Brief report: Mutations in SIV Nef that disrupt and restore tetherin downregulation

Mapping Intimacies ◽

10.1101/833137 ◽

2019 ◽

Author(s):

Nicholas J Maness ◽

Blake Schouest

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cd4 T Cells ◽

Adaptor Protein ◽

Fitness Cost ◽

Compensatory Evolution ◽

Parental Plasmid ◽

Published Report ◽

Functional Defects

AbstractThe H196 residue in SIVmac239 Nef is conserved across nearly all HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) interacting domain (ExxxLM195), and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317). Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q196, which is associated with loss of multiple AP-2 dependent functions in SIVmac239. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q196 that might compensate for functional defects associated with this mutation. Variants were engineered into the SIVmac239 parental plasmid and mutant viruses were used to test tetherin downregulatory capacity in primary CD4 T cells using flow cytometry. SIVmac251 stocks that encode a Q196 residue in Nef uniformly also encode an upstream R191 residue. We show that R191 restores the ability of Nef to downregulate tetherin in the presence of Q196. However, a published report showed Q196 commonly evolves to H196 in vivo, suggesting a fitness cost. R191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q196.

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Reconstitution of CD52-Negative CD4 T Cells after Alemtuzumab-Based T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v112.11.1182.1182 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1182-1182

Author(s):

Eva M Wagner ◽

Aline N Lay ◽

Sina Wenzel ◽

Timo Schmitt ◽

Julia Hemmerling ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Lymphocytic Leukemia ◽

Cell Depletion ◽

Down Regulation ◽

T Cell Depletion ◽

Hematopoietic Stem

Abstract The human CD52 molecule is the target of the monoclonal antibody Alemtuzumab, which is used for treating patients with chemo-refractory chronic lymphocytic leukemia as well as for T cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT). The molecule is expressed on the surface of lymphocytes, dendritic cells and to a lesser extent on blood-derived monocytes. Previously, investigators have demonstrated that the surface expression of CD52 on T cells is down-regulated after in vitro incubation with Alemtuzumab. By treating purified human CD4 T cells over 4 hours with 10 μg/mL Alemtuzumab in medium supplemented with 10% human AB serum in vitro, we observed a strong decrease of CD52 expression by flow cytometry with a maximum 3–7 days after incubation. The CD52 down-regulation was also found at weaker intensity on CD8 T cells. From previous studies in chronic lymphocytic leukemia patients, it is known that Alemtuzumab treatment also leads to a down-regulation of CD52 on T cells in vivo. However, similar experiments have not been performed in allogeneic HSCT patients receiving Alemtuzumab in vivo for T cell depletion. We therefore analyzed the expression of CD52 on human peripheral blood mononuclear cells isolated at repeated time points from 22 allogeneic HSCT patients after reduced-intensity conditioning with fludarabine and melphalan and in vivo T cell depletion with Alemtuzumab (100 mg). Half of the patients received prophylactic CD8-depleted donor lymphocyte infusions (DLI) to promote immune reconstitution. By flow cytometry, we observed that the CD52 expression on monocytes, B cells, and natural killer cells remained unaltered after transplantation and was not influenced by the application of DLI. In contrast, the majority of CD4 T cells were CD52-negative (median, 72%) after transplantation and they remained CD52-negative in patients who did not receive DLI throughout the first year after HSCT. The permanent lack of CD52 expression could not be explained by a continuous effect of Alemtuzumab, because earlier studies have shown that the antibody is not present in active plasma concentrations beyond day +60 after HSCT. In contrast, patients receiving CD8-depleted DLI demonstrated a significant increase in the proportion of CD52-positive CD4 T cells. In three of our patients (DLI: n=2, non-DLI: n=1) we analyzed the donor chimerism of CD52-positive and CD52-negative CD4 T cells sorted with high purity by flow cytometry. Three months after HSCT (before DLI), the proportion of donor T cells was clearly higher among the CD52-negative compared to the small proportion of CD52-positive cells in all patients (44% vs. 10%, 83% vs. 0%, and 100% vs. 40%). In the patient who did not receive DLI, the donor T cell chimerism remained mixed in the CD52-negative and CD52-positive fractions on days 200 (CD52-negative: 95%; CD52-positive: 15%) and 350 (CD52-negative: 92%; CD52-positive: 65%). In contrast, the two patients receiving CD8-depleted DLI showed a strong increase in the proportion of CD52-positive CD4 T cells that were of complete donor origin. Altogether, CD52 is permanently down-regulated in reconstituting CD4 T cells following HSCT with an Alemtuzumab-based TCD regimen unless DLI are applied. Our data support the idea of an active mechanism for CD52 down-regulation in CD4 T cells that is not related to B cells and natural killer cells and that appears to differently affect donor and host T cells, respectively.

