Immunocompetent Recipients Research Articles

Renal manifestations of hepatitis E among immunocompetent and solid organ transplant recipients.

Hepatitis E virus (HEV) infections are generally self-limited. Rare cases of hepatitis E induced fulminant liver failure requiring liver transplantation are reported in the literature. Even though HEV infection is generally encountered among developing countries, a recent uptrend is reported in developed countries. Consumption of unprocessed meat and zoonosis are considered to be the likely transmission modalities in developed countries. Renal involvement of HEV generally holds a benign and self-limited course. Although rare cases of cryoglobulinemia are reported in immunocompetent patients, glomerular manifestations of HEV infection are frequently encountered in immunocompromised and solid organ transplant recipients. The spectrum of renal manifestations of HEV infection include pre-renal failure, glomerular disorders, tubular and interstitial injury. Kidney biopsy is the gold standard diagnostic test that confirms the pattern of injury. Management predominantly includes conservative approach. Reduction of immunosuppressive medications and ribavirin (for 3-6 mo) is considered among patients with solid organ transplants. Here we review the clinical course, pathogenesis, renal manifestations, and management of HEV among immunocompetent and solid organ transplant recipients.

Update on COVID-19 vaccination in pediatric solid organ transplant recipients.

COVID-19 vaccination has been successful in decreasing rates of SARS-CoV-2 infection in areas with high vaccine uptake. Cases of breakthrough SARS-CoV-2 infection remain infrequent among immunocompetent vaccine recipients who are protected from severe COVID-19. Robust data demonstrate the safety, immunogenicity, and effectiveness of several COVID-19 vaccine formulations. Importantly, Pfizer-BioNTech BNT162b2mRNA COVID-19 vaccine studies have now included children as young as 5years of age with safety, immunogenicity, and effectiveness data publicly available. In the United States, emergency use authorization by the Federal Drug Administration and approval from the Centers for Disease Control/Advisory Committee on Immunization Practices have been provided for the 5- to 11-year-old age group. Members of the International Pediatric Transplant Association (IPTA) provide an updated review of current COVID-19 vaccine data with focus on pediatric solid organ transplant (SOT)-specific issues. This review provides an overview of current COVID-19 immunogenicity, safety, and efficacy data from key studies, with focus on data of importance to pediatric SOT recipients. Continued paucity of data in the setting of pediatric transplantation remains a challenge. Further studies of COVID-19 vaccination in pediatric SOT recipients are needed to better understand post-vaccine COVID-19T-cell and antibody kinetics and determine the optimal vaccine schedule. Increased COVID-19 vaccine acceptability, uptake, and worldwide availability are needed to limit the risk that COVID-19 poses to pediatric solid organ transplant recipients.

Humoral response to SARS-CoV-2 infection among liver transplant recipients

ObjectiveImmunosuppressive agents are known to interfere with T and/or B lymphocytes, which are required to mount an adequate serologic response. Therefore, we aim to investigate the antibody response to SARS-CoV-2...

A Series of BRAF- and NRAS-Driven Murine Melanoma Cell Lines with Inducible Gene Modulation Capabilities

Murine cancer cell lines are powerful research tools to complement studies in genetically engineered mouse models. We have established 21 melanoma cell lines from embryonic stem cell-genetically engineered mouse models driven by alleles that model the most frequent genetic alterations in human melanoma. In addition, these cell lines harbor regulatory alleles for the genomic integration of transgenes and the regulation of expression of such transgenes. In this study, we report a comprehensive characterization of these cell lines. Specifically, we validated melanocytic origin, driver allele recombination and expression, and activation of the oncogenic MAPK and protein kinase B pathways. We further tested tumor formation in syngeneic immunocompetent recipients as well as the functionality of the integrated Tet-ON system and recombination-mediated cassette exchange homing cassette. Finally, by deleting the transcription factor MAFG with an inducible CRISPR/Cas9 approach, we show the utility of the regulatory alleles for candidate gene modulation. These cell lines will be a valuable resource for studying melanoma biology and therapy.

Robust and Persistent B- and T-Cell Responses after COVID-19 in Immunocompetent and Solid Organ Transplant Recipient Patients.

