 15 patients, 80% did not receive any antimicrobial prophylaxis. OI included PJP pneumonia (n=4), cytomegalovirus pneumonia (n=1), varicella-zoster virus (n=1), aspergillus pneumonia (n=1), disseminated aspergillosis (n=1), cerebral toxoplasmosis (n=1), pulmonary histoplasmosis (n=1), necrotizing otitis externa due to Pseudomonas aeruginosa (n=1) and severe Clostridium difficile colitis (n=4). Risk factors for severe infection and OI that were analyzed included age, underlying disease, pre-treatment severe lymphopenia, post-treatment severe lymphopenia, number of prior therapies, use of rituximab maintenance and G-CSF use. No risk factors for severe infection or OI were identified in our analysis. OI were noted less frequently in patients with severe post-treatment lymphopenia (5/107, 4.7%) compared to those without severe lymphopenia (10/123, 8%) which may have been due to the use of antimicrobial prophylaxis in these patients, as 62% of severely lymphopenic patients received prophylaxis, compared with 49.6% of patients without severe lymphopenia (p=0.06). Conclusion: Severe lymphopenia following bendamustine therapy is common. The median 9.5 month time to count recovery in our analysis was similar compared to previous studies. Incidence of OI was lower in patients who received antimicrobial prophylaxis. OI were not limited to patients with severe post-treatment lymphopenia, which may be related to the increased use of prophylaxis in these patients. Further prospective studies are needed to confirm the utility of antimicrobial prophylaxis in the setting of bendamustine-associated severe lymphopenia. Disclosures Phillips: Seattle Genetics: Consultancy; BMS: Consultancy; Bayer: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Cardinal Health: Consultancy; Lymphoma Connect: Other; University of Michigan: Current Employment; Beigene: Consultancy; Karyopharm: Consultancy; AstraZeneca: Consultancy; Incyte: Consultancy, Research Funding.

Description: Background: In recent years, there has been a growing interest in telemedicine initiatives that maximize outcomes, reduce healthcare costs, and improve quality. The COVID-19 pandemic reduced healthcare access for many patients, including those undergoing chemotherapy, and thus accelerated these initiatives. We sought to evaluate the potential utility of telemedicine initiatives for lymphoma patients undergoing immunochemotherapy. Methods: To address this question, 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 those for which dose modifications were required. Dose modifications were defined as a change in prescribed dose from the preceding cycle, or a change in the administered dose by 10% or greater. Laboratory results, patient history, and/or physical exam findings that informed dose modifications were retrospectively identified. Results: Of the 1,290 total treatment cycles identified in 301 unique patients, 1,102 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 found that 144 cycles (11.2%) were subject to dosing adjustments. The cohort of patients that received dose adjustments was comprised of 104 unique patients, of which 87 (60.4%) were male and 57 (39.6%) were female. Average age at diagnosis was 64 years old (Range: 22-91). Our cohort represented greater than 10 different lymphoma subtypes, most commonly Diffuse Large B-cell lymphoma (66.3%), Follicular lymphoma (14.5%), and Peripheral T-cell lymphomas (7.7%). We examined the basis for each dose adjustment by reviewing the clinical records from visits immediately preceding the dose adjusted cycle. Of the 144 dose adjustments, 11% of cycles contained dose increases due to a well-tolerated previous dose noted in the clinical assessment based on a combination of laboratory findings, interim history, and physical exam. The remaining 89% of adjustments (n=128) were dose reductions. The decision to dose reduce was most commonly informed by the clinical history (n=104, 81%). The clinical history was dichotomized into newly reported patient symptoms (69/104) or interim complications (35/104), usually infectious (n=26). Clinical assessments utilized laboratory findings as a rationale for dose reductions in 33/128 (26%) of cycles, most of which were secondary to myelosuppression (28/33 cycles). In contrast, only 7/128 dose reductions were based on physical exam findings alone, all of which were due 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. Conclusions: The inability to perform a complete physical exam is a notable limitation of telemedicine initiatives. However, in an unselected group of lymphoma patients treated with immunochemotherapy, who subsequently had dose reductions to their regimens, we have found that all of the dose modifications were based on laboratory findings or the patient history, both of which are amenable to virtual visits. In stark contrast, no dose modifications were prompted by an abnormal physical exam finding alone. While further studies are needed, the data reviewed supports the implementation of telemedicine initiatives in lymphoma patients undergoing immunochemotherapy during the pandemic and potentially long term. Disclosures Phillips: Karyopharm: Consultancy; AstraZeneca: Consultancy; Beigene: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy; Bayer: Consultancy, Research Funding; BMS: Consultancy; Incyte: Consultancy, Other: travel expenses; Seattle Genetics: Consultancy; Cardinal Health: Consultancy.