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P025 Identification and validation of predictors for tofacitinib through supervised and unbiased approaches in Inflammatory Bowel Disease

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjab076.154 ◽

2021 ◽

Vol 15 (Supplement_1) ◽

pp. S141-S141

Author(s):

B Liu ◽

M Spalinger ◽

L G Perez ◽

A Machicote ◽

N Gagliani ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Signaling Pathway ◽

Cd4 T Cells ◽

Cytokine Production ◽

Stat Signaling ◽

Deficient Mice

Abstract Background Inflammatory Bowel Disease (IBD) is characterized by an overwhelming gut inflammation, where CD4+ effector T cells are main mediators of the inflammatory response. Tofacitinib, a small molecular drug recently used in IBD patients, blocks the JAK/STAT signaling pathway necessary for CD4+ effector T-cell activation. However, clinical data show that a percentage of patients do not respond to the treatment. Our main goal is to identify biomarkers predicting the response of patients to tofacitinib. Methods Tofacitinib efficacy was studied in vivo in wild type (WT) and T-cell-specific PTPN2 deficient mice (CD4-Cre;Ptpn2 floxed) in which the JAK/STAT signaling pathway is over activated. WT and PTPN2 deficient mice were gavaged with tofacitinib (50mg/kg, twice daily) or vehicle. Acute DSS-colitis was induced. Colitis development was evaluated by weight loss, colonoscopy and histology. CD4+ T cells were isolated from the colon and analyzed by flow cytometry. To study the effect of tofacitinib on T-cell differentiation, we isolated naïve T cells from mouse spleen and polarized them in vitro to different T-cell subsets with or without tofacitinib. CD4+ T cells differentiation and cytokine production were analyzed by flow cytometry. To evaluate the influence of tofacitinib on human CD4+ T cells, human peripheral blood mononuclear cells (PBMCs) from healthy donors and IBD patients were stimulated in presence of tofacitinib, and analyzed by flow cytometry. Results While no protective effect was found after tofacitinib treatment in WT mice, PTPN2 deficient mice were protected from colitis based on less weight loss, lower endoscopic and histological scores. The expression of pro-inflammatory cytokines such as IL-17 and IFN-γ by colonic CD4+ T cells was also decreased by tofacitinib. Consistent with the in vivo observations, in vitro experiments revealed a strong impact of tofacitinib on CD4+ T-cells cytokine production. In PBMCs from IBD patients, IFN-γ and TNF-α expression was strongly impacted. In contrast, in healthy donors, IL-10 was the most impacted cytokine. Finally, tofacitinib decreased the in vitro differentiation of Th1, Th2, Th17, Th22, Treg and Tr1. Conclusion In the T-cell-specific PTPN2 deficient mice, tofacitinib exerts a protective effect after DSS-induced colitis. In line with the in vivo findings, in vitro experiments show that tofacitinib has a strong impact on pro-inflammatory cytokine production, especially in the IBD patients. Taken together, these data suggest that tofacitinib might be suitable primarily for IBD patients where the JAK/STAT signaling pathway is over activated.