The development and persistence of SARS-CoV-2-specific immune response in immunocompetent (IC) and immunocompromised patients is crucial for long-term protection. Immune response to SARS-CoV-2 infection was analysed in 57 IC and 15 solid organ transplanted (TX) patients. Antibody responses were determined by ELISA and neutralization assay. T-cell response was determined by stimulation with peptide pools of the Spike, Envelope, Membrane, and Nucleocapsid proteins with a 20-h Activation Induced Marker (AIM) and 7-day lymphoproliferative assays. Antibody response was detected at similar levels in IC and TX patients. Anti-Spike IgG, IgA and neutralizing antibodies persisted for at least one year, while anti-Nucleocapsid IgG declined earlier. Patients with pneumonia developed higher antibody levels than patients with mild symptoms. Similarly, both rapid and proliferative T-cell responses were detected within the first two months after infection at comparable levels in IC and TX patients, and were higher in patients with pneumonia. T-cell response persisted for at least one year in both IC and TX patients. Spike, Membrane, and Nucleocapsid proteins elicited the major CD4+ and CD8+ T-cell responses, whereas the T-cell response to Envelope protein was negligible. After SARS-CoV-2 infection, antibody and T-cell responses develop rapidly and persist over time in both immunocompetent and transplanted patients.

Dissecting Richter's Syndrome in a Multiplexed CRISPR-Based Mouse Model Reveals Key Changes in MYC, Interferon and BCR Signaling Underlying Transformation

Richter's syndrome (RS) represents one of the foremost challenges in CLL management, and its pathogenesis remains largely undefined. We recently leveraged CRISPR-Cas9 in vivo gene editing to develop mouse models of RS by engineering multiplexed loss-of-function lesions typical of CLL (Atm, Trp53, Chd2, Birc3, Mga, Samhd1) in early stem and progenitor cells [Lineage - Sca-1 + c-kit + (LSK)] from MDR-Cd19Cas9 donor mice. These animals express Cas9-GFP in a B-cell restricted fashion and the leukemogenic MDR lesion, which mimics del(13q) when the sgRNA-transduced LSK cells are transplanted in CD45.1 immunocompetent recipients. Through these methods, we observed not only development of CLL, but also transformation into RS, and even captured a stage where CLL and RS were co-existing in the same animal (CLL/RS).We hypothesized that the molecular events underlying RS development would be markedly distinct from those of CLL and performed transcriptome analysis of FACS-sorted CLL and/or RS cells (5 CLL, 4 CLL/RS, 10 RS) and normal B cell controls from 4 age-matched wild type MDR-Cd19Cas9 mice. We identified a unique transcriptional profile of RS (ANOVA, FDR<0.1), characterized by upregulation of pathways involved in cell survival and proliferation (E2F/MYC targets, G2-M checkpoint, mitotic spindle). In contrast, genes involved in interferon gamma response, JAK-STAT and BCR signaling were predominantly downregulated.We asked whether these oncogenic circuitries would be recapitulated in human RS. By correlating the differentially expressed genes in murine RS with those of 7 human RS cases (compared to matched CLL), we identified similar pathway dysregulations with >100 commonly altered genes including upregulated cell cycle regulators (CDK1, CCNA2) and downregulated signaling adapters (ITPKB, MAP3K9).To further dissect gene regulatory networks driving transformation in the mouse, we profiled one CLL and one RS case by single cell ATAC sequencing (scATAC-seq). Consistent with the RNA-seq profiles, we detected increased chromatin accessibility of MYC-family associated transcription factor motifs (MAX, MYCN), and reduced accessibility of the pro-inflammatory STAT2 motif in RS (-log10adjP>50). Functionally, decreased interferon gamma responses were confirmed by the reduced ability of RS cells to phosphorylate STAT1 and STAT3 at 5‘ and 15‘ after IFN-gamma stimulation, compared to CLL and normal B cells (Western Blot).To define the genetic landscape underlying these changes, we performed whole genome sequencing analysis, and identified loss of chr12 and chr16 as recurrent events in RS (6/8 cases) and CLL/RS (2/2), but not in CLL cases (0/5). Among the genes encoded by these chromosomes, we identified several epigenetic drivers (Dnmt3a, Crebbp, Setd3/4), MAP kinase family members (Map4k5, Mapk1), cytoskeletal regulators (Hcls1, Rhoj), and interferon family receptors (Ifnar1/2, Ifngr2), suggesting that broad epigenetic modifications together with loss of BCR and interferon signaling molecules represent key events of transforming disease. RS cases were also characterized by a significantly higher number of full chromosome amplifications or deletions (median=6; range: 2-9), as compared to CLL or CLL/RS (1; 0-5, P=0.0008), consistent with the high degree of genomic instability observed in human disease.Finally, we asked whether the observed changes would impact RS therapeutic vulnerabilities, and exposed 15 primary murine RS splenocyte samples to 20 drugs in vitro for 24 hours, followed by CellTiter-Glo assessment of cellular viability. We observed strong sensitivity to the BRD4 inhibitor JQ1 and the mTOR inhibitor everolimus (both reported to interfere with MYC signaling, P<0.0001), and to CDK inhibitors (e.g. the CDK4/6 inhibitor palbociclib, P=0.0007), modest activity of the JAK1/2 inhibitor ruxolitinib (P=0.05), and minimal, if any, response to ibrutinib, venetoclax and fludarabine.In conclusion, we define the evolutionary trajectories and therapeutic vulnerabilities of RS in a mouse model, with unique transcriptional, genetic, and epigenetic features, indicative of broad changes in MYC, IFN and BCR signaling pathways and remarkable similarities with human disease. In-depth analyses of BCR signaling and in vivo treatment studies are underway and will refine mechanistic insights into the biology of RS. DisclosuresDavids: Surface Oncology: Research Funding; Eli Lilly and Company: Consultancy; Genentech: Consultancy, Research Funding; Takeda: Consultancy; MEI Pharma: Consultancy; Janssen: Consultancy; Verastem: Consultancy, Research Funding; Ascentage Pharma: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Merck: Consultancy; Adaptive Biotechnologies: Consultancy; Research to Practice: Consultancy; AbbVie: Consultancy; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; BeiGene: Consultancy. Letai: Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board; Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

