Mature CD4 Research Articles

Functional reconstruction of human AML reveals stem cell origin and vulnerability of treatment-resistant MLL-rearranged leukemia.

Chemoresistance remains the major challenge for successful treatment of acute myeloid leukemia (AML). Although recent mouse studies suggest that treatment response of genetically and immunophenotypically indistinguishable AML can be influenced by their different cells of origin, corresponding evidence in human disease is still largely lacking. By combining prospective disease modeling using highly purified human hematopoietic stem or progenitor cells with retrospective deconvolution study of leukemia stem cells (LSCs) from primary patient samples, we identified human hematopoietic stem cells (HSCs) and common myeloid progenitors (CMPs) as two distinctive origins of human AML driven by Mixed Lineage Leukemia (MLL) gene fusions (MLL-AML). Despite LSCs from either MLL-rearranged HSCs or MLL-rearranged CMPs having a mature CD34-/lo/CD38+ immunophenotype in both a humanized mouse model and primary patient samples, the resulting AML cells exhibited contrasting responses to chemotherapy. HSC-derived MLL-AML was highly resistant to chemotherapy and expressed elevated amounts of the multispecific anion transporter ABCC3. Inhibition of ABCC3 by shRNA-mediated knockdown or with small-molecule inhibitor fidaxomicin, currently used for diarrhea associated with Clostridium difficile infection, effectively resensitized HSC-derived MLL-AML toward standard chemotherapeutic drugs. This study not only functionally established two distinctive origins of human LSCs for MLL-AML and their role in mediating chemoresistance but also identified a potential therapeutic avenue for stem cell-associated treatment resistance by repurposing a well-tolerated antidiarrhea drug already used in the clinic.

IMMUNOLOGICAL REACTIVITY IN MEGAPOLIS RESIDENTS

Introduction: Living in an urban environment with a high population density, time pressure, increased frequency of involuntary social contacts privacy violation, lack of recreation areas may lead to not only mental health disorders, but also to somatic diseases. The evidence on the state of immune homeostasis of residents of the urbanized environment is limited. Aim: To assess immunological reactivity in residents of a megapolis. Methods: We assessed phagocytic activity, phenotypes of lymphocytes CD3+, CD5+, CD4+, CD8+, CD10+, cytokines IL-1β, IL-2, TNF-α, immunoglobulins IgA, IgM, IgE, IgG and carcinoembryonic antigen in a sample of 40-60 years old healthy residents of Moscow. Results: The analysis showed that in 83.33% of subjects the ratios of lymphoproliferation and differentiation of lymphoid populations were in balance. There was no deficiency of either CD8+ immunosuppressors or CD4+ T-helpers. Concentrations of lymphoid cells with receptors to interleukin - 2 (CD25+) were detected within the physiological standard in 98.79 % of the study participants. It was found that in 8.13 % of individuals the increased content of mature functional CD3+ lymphocytes was associated with greater concentrations of immunoglobulin E, but within physiological limits. Conclusion: The most common immune defense defect in 40-60-year-old residents of the metropolis is a deficiency in serum IgA (76,04 %) and mature T-lymphocytes (51,04 %) in peripheral blood

Long-term physical training in adolescent sprint and middle distance swimmers alters the composition of circulating T and NK cells which correlates with soluble ICAM-1 serum concentrations

PurposeIt remains unknown how different training intensities and volumes chronically impact circulating lymphocytes and cellular adhesion molecules. First, we aimed to monitor changes in NK and T cells over a training season and relate these to training load. Second, we analyzed effects of training differences between swimmers on these cells. Finally, we examined if changes in lymphocytes were associated with sICAM-1 concentrations.MethodsWe analyzed weekly training volume, training intensity, proportions of T and NK cells and serum sICAM-1 in eight sprint (SS) and seven middle-distance swimmers (MID) at three points over a 16-week training period: at the start (t0), after 7 weeks of increased training load (t7) and after 16 weeks, including 5-day taper (t16).ResultsTraining volume of all swimmers was statistically higher and training intensity lower from t0–t7 compared to t7–t16 (p = 0.001). Secondly, training intensity was statistically higher in SS from t0–t7 (p = 0.004) and t7–t16 (p = 0.015), while MID had a statistically higher training volume from t7–t16 (p = 0.04). From t0–t7, NK (p = 0.06) and CD45RA+CD45RO+CD4+ cells (p < 0.001) statistically decreased, while CD45RA−CD45RO+CD4+ cells (p = 0.024) statistically increased. In a subgroup analysis, SS showed statistically larger increases in NK cells from t7–t16 than MID (p = 0.012). Lastly, sICAM-1 concentrations were associated with changes in CD45RA−CDRO+CD4+ cells (r = − 0.656, p = 0.08).ConclusionThese results indicate that intensified training in swimmers resulted in transient changes in T and NK cells. Further, NK cells are sensitive to high training volumes. Lastly, sICAM-1 concentrations may be associated with the migration and maturation of CD4+ cells in athletes.

