ADAM17-Mediated Processing of TNF-α Expressed by Antiviral Effector CD8+ T Cells Is Required for Severe T-Cell-Mediated Lung Injury

ABSTRACT CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-γ and TNF-α production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes.

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Combinatorial Cytokine Secretion Signature of Donor-Derived T Cells Infused with the Graft: A New Potential Biomarker of Acute Graft-Versus-Host Disease in Αβt-Cell/CD19 B-Cell Depleted Hematopoietic Stem Cell Transplant Recipients

Blood ◽

10.1182/blood-2021-149948 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1684-1684

Author(s):

Raul Montiel-Esparza ◽

Giulia Barbarito ◽

Samantha Peck ◽

Magali Bazzano ◽

Rachana Patil ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Cytokine Secretion ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Tnf Α

Abstract Background: Hematopoietic stem cell graft manipulation strategies, such as αβT-cell/CD19 B-cell depleted hematopoietic stem cell transplantation (αβhaplo-HSCT), address the lack of matched donors and reduce the incidence of severe acute graft-versus-host disease (aGvHD). However, grade II-IV aGvHD still occurs in 25-30% of αβhaplo-HSCT recipients . Studies aimed at understanding the pathogenesis underlying aGVHD in αβhaplo-HSCT are lacking. We hypothesized that αβT cells adoptively transferred with the HSCT (<1x10 5/Kg) have unique combinatorial cytokine secretion signatures that may predict the occurrence of aGvHD. Here we used the IsoPlexis single-cell proteomics for CD4 + and CD8 + T cells to identify those putative signatures . Methods: Six patients with hematologic malignancies receiving fully myeloablative αβhaplo-HSCT at Lucile Packard Children's Hospital, Stanford, between 08/2018 and 05/2020 were enrolled upon signing IRB approved informed consent. Three patients developed grade II-IV aGvHD, while three did not. Aliquot of the graft and of peripheral blood collected at the time of aGvHD onset or at corresponding time points for the patients who did not develop aGvHD, were analyzed. Single sorted CD4 + and CD8 + T cells were profiled by single-cell barcode chip assay from IsoPlexis system (IsoPlexis, Branford, CT) after stimulation with PMA (50 ng/mL) and Ionomycin (1mcg/mL). Following the Human Adaptive Immune Panel, cytokines from CD4 + and CD8 + single T cells were captured by fluorescence ELISA, which measured the numbers of cytokine-producing cells (secretion frequency) and numbers of cytokines produced by individual cells across five functional groups: effector, stimulatory, chemoattractive, regulatory and inflammatory (Table 1). Polyfunctionality was defined as the secretion of 2+ cytokines from each CD4 + and CD8 + T cell. The T cell polyfunctional strength Index (PSI) was defined as the percentage of polyfunctional cells, multiplied by the sum of the mean fluorescence intensity of the proteins secreted by those cells. Additional statistical analysis was performed using the Student's t test. Results: We compared the combinatorial cytokine secretion signature of individual CD4 + and CD8 + T cells isolated from grafts infused into patients, who eventually did or didn't develop aGvHD. We are comparing the signature of post-HSCT CD4 + and CD8 + T cells isolated from patients who did or did not develop aGvHD. Collectively, we considered three variables: cytokine secretion frequency, numbers of cytokines produced by individual cells and characteristics of the cytokines secreted (functional group) upon stimulation. Single-cell functional heterogeneity evaluated by t-Distributed Stochastic Neighbor Embedding (t-SNE), showed higher CD4 + and CD8 + T-cell polyfunctionality (up to 4+ cytokines) with effector and stimulatory dominant functions in the grafts of patients who developed aGvHD, compared to those who did not develop aGvHD (Fig1). The average PSI (driven by Granzyme B, TNF-α, IFN-γ, MIP-1β, IL2, and IL-8) was found to be higher in both CD4 + and CD8 + T cells from the grafts of patients who developed aGvHD (Fig 2). Combinatorial cytokine secretion analysis showed that T cells from grafts of patients who did not develop aGvHD had unique signatures with CD4 + T cells having the predominant cytokine secretion signature of IL2 and TNF-α, and CD8 + T cells having three predominant cytokine secretion signatures: IL2, IL8, TNF-α; MIP-1β, IL8; and MIP-1β, IFN-γ (Fig3). Conclusions: Preliminary data from αβhaplo-HSCT pediatric recipients obtained using IsoPlexis single-cell functional proteomics for CD4 + and CD8 + T cells showed that an increased donor T-cell polyfunctionality with a Th1 dominant functional phenotype may be predictive of an increased risk of aGvHD, while CD4 + and CD8 + T cells infused into patients who didn't develop aGvHD, had combinations with limited cytokine secretion signatures. Ongoing analysis suggest that polyfunctional CD8 + T cells present in the graft of patients who developed aGvHD, are present at the time of aGvHD initiation, while the polyfunctional CD4 + T cell are not present at the onset of aGvHD. Correlation with ongoing studies on circulating cytokines and clonotypic analysis of αβT cells infused with the graft will be crucial to elucidate the cross talking between the donor's immune system and recipient's inflammatory milieu. Figure 1 Figure 1. Disclosures Parkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