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Blockade of the P2Y12 signaling Pathway with Clopidogrel Restrains Treg Proliferation In Vivo during Sepsis and in Vitro

Blood ◽

10.1182/blood-2018-99-114884 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2413-2413

Author(s):

Harika Vemulapalli ◽

Albayati Samara ◽

Alexander Y Tsygankov ◽

Elisabetta Liverani

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Cd4 T Cells ◽

Human Platelets ◽

Positive Staining ◽

Treg Population ◽

Population Sizes ◽

Aggregation Of Platelets

Abstract Sepsis is a complex clinical syndrome resulting from a serious bloodstream infection. With hospital mortality rates of affected patients reportedly as high as 50%, improved methods for treating sepsis are urgently needed. To begin development of new pharmacologic therapies, we investigated the effect of an antiplatelet treatment on the proliferation of regulatory T cells (Tregs) in a murine model of sepsis. Tregs are a subset of T lymphocytes that downregulate the immune response and promote the resolution of inflammation. Septic patients have elevated levels of circulating Tregs, and this increased prevalence is associated with increased patient mortality. Platelets, which regulate inflammation through cell-cell interactions and through secretion of inflammatory mediators,have been shown to alter the proliferation and activation of Tregs in vitro. However, the influence of platelets on Tregs in vivohas not been fully investigated. We propose that suppression of platelet functions during sepsis may restrain Treg proliferation, leading to the restoration of immunological homeostasis. To study the influence of platelets on Treg proliferation in vivo, we blocked the P2Y12signaling pathway and measured the resulting population sizes of Tregs in septic mice. P2Y12is a Giprotein-coupled purinergic receptor present on platelet surfaces. Stimulation of P2Y12by ADP leads to platelet aggregation and potentiation of platelet secretion. To block the P2Y12signaling pathway, we used the P2Y12antagonist clopidogrel. To induce sepsis in mice, we used cecal ligation and puncture (CLP). Clopidogrelwas administered orallywith a loading dose (30 mg/kg in PBS) one day before surgery and a maintenance dose (10 mg/kg in PBS) two hours prior to surgery. The nonseptic mice in the negative control group (sham) were treated with PBS only. Twenty-four hours after surgery, we isolated cells from the spleens of the mice in each treatment group (sham, CLP, and CLP with clopidogrel) and measured Treg population sizes by incubating the cells with anti-CD4, anti-CD25,and anti-Foxp3 antibodies. Tregs were identified by their positive staining for CD4, CD25, and Foxp3. We found that Tregpopulation sizes were reduced in the septic mice treated with clopidogrel compared with those in the untreated septic mice (Figure 1A).Additionally, we used flow cytometry (forward and side light scattering) to investigatewhether P2Y12antagonism altered the aggregation of platelets and CD4+T cells in whole blood.Platelets and CD4+T cells wereidentified by their positive staining with PE-anti CD41 and FITC-anti CD4, respectively. Events that were double positive for FITC and PE were identified as aggregates and reported as a percentage of gated CD4+T cells.We found that aggregation of platelets and CD4+T cells was reduced in the septic mice treated with clopidogrel (15 ±5 %) compared with that in the untreated septic mice (38 ±6 %) (n= 3, p<0.05 treated CLP vs. untreated CLP). We investigated the effect of blocking the P2Y12signaling pathway in vitrousing co-cultures of human platelets and T cells. Human platelets and T cells were isolated from healthy donors and cultured in the presence or absence of anti-CD3/CD28 (5 μg/mLeach) antibodies for 5 days at 37°C in a humidified atmosphere containing 5% CO2. To block the P2Y12signaling pathway in vitro, we used AR-C69931MX (100 nM). We measured Treg population sizes using flow cytometry as described above. We found that Treg population sizes increased when resting T cells were exposed to platelets, AR-C, or both (Figure 1B). In contrast, we found that Treg population sizes decreased when CD3/CD28-stimulated T cells were exposed to a combination of platelets and AR-C (Figure 1B). Our data indicate that blockade of the P2Y12signaling pathway changes how platelets influence T cells in vitro, depending on whether the T cells have been activated. In conclusion, blockade of the P2Y12signaling pathway restrains Treg proliferation in vivoand in vitro. Our study indicates that targeting platelets to control Treg proliferation and activity may be a promising strategy for treating sepsis. Disclosures No relevant conflicts of interest to declare.