'One Year Later' - SARS-CoV-2-Specific Immunity in Mild Cases of COVID-19

Background: Duration and quality of immunity to SARS-CoV-2 have significant implications for the management of COVID-19 pandemic. Here, we present a comprehensive set of immunological data from a cohort of individuals (n=43), 12 months after mild COVID-19 disease and in the absence of virus re-exposure.Methods: Serum and PBMC were collected from mild-COVID-19 convalescents 12 months after the COVID-19 positive PCR (n=43) and from healthy SARS-CoV-2-seronegative controls (n=15). Serum titers of SARS-CoV-2-specific immunoglobulins were quantified by ELISA and virus neutralisation activity was assessed using SARS-CoV-2-Spike pseudovirus particles. Frequencies of Spike and RBD-specific memory B cells were quantified by flow cytometry. Magnitude of memory T cell responses was quantified and phenotyped with an activation-induced marker assay.Findings: In the absence of re-exposure to SARS-CoV-2 Spike- and RBD-specific antibodies were present in 90% of COVID-19 convalescents 12 months post-infection. RBD-specific IgG + memory B cells were maintained in 88.9% of patients, while 62% of patients had serum neutralising activity. Functionally mature memory CD4 + and CD8 + T cells were maintained at frequencies previously reported for earlier time points post-COVID-19, indicating substantial maintenance of durable T cell responses. Interpretations: Immunity to SARS-CoV-2 persists for 12 months in mild COVID-19 convalescent patients that retain high Spike-specific antibody titres, virus neutralisation capacity and circulating RBD-specific memory B cells. Significantly, T cell immunity remained stable 12 months post-infection. This study offers vital information on the duration of natural COVID-19 immunity and its potential protective effect against SARS-CoV-2 reinfection and clinical disease, with clear implications for the ongoing management of the global pandemic. Funding Statement: This work was funded by project grants from The Hospital Research Foundation and Women’s and Children’s Foundation, Adelaide, Australia. This work has been supported by NIH contract 75N9301900065 (A.S, D.W).Declaration of Interests: A.S. is currently a consultant for Gritstone, Flow Pharma, Arcturus, Epitogenesis, Oxfordimmunotech, Caprion and Avalia. LJI has filed for patent protection for various aspects of T cell epitope and vaccine design work.Authors PGV, CMH, MGM, AELY, HB, ZAM, ZAD, AA, DA, JG, CF, SO, EMM, DJL, GM, EJG, BAJR, DS, CKL, MRB, DW, RAB, SCB and BGB declare no conflict of interest.Ethics Approval Statement: Study protocols were approved by the Central Adelaide Clinical Human Research Ethics Committee (#13050) and the Women’s and Children’s Health Network Human research ethics (protocol HREC/19/WCHN/65), Adelaide, Australia.

Comprehensive Phenotyping of Peripheral Blood T Lymphocytes in Healthy Mice.

T lymphocytes play a central role in antigen-specific immune responses. They modulate the function of different immune cells both through a direct contact (receptor binding) and through the secretion of cytokines. At the same time, they are deeply involved in the direct killing of aberrant target cells. T lymphocytes derive from a bone marrow precursor that migrates in the thymus where the main differentiation steps take place. Mature CD4 and CD8 single-positive cells, then, leave the thymus to reach the secondary lymphoid organs. T-cell subsets and their maturation steps can be identified mainly based on the expression of extracellular markers, intracellular transcription factors and cytokine production profiles. In this review, we report, from a cytometric point of view, an overview of the most important T-cell subpopulations and their differentiation state. © 2020 International Society for Advancement of Cytometry.

