P0419AUTOANTIGEN-SPECIFIC TH17 AND TH22 INFLAME THE KIDNEY IN ANCA-VASCULITIS

Abstract Background and Aims Anti-neutrophil-cytoplasmic-antibody (ANCA)-associated-vasculitis (AAV) is an autoimmune small-vessel-vasculitis. T-cells play a pivotal role in pathogenesis as drivers of autoantibody formation and vasculitic damage. However, the involvement of T-cells in renal vasculitis is understood poorly. IL-17C is secreted by epithelial parenchymal cells enhancing the pathogenicity of Th17 cells. Furthermore, IL-17C is a chemotactic factor for Th17 cells. The aim of this study was to assess the role of Th17 cells and IL-17C in the pathogenesis of ANCA-vasculitis in an animal model of AAV. Method Wistar-Kyoto-rats were immunized with myeloperoxidase (MPO) in Freunds Adjuvant to induce AAV. Control rats were immunized with Freunds Adjuvant only. Albuminuria was determined weekly and rats were culled after two, four and six weeks. At the time of harvest, renal T-cells were isolated and characterized by flow cytometry. Antigen-specificity was determined by ELiSPOT. Petechial bleedings on the lung surface reflecting pulmonary vasculitis were quantified after harvest. Gene expression in spleen, kidneys and lungs was studied by PCR and is expressed as fold change over controls. IL-17C levels were measured in sera by ELISA. Results MPO-rats developed detectable titres of anti-MPO by week two. By week six, all MPO-animals developed renal vasculitis with significant albuminuria and pulmonary vasculitis. Accordingly, MPO animals showed significant crescent formation as compared to the controls (% of affected glomeruli: 11.4 ±10.5% vs. 0.4 ±0.7%, p<0.005). The petechial bleeding score on the lung surface was higher in MPO-immunized rats than in controls at week six (68 ±13 vs. 2 ±1, p<0.05). From week two on, Th17 and Th22 cells inflamed the kidney as determined by PCR and/or flow cytometry in MPO-rats. The Th17 and Th22 infiltrate was heaviest at week six post-immunization. The intra-renal T-cell response was skewed towards Th17 as compared to the frequency of splenic Th17 cells in MPO-rats (9.1 ±4.3% vs. 1.9 ±0.6%, p<0.005). The majority of intra-renal Th17 and Th22 cells was MPO-specific. Control rats did not show renal T-cell infiltration. Renal transcripts for IL-17C were slightly decreased in week 2 (0.88 ±0.1 fold), unchanged in week four (1.0 ±0.2) and slightly increased in week six after immunization (1.7 ±0.7 fold). Pulmonary IL-17C mRNA transcripts were decreased during weeks two and four after disease induction as compared to controls (0.29 ±0.07 fold, 0.98 ±0.2 fold). During week six, pulmonary IL-17C mRNA transcripts were slightly increased over controls (1.9 ±0.4 fold). Serum levels of IL17C were unchanged during weeks two to six after disease induction comparing MPO-immunized rats and controls. Conclusion Th17 and Th22 cells are drivers of renal inflammation in ANCA-vasculitis. The role of IL-17C in this cascade needs to be determined.

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Double Negative T Cells Influence TCR VB Variablity to Induce Allotolerance.

Blood ◽

10.1182/blood.v108.11.759.759 ◽

2006 ◽

Vol 108 (11) ◽

pp. 759-759

Author(s):

Zachariah A. McIver ◽

Marcin Wlodarski ◽

Jennifer Powers ◽

Christine O’Keefe ◽

Tao Jin ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Peripheral Tolerance ◽