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Phase 1 Study of Pomalidomide Given at the Time of Early Lymphocyte Recovery after Induction Timed Sequential Chemotherapy in Newly Diagnosed Acute Myeloid Leukemia (AML) and High-Risk Myelodysplastic Syndrome (HR-MDS)

Blood ◽

10.1182/blood.v128.22.2820.2820 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2820-2820

Author(s):

Joshua F. Zeidner ◽

Raul Montiel-Esparza ◽

Hanna A. Knaus ◽

Sofia Berglund ◽

Amer M. Zeidan ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Research Funding ◽

Phase 1 ◽

Newly Diagnosed ◽

Tnf Α ◽

Grade 3

Abstract Introduction: AML pts have a poor prognosis with conventional chemotherapy regimens. Early lymphocyte recovery (ELR) following intensive timed sequential therapy (TST) induction is characterized by a dysfunctional immunosuppressive state. Pomalidomide (Pom), a small molecule immunomodulatory agent (IMiD), has direct effects on T cell co-stimulation by promoting the ubiquitination of Aiolos, an IL-2 transcriptional repressor. We hypothesized that the administration of Pom at the time of ELR after induction TST may influence T cell differentiation and enhance an anti-leukemia immune effect. Methods: A multicenter phase 1 dose escalation study was conducted to determine the safety and tolerability of Pom after intensive induction TST in newly diagnosed AML and HR-MDS pts 18-65 years. Core-binding factor AML was excluded. All pts received induction chemotherapy with AcDVP16: cytarabine 667 mg/m2/day IV continuous infusion days 1-3, daunorubicin 45 mg/m2/day IV days 1-3, etoposide 400 mg/m2/day IV days 8-10. Pom was administered at the assigned dose and schedule after day 14 and within 3 days of the total white blood cell count (WBC) reaching >0.2x109/L above nadir, defined as ELR. Three dose levels were planned (2 mg, 4 mg and 8 mg) within 2 cohorts: 10 days of Pom and 21 days of Pom, in a traditional 3+3 dose escalation design. Results: 25 pts were enrolled on this study January 2014-June 2016 across 3 institutions (Table 1). Pom administration occurred at a median of 21 days after AcDVP16 induction. There were no dose-limiting toxicities (DLTs) in the first cohort of Pom x 10 days within each dose level- 2 mg (n=3), 4 mg (n=3) and 8 mg (n=7). There were no DLTs seen at 4 mg x 21 days (n=7). Two DLTs were seen at Pom 8 mg x 21 days (Grade 3 ALT increase and Grade 3 hypoxia, respectively). Thus, Pom 4 mg x 21 days will be further expanded. Nine (36%) pts discontinued Pom early (median duration = 5 days) due to: grade 3 rash (n=3), physician discretion (decreased WBC: n=1, fever and increased creatinine: n=1), grade 3 ALT increase (n=1), grade 3 hypoxia (n=1), disease progression (n=1), and pt preference (n=1). Adverse events (AEs) possibly associated with Pom that were seen in >1 pt included fever (n=8), rash (n=7), AST/ALT increase (grade 1: n=4, grade 3: n=1), mucositis (n=2), and fatigue (n=2). All of these AEs were self-limiting with supportive care and/or discontinuation of Pom. 60-day mortality was 0%. A complete remission (CR) was achieved in 18 pts and 1 achieved CR with incomplete platelet recovery (CRp) with a combined CR + CRp = 19/25 (76%). Among pts with adverse-risk AML, 5/6 (83%) achieved CR. One pt achieved a partial remission and 5 pts were refractory to treatment. Of the 19 CRs, 15 had no evidence of minimal residual disease by cytogenetics, FISH, or flow cytometry. Among pts who completed a course of Pom (10 days or 21 days), 14/16 (88%) achieved CR. As previously reported, a dramatic decrease of Aiolos expression via flow cytometry in T cell subsets was observed in vivo for the duration of POM treatment with doses > 2 mg, but the effect was lost after Pom was stopped. Figure 1 displays the pattern of cytokine production of CD4+ T cells visualized with pie charts, and shows a significantly different subset composition at ELR in Pom-treated pts compared to the same pts at full recovery (p=0.02), and compared to control AML pts at the same time point (p=0.004). Furthermore, there was a significant increase in TNF-α production (p=0.009) and the combination of TNF-α and IL-2 production (p=0.03) in stimulated CD4+ T cells during Pom treatment, which was reduced to baseline values after Pom was discontinued at full recovery (Figure 1: data analysis performed with the SPICE software). Conclusions: Pom can be safely administered at the time of ELR after intensive induction TST. Fever and rash are the most common AEs seen after Pom administration. Inhibition of Aiolos and consequent increase in both IL-2 and TNF-α expression, as measured by flow cytometry, appear to be reliable markers of Pom-induced T cell modulation in vivo. Planned expansion of the cohort of 4 mgx 21 days will allow further evaluation of safety and activity of Pom in AML. Expression of Cytokines in CD4+ T Cells Expression of Cytokines in CD4+ T Cells Disclosures Zeidner: Takeda: Research Funding; Merck: Research Funding; Agios: Honoraria; Otsuka: Consultancy; Tolero: Research Funding. Zeidan:Celgene: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Smith:Celgene: Consultancy, Other: member of DSMB. Levis:Millennium: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding. Foster:Celgene: Research Funding.