Establishment ofin VitroDrug Sensitivity Screening Assay for Chronic Lymphocytic Leukemia (CLL) Primary Patient Samples

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. CLL is characterized by proliferation and accumulation of monoclonal, small, mature CD5+ B-cells in peripheral blood, bone marrow and secondary lymphoid organs. The current treatment regimens have improved overall survival but CLL patients will eventually experience relapse. The disease is still incurable making it a necessity to discover and screen for new drugs. Patient derived CLL specimens undergo spontaneous apoptosis when grown in culture and is a major limitation to screening for new therapeutic drugs. We have established anin vitroculture conditions which supports growth and proliferation of patient derived primary CLL cells. Champions has a bank of primary CLL patient samples across different Rai stage, genetic mutations and relapsed and refractory cases. Using the optimizedin vitroculture conditions, we tested three CLL human primary samples (models) for proliferation . All three models proliferated 1.5 to to 2-fold in the optimized media in 6 days, as measured using CellTiter-Glo®. We also examined the phenotypic changes in the cells after growing them in the established media. Flow cytometric analysis showed that the CLL cells mostly retained the primary phenotypic characteristic CD5+CD10-Cd19+CD20+ even after being in the culture for 6 days. Next, we screened for sensitivity of primary CLL patient samples (N=10) against known standard of care (SOC) drugs venetoclax, ibrutinib, idelasib, chlorambucil and cytarabine. Primary patient samples derived from peripheral blood mononuclear cells (PBMC) were cultured in 96 well plates in the enriched media and treated with respective drugs over a concentration range over 5 logs. Drug sensitivity was assessed using CellTiter-Glo® luminescent cell viability assay on day 3. Ourin vitroassay indicated that some, but not all patient samples were sensitive to approved standard of care drugs. A relapsed bendamustine pre-treated patient sample was sensitive to all the SOC drugs tested. In addition to drug response, whole exome sequencing and RNAseq are being conducted on these samples, to compare mutational analyses with drug responsiveness. With clinically annotated patient-derived CLL samples, WES and RNAseq plus drug response to standard of care provides a comprehensiveex vivoplatform for the preclinical testing of drug candidates, which may not only provide information on agent efficacy, but that can permit potential biomarker mining and exploration of patient selection criteria for investigational new agents in CLL. Disclosures No relevant conflicts of interest to declare.

Profiling peripheral B-cell subtypes most predictive for cardiac allograft rejection

Abstract Introduction B-cells have been strongly implicated in cardiac allograft rejection (CAR). Recently, however, the CTOT-11 trial showed that depleting mature CD20+ B-cells did not reduce rates of rejection in cardiac allograft recipients and unexpectedly increased the severity of allograft vasculopathy. Therefore, it can be hypothesized that differing phenotypic subtypes of B-cells correspond with different biological mechanisms relating to CAR. Though, current applications to quantify these subtypes of immune cells, i.e with immunohistochemistry or flow cytometry, are often restricted by limited cell markers and cost-burden; therefore, we demonstrate a novel deconvolution method, FARDEEP, that has been validated to accurately enumerate peripheral blood mononuclear cell-subtypes (PBMCs) in a quicker and more cost-effective manner. Purpose To better understand the association of different B-cell subtypes in CAR by identifying the B-cell subtype most predictive for pathologically defined rejection. Methods The machine learning tool, FARDEEP, was trained with the transcriptomic signatures of 29 PBMC subtypes, characterized by previous single-cell RNA experiments. FARDEEP then was used to deconvolute data-mined RNA from 259 blood samples from 98 cardiac allograft recipients enrolled in the CARGO study (GSE2445). Random forest tree (RF) was then used to analyze the levels of deconvoluted subtypes to predict the severity of rejection assessed by endomyocardial biopsy. Finally, RF was used to identify the subtypes of PBMCs most valuable in predicting rejection. Results Out of the 259 samples with consensus pathological readings, 140 had a consensus International Society of Heart and Lung Transplant grade of 0, 63 with grade 1a, 31 with grade 1b, and 25 with grade 3a or higher. We grouped biopsy samples with grade 0, 1a, and 1b as “low-risk” rejection (n=234). 3a or higher samples were grouped as “high-risk” (n=25). There were no grade 2s in the dataset. According to the dataset, blood was extracted from patients on average 72.5 days post-transplant. The RF had good performance in predicting rejection severity. (Figure 1a) CD20- plasmablast cells were stronger predictors for differentiating high-risk from low-risk compared to CD20+ B-cell populations (i.e B Naive and B Memory cells). (Figure 1b) Overall, however, dendritic cells (DCs), neutrophils, monocytes, and basophils were the strongest predictors for rejection. Conclusion Our findings support the results from the CTOT-11 trial showing that CD20+ B-cells may not contribute to CAR as significantly as seen with other PBMC subtypes. Instead, we showed that among B-cells, CD20- plasmablasts were more likely associated with CAR, possibly explaining why targeting CD20 was ineffective in preventing rejection. Thus, targeting plasmablast-associated markers could potentially be more useful to prevent CAR. Model Performance with Variables Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): 1) Society of Academic Emergency Medicine Foundation; 2) The Jewish Fund