Double Negative ◽

Clonal Deletion ◽

Tcr Repertoire ◽

Double Negative T Cells

Abstract Immune alloresponsiveness following allogeneic HSCT is influenced by the dynamics of immune reconstitution and development of allotolerance. In general, tolerance is induced by thymic clonal deletion (central) and apoptosis or suppression of alloresponsive lymphocytes by regulatory T cells in the periphery. We have recently demonstrated that the size of the TCR repertoire within the CD4 and CD8 compartments can be assessed using VB spectrum by flow cytometry, and expansions/losses of individual TCR VB families can be used as a surrogate marker of TCR variability. (Exp. Hem.32: 1010–1022; Br. J. Haematol.129:411–419). Additionally, regulatory T cells can also impact the clonal contractions and expansions within the TCR VB repertoire. Various types of regulatory T cells have been described including CD4+CD25+, CD8+, NK T−cells, and CD3+CD4/CD8− double negative T cells (DN Tregs). In our current study we investigated the role of DN Tregs on the restoration of immune repertoire diversity. We hypothesized that alloresponsiveness clinically detected as a manifestation of GvHD may be associated with oligoclonal T−cell expansions, and in this context decreased numbers of regulatory T cells suggest deficient tolerizing function by regulatory T cells including DN Tregs. Here we studied a cohort of 60 HSCT recipients (AML, CML, CLL, NHL, AA, and PV), of which 25 patients received matched unrelated donor grafts and 35 received matched sibling donor grafts. Blood was sampled between 2003–2006 at monthly intervals after HSCT, and flow cytometry for TCR repertoire in CD4 and CD8 cells as well as the numbers of DN cells were recorded. Additionally, separate samples were collected for measurement of chimerism and were included in analysis when donor lymphoid chimerism was > 60%. A subset analysis was performed based on the presence/absence of GvHD. For the 27/60 (45%) patients with episodes of GvHD, results were obtained at the time of diagnosis of GvHD (grade > 2), while for patients in whom notable GvHD was not captured, the steady−state values at corresponding times were used for analysis. For all patients serial evaluations were available. For the purpose of this study, significant VB expansions/contractions were defined as +/− 2 standard deviation over the average VB family size. Using Cox proportional hazards analysis to identify univariate risk factors for GVHD, CD8 VB TCR contractions > 14 VB families (58.3% contraction of entire CD4 VB repertoire) constituted a strong indicator for increased risk (HR=7.61, p=0.011). This observation is consistent with the fact that oligoclonality of alloreactive T cell clones is frequently accompanied by a significant contraction/loss of remaining VB families and may herald heightened alloresponsiveness as a manifestation of GvHD. Estimation for correlation by Pearson’s correlation coefficient also demonstrated that percentage of DN cells strongly correlated with a normalization of CD4 VB TCR repertoire (lower number of expansions; N=57, R= −0.51, p=0.027), supporting our hypothesis that DN cells participate in peripheral tolerance and suppress proliferative, alloresponsive CD4 clones. In summary, our results further characterize TCR variability post HSCT and define the role of DN cells in the induction of allotolerance.

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Damage to Thymic Stroma Results in Increased Production of Pathogenic Dual Receptor T Cells Associated with Chronic Graft Versus Host Disease

Blood ◽

10.1182/blood.v128.22.1156.1156 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1156-1156

Author(s):

Amritha Balakrishnan ◽

Burhan Jama ◽

Nicholas Joseph Gloude ◽

Eric Jon Anderson ◽

Edward D. Ball ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Graft Versus Host Disease ◽

Graft Versus Host ◽

Post Transplant ◽

Thymic Stroma

Abstract Evidence from clinical investigations and animal models indicate that chronic graft versus host disease (cGVHD) results from defective thymic generation of functional and self-tolerant T cell populations following hematopoietic stem cell transplantation (HSCT). We have previously demonstrated that the rare subset of T cells that naturally express 2 T cell receptors (TCRs) on the cell surface as a result of incomplete allelic exclusion are predisposed to respond to auto- and alloantigens. Dual TCR T cells disproportionately participate in pathologic alloreactivity in HSCT patients and mouse models of acute GVHD. These findings, combined with observations demonstrating that dual TCR T cells represent a physiologic reservoir of unique TCRs that evade negative selection, prompted us to examine the role of thymic selection and dual TCR T cells in cGVHD. To study the role of post-transplant thymopoiesis in generation of potentially pathogenic dual TCR T cells, we used a mouse model of syngeneic bone marrow transplantation into lethally-irradiated recipients. Radiation-induced damage to the thymic stroma was characterized by disruption of thymic architecture and loss of cortical and medullary thymic epithelial cells (TECs). This damage resulted in significantly increased generation of dual TCR T cells following transplantation of congenically-marked syngeneic T cell-depleted bone marrow. Two-fold increased production of dual TCR T cells persisted for at least 20 weeks after transplantation. These data demonstrate the hazard for production of T cells predisposed to pathogenic reactivity in the post-transplant environment, and suggest that dual TCR T cells could be a source of T cells causing cGVHD. To examine involvement of dual TCR T cells in cGVHD, we analyzed peripheral blood samples from patients after allogeneic HSCT (> 12 months post-transplant) using our previously utilized pair-wise TCRVa labeling flow cytometry approach. Flow cytometry analysis revealed that dual TCR T cells were present at increased frequencies in patients with cGVHD (n = 10, 8.3% + 1.1%, P = 0.028) compared to patients without cGVHD (n = 3, 2.5 + 1.1%) or healthy age-matched controls (n = 5, 1.9 + 0.4%). Dual TCR T cells from patients with cGVHD had an activated CD69+ phenotype as compared to T cells expressing only a single TCR from the same patient. Single-cell TCRa/TCRb sequencing confirmed the increased frequencies of dual TCR T cells specific to activated T cells in patients with cGVHD. Repertoire analysis of TCRs sequenced from single cells indicated that the increase in dual TCR T cells was polyclonal. The single-cell sequencing approach enabled multiplexed examination of T cell lineage-associated transcription factors and cytokines. Single-cell transcriptional profiling demonstrated that dual TCR T cells demonstrated predominantly pro-inflammatory and cytotoxic phenotypes with expression of Tbet and perforin. This is in contrast to T cells expressing only a single TCR from the same patient, or dual TCR T cells from healthy control patients, which had a quiescent phenotype. These data indicate a role for dual TCR T cells in mediating cGVHD. Together, these results suggest that dual TCR T cells may be an important link between post-transplant T cell development and cGVHD. Disclosures No relevant conflicts of interest to declare.