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640 Stem-like CD4 T cells in cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.640 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A669-A669

Author(s):

Maria Cardenas ◽

Nataliya Prokhnevska ◽

Caroline Jansen ◽

Viraj Master ◽

Haydn Kissick

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Single Cell ◽

Cd4 T Cells ◽

Tumor Response ◽

Ex Vivo ◽

University Hospital ◽

In Vivo Studies ◽

Endogenous Virus

BackgroundCD4 T cells can differentiate into multiple effector subsets that can mediate variable functions. In this work we aim to understand how CD4 T cells differentiate in response to tumor antigens and their respective function in the anti-tumor response.MethodsTumor tissue was collected from patients undergoing surgery at Emory University Hospital. Activated PD1+ CD45RA- tumor infiltrating CD4 T cells were sent for 10X single cell RNA-seq. Tumor samples were also processed for flow cytometry and ex vivo functional analyses. For in vivo studies, prostate cancer mouse model expressing the LCMV glycoprotein (TRAMPC1-GP) was used, as well as LCMV Armstrong infection.ResultsTo characterize the heterogeneity of CD4 T cells infiltrating kidney tumors, we performed single cell RNAseq. We found three distinct activated (PD1+ CD45RA-) CD4 T cell populations. Two effector clusters consisting of Th1-like (EOMES+) and Treg (FOXP3+) cells, and a third cluster expressing TCF1, and genes associated with stemness and survival that did not fit defined CD4 effector lineages. We further confirmed these data by flow cytometry and found the same tumor infiltrating CD4 subsets in 100 kidney cancer patients. When placed in culture under different polarization conditions, tumor TCF1+ CD4 T cells proliferated and differentiated into the Th1-like and Treg effector populations found in the tumor, in addition to other effector lineages (Th1, Tfh) given the appropriate conditions, while the Th1-like and Treg cells underwent no proliferation or phenotype changes. These data suggests that the TCF1+ CD4s act as activated unpolarized precursors to the effector subsets in the tumor. To further test this hypothesis in vivo, we adoptively transferred tumor specific (SMARTA) CD4 T cells into mice followed by TRAMPC1-GP tumor inoculation. Transferred SMARTAs activated and first acquired a TCF1+ phenotype in the TDLN prior to predominantly differentiating into Tregs in the tumor. Given their plasticity in vitro, we asked whether TCF1+ SMARTAs primed in tumors were destined to differentiate into Tregs. To test this, we transferred 4-week activated TCF1+ SMARTAs from TDLNs of TRAMPC1-GP mice into naïve mice that were immediately infected with LCMV Armstrong. We found that the transferred SMARTAs differentiated into Th1 and Tfh cells in response to the virus, similar to the endogenous virus specific CD4 T cells.ConclusionsOverall, this work shows that CD4 T cells remain in an activated phenotype in the tumor with the capacity to differentiate into non-suppressive effector lineages given the appropriate conditions that may benefit the anti-tumor response.

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