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Itacitinib, a JAK1 Selective Inhibitor Preserves Graft-Versus-Leukemia (GVL), Enhances Survival and Is Highly Efficacious in a MHC-Mismatched Mouse Model of Acute GvHD

Blood ◽

10.1182/blood-2018-99-112144 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4522-4522 ◽

Cited By ~ 1

Author(s):

Ashish Juvekar ◽

Bruce Ruggeri ◽

Sindy Condon ◽

Andrew Borkowski ◽

Reid Huber ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Inflammatory Cytokine ◽

Acute Gvhd ◽

Colon Tissue ◽

Graft Versus Leukemia ◽

Tnf Α ◽

Dosing Regimens ◽

Ifn Γ

Abstract Introduction: Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK1/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (GRAVITAS-301: NCT03139604; GRAVITAS-119: NCT03320642). Methods: Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the selective JAK1 inhibitor, itacitinib (60 mg/kg or 120 mg/kg twice daily). Engraftment was analyzed for the proportion of donor and host leukocytes (CD45+, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analysis. Colon samples were sectioned and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3 and CD3+phosphoSTAT3 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of itacitinib on preservation of Graft-versus-Leukemia (GVL) were evaluated by injecting BALB/c mice with A20 lymphoma cells that are of H-2Kd phenotype along with combination of splenocytes and T cell depleted bone marrow from C57BL/6 (H-2Kb) mice. Results: Itacitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss and improving GvHD scores. Itacitinib did not significantly impact donor engraftment as determined by CD45+/H-2Kb quantification by flow cytometry. Similar efficacy was observed with 60 mg/kg versus 120 mg/kg twice daily dosing regimens. Oral itacitinib administration achieved JAK1 IC50 coverage for 4 h and 12 h at 60 mg/kg twice daily and 120 mg/kg twice daily, respectively. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Itacitinib (120 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen with itacitinib treatment, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with significant JAK1/STAT3 inhibition as measured by reductions in normalized pSTAT3 in T cells and colonic epithelial cells. Itacitinib treatment did not negatively impact GVL responses, as evidence by T cell mediated reduction of tumor burden. Furthermore, itacitinib treatment enhanced the survival of the recipient BALB/c mice in comparison to the vehicle treated animals. Conclusions: Itacitinib, a selective JAK1 inhibitor ameliorated GvHD severity when administered prophylactically or therapeutically and had no detrimental effects on engraftment and preservation of GVL. Furthermore, itacitinib inhibited JAK1/STAT3 activation in diseased colon tissue and infiltrating T-cells, and reduced disease burden and improved survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. Disclosures Juvekar: Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

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Regulation of Acute Graft-Versus-Host Disease by MicroRNA-155

Blood ◽

10.1182/blood.v116.21.245.245 ◽

2010 ◽

Vol 116 (21) ◽

pp. 245-245

Author(s):

Nicole Stauffer ◽

Sayed Mehdi Hamadani ◽

Catherine Heaphy ◽

Ramasamy Santhanam ◽

Sukhinder Sandhu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Cd8 T Cells ◽