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Analysis of T-Cell Subpopulations in the Lymph Nodes of Patients with Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v110.11.1580.1580 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1580-1580

Author(s):

Elizabeth Sagatys ◽

Lynn Moscinski ◽

Sophie Dessureault ◽

Eduardo Sotomayor ◽

Hernani Cualing

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cell Lymphoma ◽

Rank Correlation ◽

Cell Populations ◽

Spearman Rank Correlation ◽

Spearman Rank ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell NHL characterized by a high relapse rate. Novel treatments capable of providing and/or sustaining more durable responses in MCL patients are clearly needed. Manipulation of the immune system to unleash its protective effect might induce durable responses in MCL. To effectively harness the immune system against MCL, it is important to understand how T-cells interact with malignant B-cells that reside where immune responses are normally initiated. In the present study, we evaluated the number and phenotype of T-cells present in the lymph nodes of 14 patients with MCL. Given the increasingly important role of T regulatory cells (Tregs) and the paucity of information regarding their role in MCL, we also evaluated if T-cells infiltrating the lymph node of MCL patients co-express CD4 and FoxP3. Our patients consisted of 1 woman and 13 men, age range from 49–78 (mean age 65), 6 previously treated with chemotherapy, and 8 de novo. There was heterogeneity in the CD3+ T cell populations in our patients (range 2.7–55% [mean 15.8%]). Double staining with CD4/FoxP3 (range 0.2–3.4% [mean 1.7%]) and CD8/FoxP3 (range 0–3.2% [mean 1.4%]) showed significant heterogeneity in both populations (Table 1). A significant portion of FoxP3 positive, CD4/CD8 negative cells were seen in several cases (range 0–16% [mean 4.9%]). Flow cytometry was run on all 14 cases to evaluate the T cell populations. Spearman non-Gaussian regression analysis (using GraphPad™ software) comparing the CD4/FoxP3+ and CD8/FoxP3+ cells to the total CD8 cells showed a negative correlation by both immunohistochemical and flow, confirming that as the Treg population increases the CD8 population decreases (Table 2). Correlating CD4 Treg with CD8 by flow cytometry and IHC indicated an inverse correlation in 7 of 14 cases. Only 1 case had a positive correlation. The remaining cases had no correlation. These findings suggest increasing FoxP3 populations in MCL could result in a decrease in CD8+ T cell immune response against the malignant cells. Future studies including the characterization of the non-CD4/CD8 FoxP3+ cells may help clarify the role of Tregs in MCL. Table 1: MCL T cell Populations CD3+ FLOW (%) Total FoxP3 IHC (%) CD4/FoxP3+ IHC (%) CD8/FoxP3+ IHC (%) CD4+ IHC (%) CD8+ IHC (%) CD4+ FLOW (%) CD8+ FLOW (%) 6.5 3.2 1.8 0.4 6.4 7.8 2 4 43 12.8 0.6 1 10.6 19.2 16 27 16 4.4 0.2 0.8 8.8 15.4 5 11 11 3.6 1.4 0 6.2 1.8 8 3 8.4 3.4 0.8 1.4 17.6 11.2 1.6 3.9 10 7.8 1.8 0.6 13.6 9.2 7.5 2.5 26 12.4 3.4 2.4 15.8 16.8 20 0.1 6.3 8.2 2.2 3.2 11.6 11 1.7 3.7 55 8.6 3.4 1 21 21.6 30 25 12 10.8 2.4 2 16.8 17 5.1 6 8.4 4.6 1.6 1.2 17.6 18 5.1 3.2 5.4 9.6 1.8 1.8 13.6 16 3.6 0.3 2.7 2.2 0.8 1.4 3.8 3.4 0.8 1.9 11 19 1.2 1.8 22 8.6 7.6 3.6 Table 2: Spearman Rank Correlation Group Spearman Rank Correlation 95% Confidence Interval CD4/FoxP3+ vs. CD8 (IHC) 0.2788 −0.3115 to 0.7138 CD4/FoxP3+ vs. CD8 (FLOW) −0.2614 −0.7045 to 0.3284 CD8/FoxP3+ vs. CD8 (IHC) 0.2558 −0.3337 to 0.7105 CD8/FoxP3+ vs. CD8 (FLOW) −0.2196 −0.6815 to 0.3673 CD4/FoxP3+ vs. CD4 (IHC) 0.3448 −0.2440 to 0.7479 CD4/FoxP3+ vs. CD4 (FLOW) 0.3636 −0.2237 to 0.7572 CD8/FoxP3+ vs. CD4 (IHC) 0.4044 −0.1777 to 0.7769 CD8/FoxP3+ vs. CD4 (FLOW) −0.2243 −0.6841 to 0.3630