Expression of p53 up-regulated modulator of apoptosis (PUMA) in non-melanoma skin cancer of long-term immunosuppressed solid organ transplant recipients compared to immunocompetent patients.

The risk of UV radiation (UVR)-induced non-melanoma skin cancer (NMSC) is dramatically increased in immunosuppressed organ transplant recipients compared to immunocompetent patients. In the skin, p53 up-regulated modulator of apoptosis (PUMA) is a central regulator of apoptosis in response to UVR damage and immune response regulation. Data on the expression of PUMA in patients with NMSC relative to immune status is limited To study differences in the expression and distribution of PUMA in cutaneous SCC and BCC by immunohistochemistry between immunocompetent patients and organ transplant recipients, and the effect of CsA-containing immunosuppressive maintenance regimens on this expression. PUMA expression in SCC (n = 34) and BCC (n = 20) was analysed comparatively by immunohistochemical staining in matched cohorts of 27 immunocompetent patients and 27 organ transplant recipients SCC and BCC showed unequivocal positive PUMA expression, however, there was no significant difference in NMSC between organ transplant recipients and immunocompetent patients. A 17% reduction in staining score for PUMA in SCC, but not in BCC, of organ transplant recipients treated with a cyclosporin (CsA)-containing regimen was noted compared to organ transplant recipients without chronic CsA intake (p = 0.0381) PUMA expression in SCC, but not BCC, is significantly reduced by CsA-containing therapy, suggesting a disturbance of apoptosis by iatrogenic immunosuppression with a divergent impact on SCC and BCC.

Lesion Genotype Modifies High-Fat Diet Effects on Endometriosis Development in Mice.

Endometriosis is a chronic, estrogen-dependent gynecologic disorder that affects reproductive-aged women and to a lesser extent, post-menopausal women on hormone therapy. The condition is associated with systemic and local immune dysfunctions. While its underlying mechanisms remain poorly understood, endometriosis has a genetic component and propensity for the disease is subject to environmental, nutritional, and lifestyle influences. Previously, we showed that high-fat diet (HFD) increased ectopic lesion numbers, concurrent with systemic and peritoneal changes in inflammatory and oxidative stress status, in immunocompetent recipient mice ip administered with endometrial fragments null for Krüppel-like factor 9 gene. Herein, we determined whether HFD modifies lesion parameters, when recipient peritoneal environment is challenged with ectopic wild-type (WT) endometrial fragments, the latter simulating retrograde menstruation common in women during the menstrual period. WT endometrium-recipient mice fed HFD (45% kcal from fat) showed reduced lesion incidence, numbers, and volumes, in the absence of changes in systemic ovarian steroid hormone and insulin levels, relative to those fed the control diet (CD, 17% kcal from fat). Lesions from HFD- and CD-fed recipients demonstrated comparable gene expression for steroid hormone receptors (Esr and Pgr) and cytokines (Il-6, Il-8, and CxCL4) and similar levels of DNA oxidative biomarkers. HFD moderately altered serum (3-nitrotyrosine and methionine/homocysteine) and peritoneal (reduced glutathione/oxidized glutathione) pro-oxidative status but had no effect on peritoneal inflammatory (tumor necrosis factor α and tumor necrosis factor receptor 1) mediators. Results indicate that lesion genotype modifies dietary effects on disease establishment and/or progression and if translated, could be important for provision of nutritional guidelines to women with predisposition to, or affected by endometriosis.