T Test ◽

Spleen Cells ◽

Graft Versus Host ◽

Tnf Α ◽

Donor T Cells ◽

Acute Graft

Abstract Abstract 245 Acute Graft-versus-host disease (aGVHD) is a frequent and lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT) in which donor T cells destroy HLA mismatched host tissues by secreting soluble inflammatory cytokines (TNF-α, IFN-γ) and/or inducing direct cytotoxic cellular responses. Despite recent advances, GVHD still remains a major clinical problem, underscoring the need to elucidate further its mechanisms to then develop novel therapeutic strategies. Recent studies indicate that microRNAs (miR) play critical roles in the development and function of the immune system. In particular, miR-155 is required for normal function of B and T lymphocytes. MiR-155 has been shown to be up-regulated upon B and T cell activation and mice deficient for miR-155 are immunodeficient, exhibiting T cells with attenuated INF-γ and TNF-α release in response to antigen stimulation. Based on these observations, we hypothesized that miR-155 is up-regulated in donor T cells during aGVHD and is involved in the modulation of this process. To test this hypothesis, we initially measured miR-155 expression from activated CD8 T cells obtained from an experimental animal model of aGVHD. For this, a major histocompatibility complex (MHC) mismatched HSCT model was used in which spleen cells and BM from C57BL/6 (B6) donors were transferred i.v. into lethally irradiated B6D2F1 recipient mice (n=4). Two additional groups were included as controls with one group receiving no cell infusion (radiation only, n=4); and a second group receiving T cell depleted bone marrow only (t-BM) (5×106) (n=4). GVHD scores were performed daily according to Cooke et al, Blood 1996;8:3230-9. Recipient mice were sacrificed and tissues harvested when GVHD scores were ≥7 or at the end of the experiment. After an average of 3–4 weeks, mice receiving donor spleen cells but not BM alone developed severe GVHD (scores >7) which was confirmed by histology. Activated CD8 T cells from the spleen were isolated using CD8+/CD44+ antibodies and after obtaining total RNA, miR-155 expression was measured by RT-PCR. Mice receiving donor BM plus spleen cells developed severe aGVHD and exhibited increased miR-155 expression with respect to the BM only group (fold change 5.2, t-test p<0.001). To confirm that a causal relationship exists between miR-155 and aGVHD severity, we repeated the above MHC mismatched murine experiment using BL/6 mice deficient for miR-155 expression as donors (Rodriguez et al, Science 2007;316;608-611). The groups were as follows; radiation alone (n=4), t-BM (n=8), BM + wild type (WT) spleen cells (n=8), and t-BM+ miR-155 KO spleen cells (n=8). We found that mice receiving donor spleen cells from miR-155 KO mice exhibited dramatically lower mean GVHD scores (3 Vs. 5.5, t-test p=0.0007) and improved survival compared to those receiving WT spleen cells(87% vs. 13% of mice alive at 70 days, respectively; log-rank test p<0.001). Our results showed that recipients from miR-155 KO spleen cells did not exhibit high GVHD histological scores (III-IV) in the spleen, liver or gut, while 80%, 60% and 40% of the WT did. Overall survival, GVHD scores and histological GHVD findings were similar between miR-155 KO and WT t-BM only group. Since high levels of soluble TNF-α is characteristic for aGVHD, we measured serum TNF-α by ELISA assay in mice at the time of harvest. Mice receiving miR-155 KO spleen cells had significantly lower TNF-α levels (mean 14 pg/ml) than WT controls (mean 48 pg/ml, t-test, p=0.005). Finally, to establish the relevance of this finding to the human system, we measured miR-155 expression using Locked Nucleic Acid in situ hybridization from histologically confirmed gut biopsies of aGVHD patients (n=5), and healthy controls (n=3). We found a strong up-regulation of miR-155 expression in all patients with gut aGVHD while miR-155 expression was absent in normal gut. In summary, we have shown that miR-155 expression is up-regulated in CD8 T cells from mice with aGVHD and showed that mice receiving donor lymphocyte cells deficient for miR-155 exhibited less GVHD and improved survival as compared to mice receiving WT donor lymphocytes. Finally, up-regulation of miR-155 was also found in clinical specimens from patients with gut aGVHD. Altogether our data indicate a role for miR-155 in the modulation of aGVHD, and thus point to miR-155 as a novel target for therapeutic intervention in aGVHD. Disclosures: Off Label Use: Decitabine and bortezomib in AML.