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Calcium Signaling in T Cells and Chronic Inflammatory Disorders of the Oral Cavity

Journal of Dental Research ◽

10.1177/0022034521990652 ◽

2021 ◽

pp. 002203452199065

Author(s):

S. Hasiakos ◽

Y. Gwack ◽

M. Kang ◽

I. Nishimura

Keyword(s):

T Cells ◽

T Cell ◽

Oral Cavity ◽

Calcium Signaling ◽

Signaling Pathways ◽

Chronic Inflammation ◽

Th17 Cells ◽

Inflammatory Diseases ◽

Chronic Inflammatory Diseases

Acute immune responses to microbial insults in the oral cavity often progress to chronic inflammatory diseases such as periodontitis and apical periodontitis. Chronic oral inflammation causes destruction of the periodontium, potentially leading to loss of the dentition. Previous investigations have demonstrated that the composition of oral immune cells, rather than the overall extent of cellular infiltration, determines the pathological development of chronic inflammation. The role of T lymphocyte populations, including Th1, Th2, Th17, and Treg cells, has been extensively described. Studies now propose pathogenic Th17 cells as a distinct subset, uniquely classifiable from traditional Th17 populations. In situ differentiation of pathogenic Th17 cells has been verified as a source of destructive inflammation, which critically drives pathogenesis in chronic inflammatory diseases such as diabetes, rheumatoid arthritis, and inflammatory bowel disease. Pathogenic Th17 cells resemble a Th1 penotype and produce not only interleukin 17 (IL-17) but also γ-interferon (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The proinflammatory cytokine-specific mechanisms known to induce IL-17 expression in Th17 cells are well characterized; however, differentiation mechanisms that lead to pathogenic Th17 cells are less understood. Recently, Ca2+ signaling through Ca2+ release-activated Ca2+ channels (CRAC) in T cells has been uncovered as a major signaling axis involved in the regulation of T-cell-mediated chronic inflammation. In particular, pathogenic Th17 cell–mediated immunological diseases appear to be effectively targeted via such Ca2+ signaling pathways. Pathogenic plasticity of Th17 cells has been extensively illustrated in autoimmune and chronic inflammatory diseases. Although their specific causal relationship to oral infection-induced chronic inflammatory diseases is not fully established, pathogenic Th17 cells may be involved in the underlining mechanism. This review highlights the current understanding of T-cell phenotype regulation, calcium signaling pathways in this event, and the potential role of pathogenic Th17 cells in chronic inflammatory disorders of the oral cavity.

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Research on the Key T Cells and Cytokines of aGVHD after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v124.21.3907.3907 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3907-3907

Author(s):

Yamin Tan ◽

Yi Luo ◽

Yanmin Zhao ◽

Weichao Liao ◽

Huarui Fu ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Early Diagnosis ◽

Treg Cell ◽

Th17 Cells ◽

Early Stage ◽

Serum Levels ◽

Biomarker Panel ◽

Related Mortality

Abstract Objective: Acute graft versus host disease(aGVHD) is due to one of the most important causes of treatment related mortality after allo-HSCT. T cells play the key roles in the development of aGVHD after allo-HSCT, and the different subsets play different roles. Research on the reconstruction of T cell immune function and its cytokines in the occurrence of aGVHD has become one of the hottest topic in aGVHD research. We investigated the key T lymphocyte subsets and its cytokines, to explore the relationship between the characteristics of T cell subsets and the process of aGVHD. We desire to find some biomarkers and establish early diagnosis index for aGVHD, furthermore find an important target for immunotherapy. Ultimately the strategy could reduce aGVHD related mortality and improve long-term disease-free survival in transplantation patients. Methods: 64 patients were included in the study, from May 2013 to January 2014. Blood samples of these patients were monitoring every week. Treg, Th1 and Th17 cells number were detected by flow cytometry. Serum levels of IL-2, IL-6, IL-10, IL-17, TNF-a and IFN-γwere detected by flow cytometry (CBA). ELISA technique was used for the detection of serum TGF- β level. Results: 36 patients (56.3%) suffered from II-IV aGVHD, and 10 cases (15.6%) among them was diagnosed of III°-IV° aGVHD. Before the onset of aGVHD, Treg, Th1, Th17 cells levels had no statistical difference between aGVHD patients and non aGVHD patients. In aGVHD patient, serum levels of IL-2 (P=0.0417) and TGF- β (P=0.0168) elevated in the early stage of aGVHD. IL-2 (P=0.0231), IL-10 (P=0.0089),TGF-β (P=0.0138) serum levels after aGVHD was significantly higher than that in non aGVHD patients, and Treg cellsnumber was significantly lower than that of patients without aGVHD (P=0.0150). Using ROC and logistic regression analysis, we found a composite biomarker panel (IL-2, IL-10 and Treg cell, which could optimally discriminated patients with and without aGVHD, the formula is: 1.656+0.103×IL-2|0.052 ×IL-10|0.037×Treg. ROC curve of composite panel is 0.89 (95% CI, 0.78-0.99, P=0.0002), which is larger than that of any single index. Conclusion: Increased IL-2, IL-10, TGF-β serum levels in early stage after allo-HSCT can help to establish the clinical diagnosis of aGVHD, and which is expected to provide a clinical basis for further target immunotherapy. Treg cell level was negatively related to the onset of aGVHD. Furthermore, we found a composite biomarker panel (IL-2, IL-10 and Treg cell), which could optimally discriminated patients with and without aGVHD. It could provide a reliable basis for early diagnosis of aGVHD. Disclosures No relevant conflicts of interest to declare.