Hyper-Activation of the Adenosinergic Axis in the Eμ-TCL1 Chronic Lymphocytic Leukemia Mouse Model Offers Therapeutic Opportunities

Background. Tumor immunosuppression is a major cause for treatment failure and disease relapse, both in solid tumors and leukemia. Several indications suggest that increased extracellular adenosine levels, which potently suppress T cell responses and skew macrophages towards an M2 phenotype, characterize the tumor microenvironment. Hence, there is intense investigation to identify drugs that target this axis.We previously showed that CLL cells, particularly those endowed in the proliferation centers, express CD39 and CD73, the enzymes that lead to extracellular adenosine production. We also showed that the A2A adenosine receptor is expressed at high levels in CLL lymph nodes, likely due to the effect of hypoxia in positively modulating adenosine production.Aim of the work. The main aim of this work is to study the expression and the role of the adenosinergic axis in vivo, determining the impact of this signaling pathway in CLL progression and modulation of immune responses. To this purpose, we exploited the Eμ-TCL1 (TCL1) mouse model based on the adoptive transfer of spleen-purified leukemic cells in naïve C57BL/6 immunocompetent recipients (WT).Results. Firstly, expression of the adenosinergic axis (CD39, CD73, CD26 and A2A) both on the leukemic side and on bystander immune cells was assessed by comparing cells obtained from the spleen, the bone marrow (BM) and peripheral blood (PB) of TCL1 and WT mice. Flow cytometry analysis of B220+/CD5+ leukemic cells showed that CD39 was constantly expressed at high levels, in contrast to normal circulating B cells from naïve mice. CD73 expression was more heterogeneous, with both CD73+ and CD73- leukemic clones. These data recapitulate the phenotype observed in CLL patients and sustain the hypothesis of variable levels of adenosine in tissues colonized by leukemic cells. In contrast, CD26, the adenosine deaminase-anchoring protein was poorly detected, as opposed to WT B cells. Real-time PCR data obtained from purified cell populations confirmed this expression pattern and also indicated dramatic up-regulation in the expression of the A2A receptor, suggesting that leukemia development is accompanied by increased ability to respond to extracellular adenosine. The development of CLL is usually associated with specific T cell and myeloid subset changes. Multiparametric flow cytometry analyses, with selective markers, were adopted to identify naïve, memory, central memory, effector memory and effector T cells, as well as inflammatory, intermediate or patrolling monocytes. Data obtained confirm that in TCL1 mice the naïve T cell subset was dramatically reduced compared to WT mice, with the simultaneous increase in memory, exhausted and regulatory T cell sub-populations. Both CD4+ and CD8+ lymphocyte subsets in TCL1 mice were highly CD39+ compared to the normal counterpart, as opposed to CD73 that was expressed in distinct subpopulation of cells, including regulatory T cells. No significant differences were highlighted for CD26, in all the tissues considered, while the A2A receptor became expressed at very high levels in all cellular components of the leukemic niche. In the monocyte compartment, we observed a reduction of the inflammatory subset, with a parallel increase of patrolling population, functionally similar to M2 tumor-supportive macrophages.Given the role of low oxygen tension as a critical modulator of the adenosinergic system, hypoxia levels were determined, comparing tumor-bearing and WT mice. Mice were treated with pimonidazole hydrochloride, a hypoxia marker, 2 hours prior euthanasia. Flow cytometry and immuno-histochemistry analyses indicated that leukemic engraftment is paralleled by increased hypoxia levels in the lymphoid tissues, such as spleen or BM.Conclusions.Taken together, these results show that the adenosinergic axis is over-expressed in the TCL1 mouse model and confirm that the presence of leukemic cells polarizes the host immune cells, favoring the establishment of a tumor supportive environment. This could be due, at least partly, to the presence of a highly hypoxic leukemic niche. These observations confirm what described for CLL patients, indicating that this animal model can be used to test the impact of adenosine-targeting therapies, in combination with drugs that directly target the clone. DisclosuresNo relevant conflicts of interest to declare.

Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells

Bone marrow (BM) chimeric mice are a valuable tool in the field of immunology, with the genetic manipulation of donor cells widely used to study gene function under physiological and pathological settings. To date, however, BM chimera protocols require myeloablative conditioning of recipient mice, which dramatically alters steady-state hematopoiesis. Additionally, most protocols use fluorescence-activated cell sorting (FACS) of hematopoietic stem/progenitor cells (HSPCs) for ex vivo genetic manipulation. Here, we describe our development of cell culture techniques for the enrichment of functional HSPCs from mouse BM without the use of FACS purification. Furthermore, the large number of HSPCs derived from these cultures generate BM chimeric mice without irradiation. These HSPC cultures can also be genetically manipulated by viral transduction, to allow for doxycycline-inducible transgene expression in donor-derived immune cells within non-conditioned immunocompetent recipients. This technique is therefore expected to overcome current limitations in mouse transplantation models.

Antifungal Prophylaxis for Adult Recipients of Veno-Venous Extracorporeal Membrane Oxygenation: A Cautionary Stance During the COVID-19 Pandemic.

Antifungal Prophylaxis for Adult Recipients of Veno-Venous Extracorporeal Membrane Oxygenation: A Cautionary Stance During the COVID-19 Pandemic.

Off-the-Shelf Chimeric Antigen Receptor T Cells: How Do We Get There?

Banked chimeric antigen receptor (CAR) T cells immediately available for off-the-shelf (OTS) application can solve key limitations of patient-specific CAR T-cell products while retaining their potency. The allogeneic nature of OTS cell therapies requires additional measures to minimize graft-versus-host disease and host-versus-graft immune rejection in immunocompetent recipients. In this review, we discuss engineering and manufacturing strategies aimed at minimizing unwanted interactions between allogeneic CAR T cells and the host. Overcoming these limitations will improve safety and antitumor potency of OTS CAR T cells and facilitate their wider use in cancer therapy.

52350 PKM2 mediates anti-tumor immunity and T cell dysfunction

ABSTRACT IMPACT: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and benefit to patients; our work will identify novel mediators of this process for both therapeutic potential and underlying mechanism, allowing for both potential immediate clinical utility and identification of future targets based on new mechanistic insights. OBJECTIVES/GOALS: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and clinical benefit to the majority of patients. We aim to interrogate a novel mediator of dysfunction identified from transcriptome analyses, pyruvate kinase muscle isozyme isoform 2 (PKM2), for therapeutic utility and underlying mechanism. METHODS/STUDY POPULATION: Transcriptome analyses of CD8+ lymphocytes from tumor-bearing lungs from both murine KrasG12D p53-/- and human non-small cell lung cancer (NSCLC) patients were performed, and differentially expressed genes identified. Flow cytometric analyses for PKM isoform expression and effects of target knockdown on accumulation of dysfunctional characteristics, including checkpoint and transcription factor expression, proliferation, and cytokine production, were performed using an in vitro co-culture of murine antigen-specific T (OT-I) cells and antigen-expressing NSCLC (HKP1-ova) cells. In vivo examination of the same was performed using adoptive transfer of OT-I cells into immunocompetent recipient mice with engraftment of HKP1-ova cells, and subsequent evaluation of mouse survival and T cell phenotypes. RESULTS/ANTICIPATED RESULTS: Transcriptome analyses demonstrated that PKM expression was upregulated in dysfunctional T cells from both murine and human samples. This was confirmed both in vitro with co-culture and in vivo with adoptive transfer approaches, with both activated and dysfunctional OT-I cells expressing higher levels of isoform 2 of PKM than naive OT-I cells. Expression of PKM2 mimicked the kinetics of the transcription factor Tox, a known driver of dysfunction, and knockdown of PKM2 resulted in reduced granzyme B expression, and increased proportions of progenitors with fewer terminally differentiated dysfunctional cells. Knockdown of PKM2 in adoptively-transferred OT-I cells led to enhanced tumor control; results are being extended to other tumor models, and T cells metabolically profiled with PKM2 manipulation. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work identified a novel mediator of dysfunction whose targeting has the potential to enhance anti-tumor immunity. Mechanistically, targeting PKM2 led to altered T cell differentiation to a dysfunctional state, linking metabolic phenotypes to these traits and underlining the importance and therapeutic potential of T cell metabolic pathways.

Does Transfusion of Red Blood Cells Impact Germline Genetic Test Results?