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The Role of β-Catenin in Th1 Immune Response against Tuberculosis and Profiles of Expression in Patients with Pulmonary Tuberculosis

Journal of Immunology Research ◽

10.1155/2021/6625855 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Kunlong Xiong ◽

Jinxia Niu ◽

Ruijuan Zheng ◽

Zhonghua Liu ◽

Yanzheng Song ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Cd4 T Cell ◽

Cell Frequency ◽

Tnf Α ◽

Ifn Γ

β-Catenin is a key molecule of canonical Wnt/β-catenin pathway. Its roles and expression profiles in T cells of tuberculosis (TB) remain unclear. The aim of this study was to explore the role of β-catenin in CD4+ T cells and its expression characteristics in patients with pulmonary tuberculosis (PTB). In this study, CD4+ T cell-specific β-catenin conditional knockout mice (β-CAT-cKO mice) were aerosol infected with Mycobacteria tuberculosis (Mtb) H37RV with wild-type mice as controls. Four weeks after infection, the mRNA expression of IFN-γ, TNF-α, and TCF-7 in the lungs of mice was measured. CD4, CD8, β-catenin, IFN-γ, and TNF-α in mononuclear cells from the lungs and spleens were measured by flow cytometry, and the pathological changes of lungs were also observed. Patients with PTB were enrolled, with blood samples collected and PBMCs isolated. The expressions of β-catenin, IFN-γ, TNF-α, and PD-1 in CD4+ and CD8+ T cells were measured by flow cytometry. Results showed a decreased frequency of and reduced IFN-γ/TNF-α mRNA expression and secretion by CD4+ T cells in the lungs of infected β-CAT-cKO mice compared with infected wild-type controls, and only slightly more inflammatory changes were observed in the lungs. β-catenin expressions in CD4+ and CD8+ T cells were significantly decreased in blood cells of patients with severe PTB compared with those in mild PTB. The stimulation of peripheral blood mononuclear cells (PBMCs) with lithium chloride (LiCl), a stimulant of β-catenin, resulted in the increase in CD4+ T cell frequency, as well as their secretion of IFN-γ and TNF-α. β-Catenin demonstrated a moderately positive correlation with PD-1 in CD4+ T cells. β-Catenin along with PD-1 and IFN-γ in CD4+ T cells had a high correlation with those in CD8+ T cells. In conclusion, β-catenin may be involved in the regulation of Th1 response and CD4+ T cell frequency in TB.

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Tumor Necrosis Factor Receptor-1 (p55) Deficiency Attenuates Tumor Growth and Intratumoral Angiogenesis and Stimulates CD8+ T Cell Function in Melanoma

Cells ◽

10.3390/cells9112469 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2469

Author(s):

Yamila I. Rodriguez ◽

Ludmila E. Campos ◽

Melina G. Castro ◽

Nadia Bannoud ◽

Ada G. Blidner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Necrosis Factor ◽

Tumor Cell ◽

Cd8 T Cells ◽

Tumor Necrosis ◽

Cd8 T Cell ◽

Tnf Α ◽

The Impact ◽

Necrosis Factor

The role of tumor necrosis factor-α (TNF-α) in shaping the tumor microenvironment is ambiguous. Consistent with its uncertain role in melanoma, TNF-α plays a dual role, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. TNF-α signals via two cognate receptors, namely TNFR1 (p55) and TNFR2 (p75), which mediate divergent biological activities. Here, we analyzed the impact of TNFR1 deficiency in tumor progression in the B16.F1 melanoma model. Tumors developed in mice lacking TNFR1 (TNFR1 knock-out; KO) were smaller and displayed lower proliferation compared to their wild type (WT) counterpart. Moreover, TNFR1 KO mice showed reduced tumor angiogenesis. Although no evidence of spontaneous metastases was observed, conditioned media obtained from TNFR1 KO tumors increased tumor cell migration. Whereas the analysis of tumor-associated immune cell infiltrates showed similar frequency of total and M2-polarized tumor-associated macrophages (TAMs), the percentage of CD8+ T cells was augmented in TNFR1 KO tumors. Indeed, functional ex vivo assays demonstrated that CD8+ T cells obtained from TNFR1KO mice displayed an increased cytotoxic function. Thus, lack of TNFR1 attenuates melanoma growth by modulating tumor cell proliferation, migration, angiogenesis and CD8+ T cell accumulation and activation, suggesting that interruption of TNF-TNFR1 signaling may contribute to control tumor burden.