The Role of Antibodies in the Pathogenesis of Multiple Sclerosis.

The presence of persistent intrathecal oligoclonal immunoglobulin G (IgG) bands (OCBs) and lesional IgG deposition are seminal features of multiple sclerosis (MS) disease pathology. Despite extensive investigations, the role of antibodies, the products of mature CD19+ B cells, in disease development is still controversial and under significant debate. Recent success of B cell depletion therapies has revealed that CD20+ B cells contribute to MS pathogenesis via both antigen-presentation and T-cell-regulation. However, the limited efficacy of CD20+ B cell depletion therapies for the treatment of progressive MS indicates that additional mechanisms are involved. In this review, we present findings suggesting a potential pathological role for increased intrathecal IgGs, the relation of circulating antibodies to intrathecal IgGs, and the selective elevation of IgG1 and IgG3 subclasses in MS. We propose a working hypothesis that circulating B cells and antibodies contribute significantly to intrathecal IgGs, thereby exerting primary and pathogenic effects in MS development. Increased levels of IgG1 and IgG3 antibodies induce potent antibody-mediated cytotoxicity to central nervous system (CNS) cells and/or reduce the threshold required for antigen-driven antibody clustering leading to optimal activation of immune responses. Direct proof of the pathogenic roles of antibodies in MS may provide opportunities for novel blood biomarker identification as well as strategies for the development of effective therapeutic interventions.

Unique immunological profile in patients with COVID-19

The relationship between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and host immunity is poorly understood. We performed an extensive analysis of immune responses in 32 patients with severe COVID-19, some of whom succumbed. A control population of healthy subjects was included. Patients with COVID-19 had an altered distribution of peripheral blood lymphocytes, with an increased proportion of mature natural killer (NK) cells and low T-cell numbers. NK cells and CD8+ T cells overexpressed T-cell immunoglobulin and mucin domain-3 (TIM-3) and CD69. NK cell exhaustion was attested by increased frequencies of programmed cell death protein 1 (PD-1) positive cells and reduced frequencies of natural killer group 2 member D (NKG2D)-, DNAX accessory molecule-1 (DNAM-1)- and sialic acid-binding Ig-like lectin 7 (Siglec-7)-expressing NK cells, associated with a reduced ability to secrete interferon (IFN)γ. Patients with poor outcome showed a contraction of immature CD56bright and an expansion of mature CD57+ FcεRIγneg adaptive NK cells compared to survivors. Increased serum levels of IL-6 were also more frequently identified in deceased patients compared to survivors. Of note, monocytes secreted abundant quantities of IL-6, IL-8, and IL-1β which persisted at lower levels several weeks after recovery with concomitant normalization of CD69, PD-1 and TIM-3 expression and restoration of CD8+ T cell numbers. A hyperactivated/exhausted immune response dominate in severe SARS-CoV-2 infection, probably driven by an uncontrolled secretion of inflammatory cytokines by monocytes. These findings unveil a unique immunological profile in COVID-19 patients that will help to design effective stage-specific treatments for this potentially deadly disease.

Phosphate-modif ied CpG oligonucleotides induce in vitro maturation of human myeloid dendritic cells.