Purpose: molecular testing is often indicated for recently transfused patients. However, there are no guidelines regarding the potential interference from donor DNA or whether it is necessary to wait for a period of time post-transfusion prior to genetic testing. While the majority of patients are transfused in the non-trauma setting using leukoreduced (LR) red blood cell products, the degree of leukoreduction varies among centers and is not universally practiced. Methods: whole blood units collected from anonymous donors were used in an in vitro transfusion model. One unit was split: half being leukoreduced simulating a leukopenic recipient and half left untreated. Donors were simulated by leukoreduced, partially leukoreduced (PLR), or non-leukoreduced units, transfused in 2, 5, or 16 unit equivalents. DNA from the combinations were subjected to short tandem repeat (STR) analysis for chimerism detection. Results: donor DNA was not detectable in any of the LR combinations, but detected in the PLR combinations, ranging from 0.1 to 1.5% donor DNA in the immunocompetent recipient and 6.3–27.8% in the leukopenic recipient. Non-LR donor DNA was also detected (13–95%). Conclusion: donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients but may interfere in immunocompromised recipients.

Abstract 2752: The gain-of-function STAT5BN642H mutation as a driver of mature T cell leukemia/lymphoma

Abstract Mature T cell leukemias/lymphomas (MaTCL) are a group of rare hematological malignancies of mostly incurable prospects due to limited efficient therapies and a lack of faithful pre-clinical models. STAT5 transcription factors are critical downstream effectors of cytokine and growth factor signaling through the JAK/STAT pathway, particularly in the hematopoietic compartment. The two gene products, STAT5A and STAT5B, play important roles in cell proliferation, survival and differentiation. It is increasingly evident that hyperactive JAK/STAT signaling plays a role in these MaTCL diseases. Notably, a hotspot gain-of-function (GOF) mutation in STAT5B, N642H, has been found in over 100 patients with T cell neoplasia, and is associated with more aggressive disease, therapy resistance and worse prognosis. To investigate the role of STAT5BN642H in MaTCL, we utilized our transgenic mouse model harboring moderate expression of human STAT5BN642H in the hematopoietic compartment. Notably, STAT5BN642H transgenic mice rapidly develop aggressive mature CD8+ T cell disease, suggesting that CD8+ T cells are particularly susceptible to transformation by this mutation. Further examination of these mice revealed prominent infiltration of neoplastic T cells into peripheral organs including lung, skin, brain and liver. Interestingly, in addition to CD8+ T cells, infiltration of CD4+ as well as γδ T cells was also observed in various organs of the STAT5BN642H mice, suggestive of STAT5BN642H-driven transformation of other T cell lineages. This would be more consistent with human patients carrying this mutation, who suffer predominantly from MaTCL of CD4+ or aggressive γδ T cell subtypes. Therefore, we isolated these T cell subsets from the transgenic mice and performed syngeneic transplant experiments to assess T cell transformation. Indeed, transplants of γδ or CD4+ T cells from STAT5BN642H transgenic mice into immunocompetent recipients resulted in the development of γδ or CD4+ T cell neoplasia, respectively, demonstrating the full transforming capacity of STAT5BN642H in multiple T cell lineages. From this, we have generated a novel γδ T cell line harboring the STAT5BN642H mutation, which we are utilizing together with the mouse models to interrogate oncogenic mechanisms of STAT5B (through immunophenotyping and sequencing efforts) as well as to screen and test new therapeutic options for these diseases. Overall, these data highlight the aggressive nature of the STAT5BN642H driver mutation in MaTCL. We report on novel pre-clinical models for aggressive γδ or CD4+ MaTCL that more closely recapitulate human disease. These models will be valuable for understanding STAT5BN642H-driven T cell disease and for testing new, urgently needed MaTCL treatment strategies. Citation Format: Heidi A. Neubauer, Tobias Suske, Susann Schönefeldt, Simone Tangermann, Auke Boersma, Thomas Rülicke, Vasileios Bekiaris, Lukas Kenner, Richard Moriggl. The gain-of-function STAT5BN642H mutation as a driver of mature T cell leukemia/lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2752.

Engineered off-the-shelf therapeutic T cells resist host immune rejection.