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Impact of Donor Serostatus on CMV Reactivation and Reconstitution of Multi-Function CMV-Specific T Cells in CMV-Positive Transplant Recipients

Blood ◽

10.1182/blood.v112.11.4339.4339 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4339-4339

Author(s):

Wendi Zhou ◽

Jeff Longmate ◽

Simon F Lacey ◽

Joycelynne Palmer ◽

Ghislaine Gallez-Hawkins ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Donor Group ◽

Functional Profile ◽

Tnf Α ◽

Ifn Γ ◽

Cmv Reactivation

Abstract CMV reactivation remains a significant cause of morbidity and mortality due to the extended period of immunodeficiency after allogeneic hematopoietic stem cell transplantation (HCT), despite great strides in management of the infection in the past two decades. Reconstitution of cytomegalovirus (CMV)-specific CD8+ T cells is essential to control of CMV infection in CMV-seropositive recipients (R+) after HCT. The CMV serologic status of the recipient before HCT has a strong influence on outcome. Key questions addressed in this study are the impact of donor CMV serostatus on the reconstitution of effective CMV immunity or risk of CMV reactivation and GCV usage in CMV R+ recipients. Betts and colleagues have reported that HIV-specific CD8+ T-cells which simultaneously degranulated and produced IFN-γ, TNF-α, MIP-1β and IL2 were associated with lower viral load and HIV long term non-progressor status. These findings in the context of HIV infection motivated us to investigate whether levels of multi-functional CMV-specific CD8+ T-cells in HCT recipients correlated with the CMV serostatus of the donor and the differentiation state of transferred CMV-specific memory T-cells. We hypothesize that a mature CD8+ T-cell functional profile leads to a lower incidence of CMV viremia in R+ recipients of T-cell replete HCT with a D+ donor. A total of 183 R+ HCT recipients were enrolled. CMV reactivation was defined as any positive CMV blood culture or plasma PCR obtained during a monitoring period that began at d40 post-HCT and continued thereafter twice weekly until d100. After d100, CMV in plasma was monitored in patients at high risk because of GVHD or immunosuppressive medication. Peripheral blood mononuclear cells from R+ HCT recipients were collected at intervals between d90 and d360 post-HCT. A subset (n=123) of the subjects limited only by sample availability were evaluated for CMV-specific CD8+ T-cells producing IFN-g (IFN-γ-CD8+). We used a well-described pp65 peptide library as a stimulatory antigen to evaluate the ex vivo functional profile of pp65-specific CD8+ T-cells from R+ recipients either receiving CMV seropositive (D+/R+) or seronegative (D−/R+) T-cell replete donor grafts. D+ status was associated with higher IFN-γ-CD8+ during the sampling time-course by fitting linear generalized estimating equation models on a logarithmic scale (p=0.0004). A significant interaction was found between prior CMV reactivation and donor serostatus on IFN-γ-CD8+ levels (p=0.0001). Comparing the subset of IFN-γ-CD8+ measurements prior to reactivation, the D− donor group produced lower levels of IFN-γ-CD8+ than the D+ donor group (p=0.0002), although both donor groups have similar levels of IFN-γ-CD8+ post-reactivation. Similar results were obtained when adjusting for the 9 pre-transplant covariates, and none were associated with IFN-γ-CD8+ levels, except donor serostatus and its interaction with CMV reactivation. Six-color flow cytometry was used to assess the functional profile of CMV-specific CD8+ T-cells in 62 of 183 HCT recipients prospectively followed for CMV reactivation. R+ recipients receiving grafts from D− donors (D−/R+) reconstituted fewer multi-functional CD8+ T-cells expressing IFN-γ, TNF-α, MIP-1β and CD107 compared to D+/R+ recipients. Unlike mono-functional CD8+ T-cells secreting IFN-g, which were abundantly generated during CMV reactivation in D−/R+, the relative lack of multi-functional CD8+ T-cells persisted until at least one year post-HCT. In addition, D−/R+ recipients had more CMV reactivation than D+/R+ recipients. D+/R+ transplants have elevated levels of multi-functional CD8+ T cell levels and lower hazard for CMV reactivation. Virologic and immunologic outcomes were robust to adjustment for pre-transplant factors affecting HCT, including donor type, stem cell source, recipient age, and preparative regimen. Statistical modeling to account for pre- and post-transplant factors including GVHD and its treatment by steroids had minimal effect on the contribution of serostatus to the risk of CMV reactivation and differences in multi-functional CD8+ T cell levels.