Myeloid dendritic cells (DCs) play an important role in the immune response; therefore, the search for compounds that can effectively activate DCs is a needful goal. This study was aimed to investigate the effect of synthetic CpG oligodeoxynucleotides (CpG-ODN) on the maturation and allostimulatory activity of myeloid DCs in comparison with other PAMP and DAMP molecules. For the research, we synthesized known CpG-ODN class C (SD-101 and D-SL03) containing thiophosphate internucleotide groups, and their original phosphate-modified analogues (SD-101M and D- SL03M) with mesylphosphoramide internucleotide groups (M = μ-modification). The effects of CpG-ODN and other activators were evaluated on DCs generated from blood monocytes in the presence of GM-CSF and IFN-α (IFN-DC) or IL-4 (IL4-DC). Evaluation of the intracellular TLR-9 expression showed that both types of DCs (IFN-DC and IL4-DC) contained on average 52 and 80 % of TLR-9-positive cells, respectively. The CpG-ODNs studied enhanced the allostimulatory activity of IFN-DCs, and the effect of μ-modified CpG-ODNs was higher than that of CpG-ODNs with thiophosphate groups. The stimulating effect of CpG-ODN at a dose of 1.0 μg/ml was comparable (for D-SL03, D-SL03M, SD-101) with or exceeded (for SD-101M) the effect of LPS at a dose of 10 μg/ml. At the same time, IFN-DCs were characterized by greater sensitivity to the action of CpG-ODNs than IL4-DCs. The enhancement of DC allostimulatory activity in the presence of CpG-ODNs was associated with the induction of final DC maturation, which was confirmed by a significant decrease in the number of CD14+DC, an increase in mature CD83+DC and a trend towards an increase in CD86+DC. Interestingly, the characteristic ability of LPS to enhance the expression of the co-stimulatory molecule OX40L on DCs was revealed only for the μ-analogue SD-101M. In addition, CpG-ODNs (SD-101 and SD-101M) had a stimulatory effect on IFN-γ production comparable to the action of LPS. The data obtained indicate a stimulating effect of CpG-ODN on the maturation and allostimulatory activity of human myeloid DCs, which is more pronounced for μ-modified analogs.

IL-32γ potentiates tumor immunity in melanoma.

Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti–PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti–PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non–T cell–inflamed TME.

Ascorbic Acid Promotes KIR Demethylation during Early NK Cell Differentiation.

Variegated expression of killer Ig-like receptors (KIR) in human NK cells is a stochastic process exclusive to subsets of mature NK cells and CD8+ T cells. Allele-specific KIR expression is maintained by DNA methylation within the proximal promoter regions. Because KIR genes are densely methylated in NK cell progenitors, there is an implied stage of human NK cell development in which DNA demethylation takes place to allow for active transcription. When and how this process occurs is unknown. In this study, we show that KIR proximal promoters are densely methylated in less mature CD56bright NK cells and are progressively demethylated in CD56dim NK cells as they mature and acquire KIR. We hypothesized that ten-eleven translocation (TET) enzymes, which oxidize 5mC on DNA could mediate KIR promoter demethylation. The catalytic efficiency of TET enzymes is known to be enhanced by ascorbic acid. We found that the addition of ascorbic acid to ex vivo culture of sorted CD56bright NK cells increased the frequency of KIR expression in a dose-dependent manner and facilitated demethylation of proximal promoters. A marked enrichment of the transcription factor Runx3 as well as TET2 and TET3 was observed within proximal KIR promoters in CD56bright NK cells cultured with ascorbic acid. Additionally, overexpression of TET3 and Runx3 promoted KIR expression in CD56bright NK cells and NK-92 cells. Our results show that KIR promoter demethylation can be induced in CD56bright, and this process is facilitated by ascorbic acid.

Formaldehyde inhibits development of T lymphocytes in mice

Formaldehyde (FA) is a ubiquitous environmental pollutant and a group 1 carcinogen. CD8+ T cells are specific tumor killers in the adaptive immune system. In this study, 2.0 mg/m3 respiratory FA exposure to male mice was found to selectively reduce mature CD8+ T cells in the spleen and the CD8 T cells in the thymus. In order to monitor the exposure of FA in vitro, a fluorescent FA donor was utilized in the treatment of murine thymocytes. Cytotoxicity induced by FA was found to be not selective at single positive stage. Significant inhibition of the runt-related transcription factor-3, the critical transcriptional factors for CD8 lineage commitment, was found in FA-treated double positive cells. ThPOK, the transcriptional factor for CD4 commitment, was stimulated by FA in double positive cells. Increased expression of special AT-rich sequence-binding protein-1 and decreased expression of B-cell lymphoma/leukemia 11B, which are the upstream regulators of ThPOK and the runt-related transcription factor-3, were also observed in FA-treated double positive cells. These results demonstrate that FA could selectively inhibit the development of CD8+ T cells via the alteration of transcriptional factor expressions of thymocytes at double positive stages, which would potentially interfere with the immune defense against cancers.