Engineered T cells are effective therapies against a range of malignancies, but current approaches rely on autologous T cells, which are difficult and expensive to manufacture. Efforts to develop potent allogeneic T cells that are not rejected by the recipient's immune system require abrogating both T- and natural killer (NK)-cell responses, which eliminate foreign cells through various mechanisms. In the present study, we engineered a receptor that mediates deletion of activated host T and NK cells, preventing rejection of allogeneic T cells. Our alloimmune defense receptor (ADR) selectively recognizes 4-1BB, a cell surface receptor temporarily upregulated by activated lymphocytes. ADR-expressing T cells resist cellular rejection by targeting alloreactive lymphocytes in vitro and in vivo, while sparing resting lymphocytes. Cells co-expressing chimeric antigen receptors and ADRs persisted in mice and produced sustained tumor eradication in two mouse models of allogeneic T-cell therapy of hematopoietic and solid cancers. This approach enables generation of rejection-resistant, 'off-the-shelf', allogeneic T-cell products to produce long-term therapeutic benefit in immunocompetent recipients.

137 Decreased cytotoxic T cells, decreased cytotoxic/regulatory T-cell ratio, and decreased TCR clonality are associated with increased numbers of primary cutaneous squamous cell carcinomas in solid organ transplant recipients

Background: We developed a novel analytical method to define tumor infiltrating lymphocyte (TIL) phenotype in immune competent vs. immune suppressed cSCC patients based on data obtained from single cell sequencing and gene expression. Methods: CD8+ TILs (n = 34,399 for cSCC, n = 14,902 for TSCC) obtained from fresh tumor specimens from immunocompetent (n = 5) versus organ transplant recipients (OTRs; n=6) were subject to single-cell RNA profiling matched with T-cell receptor (TCR) sequencing via barcoding. Data were analyzed using iCellR, a custom R packaged we developed for single cell sequencing analysis. Antigens recognized by the top 10 clonotypes for each sample were assessed using McPAS. Results: Gene expression analysis showed TSCC had fewer cytotoxic cells (56% vs. 66%, p < 0.0001), fewer naïve cells (18% vs. 26% p < 0.0001), similar numbers of regulatory cells (8% vs. 5%) and similar numbers of exhausted T cells (9% vs. 10%). CD8+ TILs from TSCC exhibited more homogeneous gene expression compared with immunocompetent patients. TILs from both OTRs and immunocompetent patients showed clonality. However, fewer TCR clonotypes were observed in immune suppressed transplant patients (mean = 544 vs. 1140, p < 0.05). Many of the TCR sequences represent antigens previously reported in melanoma, other carcinomas and viral infections. The majority of TCR sequences for the top 10 clonotypes of each sample have known antigens, but up to 24% of these sequences recognize putative neoantigens. Solid organ transplant recipients in our study showed increased numbers of cSCC events over 12 months (6.2 vs. 1.2, p < 0.01). Conclusion: TSCC TILs tend to show decreased cytotoxic CD8+ TIL, decreased cytotoxic/regulatory T cell ratio and increased exhausted and naïve T cells and decreased cytotoxic T cells. This may contribute to the higher numbers of primary cSCC seen in OTRs.

1685-P: Islet Encapsulation with Tannic Acid Delays Allogeneic Islet Graft Rejection

Type 1 diabetes (T1D) is defined by the destruction of insulin-producing cells. Islet transplantation can restore glucose homeostasis and prevent serious hypoglycemic events; however, long-term graft survival is low. Oxidative stress can mediate islet transplantation rejection and suppressing oxidative stress with nanothin islet encapsulation materials comprised of alternating layers of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), a scaffold polymer, may allow for improved graft survival without systemic immunosuppression. We hypothesize that (PVPON/TA)-encapsulation of islets will prolong graft function following allotransplantation into immunocompetent recipients without utilizing systemic immunosuppression. Allogeneic transplantation of encapsulated C57BL/6 islets into diabetic non-obese diabetic (NOD) mice (n=12) significantly delayed graft failure by more than 90 days compared to controls and significantly reduced free radical-adduct formation produced by neutrophils and macrophages at the graft site. Gene expression displayed a 5- and 20-fold increase of anti-inflammatory genes Retnla and Arg1, respectively, and 3-fold reductions in Ccl5, Cxcl10, Prf1, and Grzmb mRNA accumulation in (PVPON/TA) versus control grafts. Flow cytometry analysis of encapsulated grafts contained fewer numbers of infiltrating dendritic cells and CD8 T cells and reduced frequencies of CCL5+, CXCR3+, MHC-II+ macrophages and perforin+ CD8 T cells by 3-fold compared to controls. Encapsulated grafts also reduced the frequencies of TNF+ and MHC-II+ dendritic cells by 2-fold. Additionally, encapsulation increased the frequency of arginase-1+ and CD206+ alternatively-activated macrophages 4-fold, suggesting that (PVPON/TA)-encapsulation may be a promising technique to prolong clinical islet graft function by skewing macrophage phenotypes and suppressing cytotoxic T cell effector responses in the absence of systemic immunosuppression. Disclosure J. Barra: None. V. Kozlovskaya: None. E. Kharlampieva: None. H.M. Tse: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK099550); JDRF (SRA-2016-270-S-B, JDRF 2-SRA-2019-692-S-B); National Science Foundation (NSF-DMR 1608728); National Institute of General Medical Sciences (T32GM008111)