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The Trend of TIM3 Expression on T Cells in Patients With Nontuberculous Mycobacterial Lung Disease: From Immune Cell Dysfunction to Clinical Severity

Frontiers in Immunology ◽

10.3389/fimmu.2021.738056 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ping-Huai Wang ◽

Ming-Fang Wu ◽

Chi-Yu Hsu ◽

Sheng-Wei Pan ◽

Chin-Chung Shu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lung Disease ◽

Cd8 T Cells ◽

Cell Apoptosis ◽

Interleukin 2 ◽

Clinical Severity ◽

Cd4 Cells ◽

Nontuberculous Mycobacterial Lung Disease ◽

Tnf Α

BackgroundThe incidence of nontuberculous mycobacterial lung disease (NTM-LD) is increasing worldwide. Immune exhaustion has been reported in NTM-LD, but T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), a co-inhibitory receptor on T cells, has been scarcely studied.MethodsPatients with NTM-LD and healthy controls were prospectively recruited from July 2014 to August 2019 at three tertiary referral centers in Taiwan. We examined TIM3 expression on the T cells from the participants using flow cytometry. TIM3 expression was analyzed for different disease statuses and after treatment. The apoptosis and cytokine profiles were analyzed according to the TIM3 expression.ResultsAmong enrolled subjects (47 patients and 46 controls), TIM3 on CD4+ cells (6.44% vs. 4.12%, p = 0.028) and CD8+ cells (18.47% vs. 9.13%, p = 0.003) were higher in NTM-LD patients than in the controls. The TIM3 level on CD4+ and CD8+ T cells was positively associated with T-cell apoptosis in the NTM-LD patients. In stimulating peripheral blood mononuclear cells using PMA plus ionomycin, a high TIM3 level on T cells correlated with low interleukin-2 and tumor necrosis factor-alpha (TNF-α) on CD4+ cells and interferon-gamma and TNF-α on CD8+ T cells. For clinical manifestation, low body mass index (BMI), positive sputum acid-fast smear, and high radiographic score correlated with high TIM3 expression on T cells. After NTM treatment, TIM3+ decreased significantly on CD4+ and CD8+ T cells.ConclusionsIn patients with NTM-LD, TIM3+ expression increased over CD4+ and CD8+ T cells and correlated with cell apoptosis and specific cytokine attenuation. Clinically, TIM3+ T cells increased in patients with low BMI, high disease extent, and high bacilli burden but decreased after treatment.

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Assessment of the Phenotype and Functionality of Porcine CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus (CSFV) and Virulent CSFV Challenge

Clinical and Vaccine Immunology ◽

10.1128/cvi.00415-13 ◽

2013 ◽

Vol 20 (10) ◽

pp. 1604-1616 ◽

Cited By ~ 24

Author(s):

Giulia Franzoni ◽

Nitin V. Kurkure ◽

Daniel S. Edgar ◽

Helen E. Everett ◽

Wilhelm Gerner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Classical Swine Fever Virus ◽

T Cell Responses ◽

Classical Swine Fever ◽

Cd8 T Cell ◽

Cell Responses ◽

Tnf Α ◽

Ifn Γ

ABSTRACTVaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3+CD4−CD8hiT cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3+CD4+CD8α+) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44hiCD62L−and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ+CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.

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Arsenic Trioxide Encapsulated in MSC-Derived Extracellular Vesicles: A Novel Therapy for aGVHD in Mice

Blood ◽

10.1182/blood-2018-99-114904 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5673-5673

Author(s):

Xiao Liu ◽

Ya-Nan Wang ◽

Gao-chao Zhang ◽

Yan Su ◽

Qiu-sha Huang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Treatment Group ◽