BK Polyomavirus-Specific CD8 T-Cell Expansion In Vitro Using 27mer Peptide Antigens for Developing Adoptive T-Cell Transfer and Vaccination.

BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells. Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers. BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1-) and cytotoxic. Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation.

Decreased Thymic Output Contributes to Immune Defects in Septic Patients.

Background: Prolonged immunosuppression and hypoinflammation, termed compensatory anti-inflammatory response syndrome (CARS), contribute to high morbidity and mortality in the late phase of sepsis. Although apoptosis is a well-known cause of lymphopenia in sepsis, the contribution of thymic output to immune alterations in sepsis and potential compensatory mechanisms are largely unknown. Methods: We investigate the release of CD4+ T cells from the thymus and their peripheral proliferation by evaluating T-cell receptor excision circles (TREC) and the expression of CD31 as markers for recent thymic emigrants (RTE) and their proliferative offspring in septic patients with relevant lymphopenia in the CARS phase. Moreover, we determine the aging of T cells by measuring telomere characteristics. Results: In septic patients, we found decreased CD4+ T-helper cell numbers, while CD8+ T cell numbers were unchanged. As a possible cause, we detected increased apoptosis of CD4+ T-helper cells and decreased levels of IL-7, which promotes the maturation of T cells in the thymus. Accordingly, the relative number of mature CD4+ T cells, TREC-containing CD4+ T cells, and CD31+ RTEs (characteristic of thymic output) was decreased, while the relative number of CD31-T cells (peripherally expanded naïve T cells) was increased. Furthermore, the telomere length decreased, although telomerase activity and markers for the shelterin complex were increased specifically in CD4+ but not in CD8+ T cells. Conclusion: We thus conclude that, in addition to T-cell apoptosis, decreased thymic output and increased aging of CD4+ T cells may contribute to lymphopenia and immunosuppression in sepsis. Increased proliferation of peripheral T cells cannot compensate for these effects.

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.

Activated HoxB4-induced hematopoietic stem cells from murine pluripotent stem cells via long-term programming

Hematopoietic stem cells (HSCs) are multipotent cells that form the entire blood system and have the potential to cure several pathogenic conditions directly or indirectly arising from defects within the HSC compartment. Pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs) can give rise to all embryonic cell types; however, efficient in vitro differentiation of HSCs from PSCs remains challenging. HoxB4 is a key regulator orchestrating the differentiation of PSCs into all cells types across the mesodermal lineage, including HSCs. Moreover, the ectopic expression of HoxB4 enhances the in vitro generation and expansion of HSCs. However, several aspects of HoxB4 biology including its regulatory functions are not fully understood. Here, we describe the role of HoxB4 in indirectly inhibiting the emergence of mature CD45+ HSCs from iPSCs in vitro. Forced activation of HoxB4 permitted long-term maintenance of functional hematopoietic stem and progenitor cells (HSPCs), which efficiently reconstituted hematopoiesis upon transplantation. Our method enables an easy and scalable in vitro platform for the generation of HSCs from iPSCs, which will ultimately lead to a better understanding of HSC biology and facilitate preparation of the roadma for producing an unrestricted supply of HSCs for several curative therapies.