Abstract B39: Regulation and function of IL-1b in immune modulation of pancreatic cancer

Abstract Pancreatic intraepithelial neoplasms (PanINs) are well-defined precursor lesions of pancreatic ductal adenocarcinoma (PDA). The initiation and progression of PanINs is marked by dynamic stromal reconstruction and localized acute inflammation. Using an orthotopic transplantation model of early and advanced pancreatic neoplasia in immunocompetent recipient mice, we have uncovered a crucial role for the cytokine IL-1b in KRasG12D-driven PanIN evolution. Tumor cell-derived IL-1b¢ was found to be critical for shaping the protumorigenic PanIN microenvironment by promoting activation of quiescent pancreatic stellate cells and establishing an immunosuppressive milieu mediated by M2 macrophages, MDSCs and CD1dhiCD5+ regulatory B cells. Furthermore, loss of IL-1 signaling in tumor stroma activates the cytotoxic T-cell immune response, significantly attenuates PanIN growth and confers survival advantage to PDAC-bearing mice. Tumor cell expression of IL-1b in vivo is driven by upregulation of TLR4 signaling and activation of the NLRP3 inflammasome. KrasG12D enhances surface TLR4 expression, sensitizing transformed cells to TLR4 ligands in the microenvironment, including the pancreatic microbiome. Collectively, our study describes a complex tumor-stromal interplay orchestrated by the TLR4-NLRP3-IL-1b signaling axis, which is critical for shaping the tumor microenvironment and driving pancreatic tumorigenesis. Citation Format: Shipra Das, Sandra Vogt, Christina Hadju, Dafna Bar-Sagi. Regulation and function of IL-1b in immune modulation of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B39.

Hypoimmunogenic Derivatives of Induced Pluripotent Stem Cells Evade Immune Rejection in Fully Immunocompetent Allogeneic Recipients

Induced pluripotent stem cells (iPSCs) are promising candidates for regenerative cell-based therapies. Because autogenic iPSCs are not available for acute treatment procedures, allogeneic strategies have to be developed which evade immune rejection. We showed in this study that both mouse and human iPSCs lose their immunogenicity when major histocompatibility complex (MHC) class I and II genes are inactivated and CD47 is over-expressed. Hypoimmunogenic mouse (β2m-/-Ciita-/-Cd47tg miPSCs) and human (B2M-/-CIITA-/-CD47tg hiPSCs) iPSCs were generated via CRISPR/Cas9 technology and lentiviral overexpression. Unmodified and β2m-/-Ciita-/-Cd47tg miPSCs as well as their cell grafts were injected into syngeneic C57BL/6 or allogeneic BALB/c mice. Unmodified and B2M-/-CIITA-/-CD47tg hiPSCs and their cell grafts were injected into humanized mice. The cellular and antibody response was quantified and in vivo bioluminescence imaging were performed to assess cell survival. FACS analysis showed normal expression of MHC I and MHC II on unmodified cells and depleted MHC I and MHC II on hypoimmunogenic cells. CD47 showed a 3-4 time increase in hypoimmunogenic cells compared to unmodified cells. After allogeneic transplantation, hypoimmunogenic cells showed a significantly decreased immune response in ELISPOT assays, comparable to the level of syngeneic transplants. No donor-specific antibodies were detected and the hypoimmunogenic cells were protected against innate response in in vivo innate cytotoxicity assays and in Xcelligence assays. Additionally, 100% graft survival was achieved with hypoimmunogenic cells in vivo with an observation period of 50 days. Our results clearly demonstrate that the lack of immunogenicity is from MHC I and MHC II antigens and that the overexpression of the CD47 molecule prevents against innate immune responses. Thus, hypoimmunogenic iPSCs might serve as an unlimited cell source for the generation of universally-compatible "off-the-shelf" cells grafts or tissues in future clinical applications.