Arsenic Trioxide ◽

Extracellular Vesicles ◽

Cd8 T Cells ◽

Notch Pathway ◽

Control Group ◽

Tnf Α ◽

Ifn Γ

Abstract Introduction: Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation. Mesenchymal stem cells (MSCs) can modulate immune response and have been used as a treatment for aGVHD. The immune-modulating factors of MSCs are secreted and reside in supernatant fractions that are enriched for extracellular vesicles (EVs). MSC-derived EVs (MSC-EVs) also exhibit immunosuppressive activity, providing many advantages compared to MSCs and have been proven therapeutic in aGVHD. Arsenic trioxide (ATO) exhibits potent antitumor effects and increasing studies indicate its immunosuppressive effects. However, ATO at high concentrations can cause severe adverse effects. If encapsulated in some kind of drug vehicles, ATO can be made less toxic. Therefore, we believed that the combination of MSC-EVs with a low dose of ATO would be an effective therapy for aGVHD. Methods: We used a classical GVHD model (BALB/c→B6) and developed 4 groups: the control group (TCD-BM), the GVHD control group (TCD-BM + spleen T cells), the MSC-EVs treatment group and the MSC-ATO-EVs (MSC-derived ATO-encapsulating EVs) treatment group. OS, GVHD clinical and histological scores were evaluated. A20-luc lymphoma cells were injected to generate the GVL model. Using flow cytometry analysis, we analyzed Th cell subsets, cytokines and transcription factors (Th1*IFN-γ/TNF-α*T-bet, Th2*IL-4*GATA3, Th17*IL-17*RORγt, Treg*IL-10*Foxp3) and sorted CD8+ SLECs, and CD8+ MPECs in BM and spleen of recipients. Dll4 expression was analyzed on DCs. B6 cells were incubated with or without BALB/c spleen cells and complete medium alone, with 10 mM ATO alone. T cell apoptosis was determined with Yopro-1 staining. We used MLR assays to examine Th subsets, cytokines and notch targeted genes with or without ATO or neutralizing Ab specific to Dll4 (anti-Dll4). Results: BALB/c mice receiving B6 TCD-BM alone developed no sign of GVHD, whereas all BALB/c mice receiving B6 donor TCD-BM + spleen T cells died of GVHD. In contrast, injection of MSC-EVs and MSC-ATO-EVs inhibited GVHD in T cell recipients, with 20% and 29% of them surviving without severe GVHD, respectively. These survival rates were accompanied by significantly lower clinical and histological scores. GVL effects mediated by MSC-EVs and MSC-ATO-EVs were comparable to those obtained in the GVHD control group. Compared to the control group, CD4+T and CD8+T cells increased substantially in T cell recipients, resulting in severe GVHD. In contrast, treatment with MSC-ATO-EVs significantly reduced the number of CD4+T and CD8+T cells, while MSC-EVs recipients retained approximately the same number of T cells as the GVHD group. Compared to the GVHD control group, Th2 and Treg cells derived from the spleen increased, while Th1 and Th17 cells were reduced significantly in both the MSC-EVs and MSC-ATO-EVs groups. We also detected lower serum levels of TNF-α and IFNγ as well as lower expression of RORγt and T-bet in blood and BM CD4+ T cells in these two groups, while the expression of GATA3 and Foxp3 increased significantly. Treatment with MSC-ATO-EVs markedly raised the MPEC/SLEC ratio compared to the MSC-EVs and GVHD control groups. We also examined Dll4high DCs in different organs and different groups and found that only MSC-ATO-EVs significantly reduced the Dll4high DCs, especially in the spleen and intestine. Treatment of stimulated B6 CD4+ T and CD8+ T cells with ATO increased production of H2O2. Yopro-1 staining of activated B6 CD4+ T and CD8+ T cells indicated that ATO dramatically triggered apoptosis in those cells. DCs were isolated and cultured with B6 mouse-derived CD4+ T or CD8+ T cells, with or without addition of ATO or anti-Dll4. ATO and anti-Dll4 both led to significant reduction of IFN-γ and TNF-α, while IL-4 and IL-10 increased slightly. We next assessed the notch pathway targeted genes in T cells and found there were significantly increased GATA3 and reduced Dtx expression levels. Conclusion: Altogether, our findings demonstrate that MSC-ATO-EVs might be a highly promising therapy for aGVHD through reducing T cell amounts and modulating Th subsets and CD8+ T cell differentiation. These effects can be explained with the inhibition of the Dll4-notch pathway by ATO. Therefore, further exploitation of the potential application of ATO in aGVHD and the mechanisms of action of ATO may improve outcomes after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

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