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3280 Mycobacterium bovis Bacille-Calmette-Guérin infection aggravates atherosclerosis

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.251 ◽

2019 ◽

Vol 3 (s1) ◽

pp. 110-110

Author(s):

Moises Huaman ◽

Joseph E. Qualls ◽

David Kuhel ◽

Shinsmon Jose ◽

Eddy Konaniah ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Aortic Root ◽

Plasma Cholesterol ◽

Cd4 T Cell ◽

Atherosclerotic Lesions ◽

En Face ◽

Atherosclerosis Development

OBJECTIVES/SPECIFIC AIMS: The study aimed at assessing whether M. bovis BCG infection and inflammation exacerbates the development of atherosclerosis in Ldlr-/- mice. METHODS/STUDY POPULATION: Twelve-week old male Ldlr-/- mice (n=10) were infected with M. bovis BCG (0.3–3.0x10^6 colony-forming units (CFUs)) via the intranasal route, to simulate a natural respiratory route of infection. Mice were subsequently fed a western-type diet (WD) containing 21% fat and 0.2% cholesterol for 16 weeks. Age-matched uninfected Ldlr-/- mice (n=10) fed with an identical WD served as controls. Mice were euthanized after 16 weeks of WD to examine atherosclerotic lesions in aortic root sections and en face aorta using Oil Red O staining. Plasma cholesterol and triglyceride levels were measured by enzymatic assays and lipoprotein distribution was assessed using fast protein liquid chromatography. Because of the important role of T cells and monocytes in atherosclerosis development, we assessed these cell subsets in blood using flow cytometry at 8 and 16 weeks. Experiments were conducted in duplicate. We used unpaired Student’s t-test for group comparisons of numeric variables and flow cytometry data. RESULTS/ANTICIPATED RESULTS: M. bovis BCG infection significantly increased atherosclerotic lesions in en face aorta (plaque size per aorta area ratio; 0.15±0.13 vs. 0.06±0.02; P<0.01), but not in the aortic root. There were no significant differences in plasma cholesterol (1,160 mg/dL vs. 1,278 mg/dL; P = 0.36), triglycerides (340 mg/dL vs. 413 mg/dL; P = 0.28), or lipoprotein profiles between infected vs. uninfected mice at 16 weeks. M. bovis BCG increased circulating T lymphocytes (1,490 cells/uL vs. 1,227 cells/uL; P = 0.03) and monocytes (901 cells/uL vs. 414 cells/uL; P<0.01) within 8 weeks post-infection. When we assessed T lymphocyte subsets, M. bovis BCG infection increased total CD4+ T cell counts (556 cells/uL vs. 416 cells/uL; P<0.01) but not CD8+ T cells. No differences in the proportion of CD44+CD25+ activated T lymphocytes were noted between groups. When we assessed monocyte subsets, M. bovis BCG infection increased the numbers of Ly6Chigh (709 cells/uL vs. 362 cells/uL; P<0.01) and Ly6Clow (145 cells/uL vs. 35 cells/uL; P<0.01) monocytes. Infection was associated with an increased proportion of Ly6Clow monocytes at week 8 (17% vs. 8%; P<0.01) and week 16 (19% vs. 5%; P<0.01), compared to uninfected mice. DISCUSSION/SIGNIFICANCE OF IMPACT: M. bovis BCG infection increased the extent of atherosclerosis formation in the aortas of WD-fed hyperlipidemic Ldlr-/- mice after 16 weeks. Lipid profiles were similar between infected and uninfected mice, and therefore do not explain the observed differences in atherosclerosis. Compared to uninfected controls, M. bovis BCG-infected mice exhibited increased CD4+ T cell and monocyte driven inflammation. Interestingly, M. bovis BCG-infected mice had a higher proportion of non-classical Ly6Clow monocytes, suggesting a pro-atherogenic contribution of these cells in our model. Overall, our results support a pathogenic role of mycobacterial infection in atherosclerosis development and ASCVD.