THE STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF VISCERALES LYMPH NODES IN THE HUMAN OF OLD AGE

The problem of age-related involution of the structures of the immune system is now becoming more and more urgent. The aim of this study is the structural and functional characteristics of the visceral lymph nodes of an elderly person. Using immune morphological methods, the structure and cellular composition of the subcarinal and mesenteric lymph nodes of an elderly person were studied. A pronounced development of fibrous connective tissue is shown, which leads to fragmentation of the parenchyma of the nodes and a reduction in the area of the cortex. In the cortex, small lymphoid nodules and a narrow para-cortical zone are revealed. In lymphoid nodules, the absence of light centers with dividing and poorly differentiated lymphocytes and a high concentration of mature CD20 B-lymphocytes are noted. In the para-cortical zone, CD4 T-helpers are not detected. In all structural components of the lymph node, Ki67-positive cells are absent, which indicates the extinction of lymphocyte-poietic function, which is due to the replacement of the reticular tissue of the microenvironment with fibrous connective tissue and the absence of CD4 T-lymphocytes, which regulate the reactions of cellular and humoral immunity. Destruction of the reticular stroma in the sinus system, which is a biological filter, leads to impaired lymph purification function.

Phosphate Transporter Profiles in Murine and Human Thymi Identify Thymocytes at Distinct Stages of Differentiation

Thymocyte differentiation is dependent on the availability and transport of metabolites in the thymus niche. As expression of metabolite transporters is a rate-limiting step in nutrient utilization, cell surface transporter levels generally reflect the cell's metabolic state. The GLUT1 glucose transporter is upregulated on actively dividing thymocytes, identifying thymocytes with an increased metabolism. However, it is not clear whether transporters of essential elements such as phosphate are modulated during thymocyte differentiation. While PiT1 and PiT2 are both phosphate transporters in the SLC20 family, we show here that they exhibit distinct expression profiles on both murine and human thymocytes. PiT2 expression distinguishes thymocytes with high metabolic activity, identifying immature murine double negative (CD4−CD8−) DN3b and DN4 thymocyte blasts as well as immature single positive (ISP) CD8 thymocytes. Notably, the absence of PiT2 expression on RAG2-deficient thymocytes, blocked at the DN3a stage, strongly suggests that high PiT2 expression is restricted to thymocytes having undergone a productive TCRβ rearrangement at the DN3a/DN3b transition. Similarly, in the human thymus, PiT2 was upregulated on early post-β selection CD4+ISP and TCRαβ−CD4hiDP thymocytes co-expressing the CD71 transferrin receptor, a marker of metabolic activity. In marked contrast, expression of the PiT1 phosphate importer was detected on mature CD3+ murine and human thymocytes. Notably, PiT1 expression on CD3+DN thymocytes was identified as a biomarker of an aging thymus, increasing from 8.4 ± 1.5% to 42.4 ± 9.4% by 1 year of age (p < 0.0001). We identified these cells as TCRγδ and, most significantly, NKT, representing 77 ± 9% of PiT1+DN thymocytes by 1 year of age (p < 0.001). Thus, metabolic activity and thymic aging are associated with distinct expression profiles of the PiT1 and PiT2 phosphate transporters.

GPA33: A Marker to Identify Stable Human Regulatory T Cells.

FOXP3-expressing regulatory T (Treg) cells safeguard immunological tolerance. Treg cells can be generated during thymic development (called thymic Treg [tTreg] cells) or derived from mature conventional CD4+ T cells that underwent TGF-β-mediated conversion in the periphery (called peripheral Treg [pTreg] cells). Murine studies have shown that tTreg cells exhibit strong lineage fidelity, whereas pTreg cells can revert into conventional CD4+ T cells. Their stronger lineage commitment makes tTreg cells the safest cells to use in adoptive cell therapy, increasingly used to treat autoimmune and inflammatory disorders. Markers to distinguish human tTreg cells from pTreg cells have, however, not been found. Based on combined proteomic and transcriptomic approaches, we report that the Ig superfamily protein GPA33 is expressed on a subset of human Treg cells. GPA33 is acquired late during tTreg cell development but is not expressed on TGF-β-induced Treg cells. GPA33 identifies Treg cells in human blood that lack the ability to produce effector cytokines (IL-2, IFN-γ, IL-17), regardless of differentiation stage. GPA33high Treg cells universally express the transcription factor Helios that preferentially marks tTreg cells and can robustly and stably be expanded in vitro even without rapamycin. Expanded GPA33high Treg cells are suppressive, unable to produce proinflammatory cytokines, and exhibit the epigenetic modifications of the FOXP3 gene enhancer CNS2, necessary for indelible expression of this critical transcription factor. Our findings thus suggest that GPA33 identifies human tTreg cells and provide a strategy to isolate such cells for safer and more efficacious adoptive cell therapy.