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Bone Marrow GZMK +IL7R + Progenitor-Exhausted CD8 + T Cells Correlate with Sustained Clinical Remission in Patients with Acute Myeloid Leukemia (AML) Undergoing Chemotherapy

Blood ◽

10.1182/blood-2021-149150 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 521-521

Author(s):

Francesco Mazziotta ◽

Luca Biavati ◽

Rupkatha Mukhopadhyay ◽

Hanna A. Knaus ◽

Ivan M. Borrello ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Myeloid Leukemia ◽

Cd8 T Cell ◽

T Cell Subsets ◽

Spectral Flow

Abstract Introduction The role of T cells in chemotherapy response and maintenance of remission in acute myeloid leukemia (AML) patients is not fully understood. In solid tumors and chronic infections, exhaustion is a multistep process ranging from less differentiated progenitor exhausted (Tpex) to intermediate and terminally exhausted T cells (Beltra et al. 2020). High frequencies of Tpex correlate with response to immune-checkpoint blockade in solid tumors (Miller et al. 2019). In AML, where the backbone of treatment is chemotherapy, the role of dysfunctional T-cell subsets has yet to be elucidated. Methods Serial bone marrow (BM) samples from 16 AML patients (10 complete responders (Res) and 6 non-responders (NonRes)) at diagnosis and at response assessment after induction chemotherapy and 12 healthy donors (HD) were analyzed by flow cytometry using a 13-color panel. Moreover, we performed single-cell RNA sequencing (scRNAseq) (10X Genomics) on BM samples from 2 HD and 5 AML patients (3 Res, 2 NonRes) at baseline and after chemotherapy. Subsequently, we used a scRNAseq-guided 26-color spectral flow cytometry panel and explored T-cell phenotypes on BM of 22 AML patients (12 Res and 10 NonRes). Custom-made R scripts were employed for high-dimensional flow cytometry and scRNAseq analysis. Results Initial flow-cytometry analysis showed a significant increase in BM PD1 +CD28 + CD8 + T cell subset (p<0.01) in Res vs NonRes at baseline and post-chemotherapy (data not shown). To further investigate these results, we performed 5' VDJ scRNAseq and used gene signatures mapped in two dimensions via UMAP to annotate the T-cell clusters as naive, Tpex, T effector CX3CR1 + (Teff CX3CR1pos), Terminally exhausted 1 (Term_exh1) and Terminally exhausted 2 (Term_exh2) (Fig 1A). Of note, the two most upregulated genes in Tpex were GZMK and IL-7R. We then performed differential abundance analysis to investigate differences in terms of clusters' frequencies across the three conditions (Res, NonRes, HD). At both timepoints Res had an increased frequency of Tpex and Teff CX3CR1pos compared to NonRes. Conversely, Term_exh2 cells were more abundant in NonRes (Fig. 1B). Next, we measured the magnitude of clonal expansion in antigen-experienced CD8 + T cells in Res and NonRes generating an overlay of the position of clonally expanded cells projected onto the UMAP. The most clonally expanded subsets were Tpex and Teff CX3CR1pos in Res (Fig. 1C) and Term_exh2 in NonRes (Fig. 1D) revealing a strong relationship between abundance and clonal expansion of the CD8 + T-cell subsets. Our scRNAseq results were then confirmed at the protein level with spectral flow-cytometry. The FlowSOM algorithm identified a CD8 + GZMK +CD127 + subset to be increased at baseline in Res vs NonRes (Fig. 1E). Remarkably, this cluster was also characterized by the expression of TIGIT, PD1 and TCF-1. These results were subsequently reproduced by manual gating of the GZMK +CD127 + subset which was significantly enriched (p<0.01) in Res vs NonRes (Fig. 1F). Of note, patients with a higher-than-median frequency of GZMK +CD127 +CD8 + T cells experienced significantly (p<0.02) prolonged overall survival after therapy (Fig. 1G). Conclusion Improving our understanding of the immune microenvironment in AML is critical for the rational integration of novel treatment strategies that seek to increase the response rate and/or maintain remission. We identified GZMK +IL7R + CD8 + cells as a distinct entity in the early differentiated CD8 + memory T cell pool that is clonally expanded and more abundant in Res compared to NonRes. This subset has a stem-like signature and may be associated with longer in vivo CD8 + T cell persistence and long-term AML control. An in-depth functional characterization with in vitro experiments and in vivo mouse models is currently ongoing. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Endothelial Cell Dysfunction Is Involved in the Progression of Myelodysplastic Syndromes

Blood ◽

10.1182/blood-2021-147663 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3668-3668

Author(s):

Tong Xing ◽

Zhong-Shi Lyu ◽

Cai-Wen Duan ◽

Qi Wen ◽

Hong-Yan Zhao ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Endothelial Cells ◽

T Cell ◽

Myelodysplastic Syndromes ◽

T Cell Subsets ◽

Leukemia Cells ◽

Rna Seq

Abstract Background: Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis, refractory anemia, and a tendency to transform to acute myeloid leukemia (AML). Ineffective hematopoiesis progression and immune deregulation are dominating pathophysiological process of MDS. Emerging evidences showed the role of bone marrow (BM) microenvironment in MDS. In MDS murine model, integral BM microenvironment contributes to inferior hematopoietic function and disease progression. As an important component of BM microenvironment, the relationship between endothelial cells (ECs) and MDS progression remains largely unknown. Although ECs from MDS patients have been identified to have decreased supporting ability to normal hematopoietic stem cells (HSCs), the supporting ability of ECs in different clinical stages of MDS remains to be elucidated. In addition, the role of BM ECs from MDS patients in supporting leukemia cells and their immunomodulatory ability remains unclear. Aims: To determine the number and functions of BM ECs in different subtypes of MDS patients. Moreover, to explore the correlation between BM ECs and MDS progression, which may represent a potential therapeutic target for MDS patients. Methods: In the prospective cohort study, patients with multilineage dysplasia (MDS-MLD, N=15), MDS with excess blasts (MDS-EB, N=15), or AML(N=15) and healthy donors (HD, N=15) were enrolled. BM ECs were analyzed in HD and patients by flow cytometry and in situ histological analyses. The functions of BM ECs were analyzed by migration, angiogenesis capacities, levels of apoptosis and reactive oxygen species (ROS). To evaluate the supporting abilities of BM ECs on HSCs, leukemia cells and T cells, in vitro co-culture strategies were used. The levels of apoptosis, ROS and colony-forming unit-plating (CFU) efficiency of CD34+ and HL-60 cells were investigated. T cell subsets were analyzed by flow cytometry as previously reported. To further investigate the underlying mechanism of dysfunctional ECs, RNA sequencing (RNA-Seq) analyses and real time-PCR (qRT-PCR) were performed in BM ECs from HD and MDS patients with different subtypes. Results: In the current study, gradually increased BM ECs were observed from MDS-MLD, MDS-EB to AML patients. Furthermore, dysfunctional BM ECs were found with MDS progression, characterized by increased levels of migration, angiogenesis capacities, apoptosis and ROS. More importantly, BM ECs from MDS patients exhibited decreased supporting ability of HSCs whereas increased supporting ability of leukemia cells in vitro with MDS progression. After coculture with ECs, levels of apoptosis and ROS in CD34+ cells were increased whereas their CFU efficiency reduced. On the other hand, levels of apoptosis and ROS of HL-60 cells were decreased. The proliferation capacity and leukemia CFU efficiency of HL-60 cells after co-cultured with ECs were enhanced with MDS progression. Furthermore, following coculture with BM ECs, deregulated differentiation was demonstrated in T cell subsets, characterized by elevating proportion of Th2 and Treg and decreasing proportion of Th1 and Th17 with MDS progression. RNA-Seq showed that the expression profile of BM ECs from MDS-EB was closer to MDS-MLD, whereas that of MDS-EB was closer to AML. Different gene expression profiles indicated the expression of hematopoiesis and immune related genes increased in BM ECs with MDS progression. Mechanistically, the mRNA levels of CX CL12, SCF and NFKB of ECs were increased with MDS progression. Summary/Conclusion: In summary, the number of BM ECs gradually increased, BM EC dysfunction more and more severe, and the supporting abilities of BM ECs on HSCs decreased, whereas on leukemia cells increased with MDS progression. Moreover, ECs regulated the differentiation of T cells into immune tolerant cells with MDS progression. Although further validation is required, these findings indicated that the improvement of BM ECs may represent a potential therapeutic approach for MDS patients. Keywords: Myelodysplastic syndromes, endothelial cells, disease progression, ineffective hematopoiesis, immune deregulation Disclosures No relevant conflicts of interest to declare.

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Regeneration of insulin-producing [beta]-cells during recovery from disease: a cure for Type 1 Diabetes

10.32469/10355/69851 ◽

2018 ◽

Author(s):

◽

Tobechukwu Kenneth Ukah

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

T Cell ◽

Combination Therapy ◽

Beta Cells ◽

Th17 Cells ◽

Nod Mice ◽

I Cells

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Type 1 diabetes (T1D) is a chronic disease condition characterized by destruction of the insulin-producing [beta]-cells by self-reactive lymphocytes of the immune system. While some immunotherapeutic approaches against T1D directly target and modulate diabetogenic specific T cells or the entire T cell repertoire, other efforts utilize antigen presenting cells or T cell-regulating molecules to control the T cells. In chapter II, we set out to determine the role of regulatory cytokines, IL-4 and IL-13 in T1D progression. IL-4 and IL-13 are widely reported as anti-inflammatory cytokines, and both can signal via the IL-4R[alpha]/IL-13R[alpha]1 heteroreceptor (HR). To determine the role of these cytokines in T1D development, we generated NOD mice in which the IL-13R[alpha]1 arm of the HR is deleted, thereby rendering the HR nonfunctional. Surprisingly, the findings indicate that NOD mice lacking the HR (13R-/-) display resistance to T1D as the rise in blood glucose level (BGL) and islet inflammation were significantly delayed in these HR-deficient relative to HR-sufficient (13R+/+) mice. In fact, the frequency and spleen-to-pancreas dynamics of both Th1 and Th17 cells were affected in 13R-/- mice. This outcome is likely due to an increase in the frequency of mTGF[beta][subscript +]Foxp3[subscript int] regulatory T cells and persistence of CD206[subscript +] macrophage in the pancreas as both types of cells confer resistance to T1D upon transfer to 13R+/+ mice. These findings reveal new insights as to the role environmental IL-4/IL-13 and the HR play in peripheral tolerance and the development of T1D. In chapter III, we investigate the source of newly formed Î²-cells during recovery from overt T1D under a combination therapy that involves an immunoglobulin chimera, Ig-GAD2 and bone marrow cells transfer. This combination therapy proved effective in driving immune modulation of diabetogenic-specific T cells and repair of the islet vasculature leading to the formation of new endogenous [beta]-cells that were able to thrive and restore long-lasting normoglycemia. Our new findings reveal and suggest that the combination therapy leads to the formation of healthy islets by inducing division of residual Î²-cells and differentiation of precursor cells. Furthermore, while the pancreas is cleared of immune infiltration during recovery from disease, both the lymph nodes and spleen displayed a significant reduction in Th17 cells, and the disease did not rebound. These circumstances are relevant to humans as intervention could be made at early as well as late stages after diagnosis. Overall, these results provide insights on future immunotherapeutic measures of T1D using regulatory cytokines or intervention with an antigen-specific therapy.

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Infrequent HIV Infection of Circulating Monocytes during Antiretroviral Therapy

Journal of Virology ◽

10.1128/jvi.01174-19 ◽

2019 ◽

Vol 94 (1) ◽

Cited By ~ 6

Author(s):

Marta Massanella ◽

Wendy Bakeman ◽

Pasiri Sithinamsuwan ◽

James L. K. Fletcher ◽

Nitiya Chomchey ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Antiretroviral Therapy ◽

Hiv Infection ◽

Cell Sorting ◽

Hiv Reservoirs ◽

Hiv Dna ◽

Monocyte Infection

ABSTRACT Whereas human immunodeficiency virus (HIV) persists in tissue macrophages during antiretroviral therapy (ART), the role of circulating monocytes as HIV reservoirs remains controversial. Three magnetic bead selection methods and flow cytometry cell sorting were compared for their capacity to yield pure CD14+ monocyte populations. Cell sorting by flow cytometry provided the purest population of monocytes (median CD4+ T-cell contamination, 0.06%), and the levels of CD4+ T-cell contamination were positively correlated with the levels of integrated HIV DNA in the monocyte populations. Using cell sorting by flow cytometry, we assessed longitudinally the infection of monocytes and other cell subsets in a cohort of 29 Thai HIV-infected individuals. Low levels of HIV DNA were detected in a minority of monocyte fractions obtained before and after 1 year of ART (27% and 33%, respectively), whereas HIV DNA was readily detected in CD4+ T cells from all samples. Additional samples (2 to 5 years of ART) were obtained from 5 individuals in whom monocyte infection was previously detected. Whereas CD4+ T cells were infected at high levels at all time points, monocyte infection was inconsistent and absent in at least one longitudinal sample from 4/5 individuals. Our results indicate that infection of monocytes is infrequent and highlight the importance of using flow cytometry cell sorting to minimize contamination by CD4+ T cells. IMPORTANCE The role of circulating monocytes as persistent HIV reservoirs during ART is still controversial. Several studies have reported persistent infection of monocytes in virally suppressed individuals; however, others failed to detect HIV in this subset. These discrepancies are likely explained by the diversity of the methods used to isolate monocytes and to detect HIV infection. In this study, we show that only flow cytometry cell sorting yields a highly pure population of monocytes largely devoid of CD4 contaminants. Using this approach in a longitudinal cohort of HIV-infected individuals before and during ART, we demonstrate that HIV is rarely found in monocytes from untreated and treated HIV-infected individuals. This study highlights the importance of using methods that yield highly pure populations of cells as flow cytometry cell sorting to minimize and control for CD4+ T-cell contamination.

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