Elevated Expression of T-Cell Immunoglobulin and Mucin Domain 3 on T Cells from Peripheral Blood in Patients with Cervical Carcinoma

2020 ◽  
pp. 1-8
Author(s):  
Juan Li ◽  
Xiao-fei Sun ◽  
Ying Shen ◽  
Qing Yang ◽  
Shu-yan Dai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cervical Carcinoma ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Control Group ◽  
Healthy Controls ◽  
Novel Therapy ◽  
Domain 3

<b><i>Objective:</i></b> To investigate the expression of T-cell immunoglobulin and mucin domain 3 (TIM-3) on peripheral T cells of cervical carcinoma patients. <b><i>Methods:</i></b> Peripheral blood samples from 15 high-grade cervical squamous intraepithelial lesion (HSIL) patients, 24 cervical carcinoma patients, and 21 healthy controls were collected. TIM-3 expressions on the surface of peripheral CD4+ T cells and CD8+ T cells were analyzed with flow cytometry. <b><i>Results:</i></b> There was significantly lower expression of CD4+ T cells and CD8+ T cells in HSIL patients and cervical carcinoma patients compared with healthy controls. We also found that TIM-3 expression on peripheral CD4+ T and CD8+ T cells of both HSIL patients and cervical carcinoma patients was significantly increased compared to the control group. Further analyses revealed that the expression of TIM-3 on peripheral CD4+ T and CD8+ T cells significantly increased in stage III–IV cervical carcinoma patients compared to stages I–II. <b><i>Conclusion:</i></b> The increased expression of TIM-3 on CD4+ T cells and CD8+ T cells of patients with cervical carcinoma and HSIL suggests the potential role of TIM-3 in the development and progression of cervical carcinoma, which may be a novel therapy target for cervical carcinoma.

Relation of CD4+CD25+ Regulatory T Cells to Immune Status in Patients with HIV Infection.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3106-3106
Author(s):  
Sachi Tsunemi ◽  
Tsuyoshi Iwasaki ◽  
Takehito Imado ◽  
Satoshi Higasa ◽  
Eizo Kakishita ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Hiv Infection ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Healthy Controls ◽  
Cell Counts ◽  
Hiv 1

Abstract Human immunodeficiency virus (HIV) infection is characterized by marked defects in CD4+ helper T cell (Th) functions that commonly progress to a substantial decline in peripheral CD4+ T cell counts. However, the mechanisms responsible for the loss of Th functions in HIV-infected patients independent of CD4+ T cell counts remains unclear. CD4+CD25+ regulatory T cells (T Reg) are essential for down-regulation of both autoreactive and alloreactive T cells. Therefore, we decided to investigate the role of T Reg in immune status of HIV-infected patients. We examined the expression of cell surface CD25, cytoplasmic IL-4 and cytoplasmic IFN-gamma in peripheral blood CD4+ T cells from both healthy controls (n=9) and HIV-infected patients (n=43). We also compared T Reg functions between the 2 groups. CD4+CD25+ T Reg isolated from both HIV-infected patients and healthy controls strongly expressed CD45RO, HLA-DR, and FoxP3, and suppressed the proliferation of CD4+CD25− T cells, suggesting that CD4+CD25+ T cells from both healthy controls and HIV-infected patients possess phenotypic and functional characteristics of Treg. CD4+CD25high T cells are a subset of circulating CD4+CD25+ T cells in normal humans and exhibit strong in vitro regulatory functions similar to those reported for murine CD4+CD25+ T Reg. We measured the frequency of CD4+CD25high T Reg by analysis of surface CD25 on CD4+ T cells in peripheral blood samples. We also examined Th1 and Th2 frequencies by analysis of cytoplasmic IFN-gamma and IL-4 levels in CD4+ T cells. T Reg from HIV-infected patients with detectable plasma HIV-1 RNA showed a statistically significant increase in CD4+CD25high cell frequency (p<0.05) compared to healthy controls, with T Reg frequencies inversely proportional to CD4+ T cell numbers (p<0.01). However, in HIV-infected patients with undetectable plasma HIV-RNA, frequencies of CD4+CD25high T Reg were not increased and not related to CD4+ T cell numbers. In both HIV-infected patient groups, T Reg frequency was inversely related to Th1 frequency (detectable: p<0.05, undetectable: p<0.001), but positively related to Th2 frequency (detectable: p<0.01, undetectable: p<0.001). Our results indicate that increased frequencies of peripheral blood T Reg were related to disease progression as measured by detectable plasma HIV-1 RNA, decreased peripheral blood CD4+ T cell counts, and polarization toward Th2 immune responses in HIV-infected patients. HIV infection may lead to induction of T reg that inhibit antiviral immune responses, resulting in the progression of the disease. Manipulation of T Reg could help restore antiviral immune responses in HIV infection, and prevent the progression of HIV infection.

Definition of a Diagnostic Window Prior to the Onset of Clinically Apparent Acute Graft-Versus-Host Disease.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3746-3746
Author(s):  
Carina A Bäuerlein ◽  
Simone S Riedel ◽  
Brede Christian ◽  
Ana-Laura Jordán Garrote ◽  
Agnes Birner ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Critical Time ◽  
Time Points ◽  
T Cell Migration ◽  
Donor T Cells

Abstract Abstract 3746 Acute graft-versus-host disease (aGvHD) is an immune syndrome after allogeneic hematopoietic cell transplantation (allo-HCT) caused by alloreactive donor T cells that attack the gastrointestinal tract, liver and skin. Thus, early T cell migration patterns to these organs could provide first cues for the onset of aGvHD. Hence, a unique surface marker profile of donor T cells at early time points after allo-HCT may be an indicator for patients at risk of aGVHD. Therefore, we analyzed the course of donor T cell activation, proliferation and homing in a clinical relevant murine MHC minor mismatch (miHAg) allo-HCT model to define critical time points and marker profiles for the detection of alloreactive T cells. Luciferase-labeled C57Bl/6 (H-2b) T cells plus bone marrow cells were transplanted into conditioned (8 Gy) MHC major mismatched Balb/c (H-2d) or miHAg Balb/b (H-2b) recipients. Donor T cell migration was visualized by in vivo bioluminescence imaging (BLI) and cells were characterized by multiparameter flow cytometry for 30 consecutive days after allo-HCT. GVHD scoring was performed by histopathology. Donor T cells proliferated exclusively in secondary lymphoid organs until day+3 (initiation phase) before migrating via the peripheral blood into target organs (effector phase). This occured in both models, MHC major mismatch and miHAg allo-HCT, which resulted in hyper-acute (starting at day+6) or acute GVHD (starting at day+21), respectively. In the hyper-acute scenario one wave of T cell migration starting at day+4 sufficed to cause lethal aGVHD. We detected a 4000-fold increase in CD4 and a 1500-fold increase in CD8 donor T cell numbers in the peripheral blood between day+3 and day+6 in this model. In contrast, in the more clinical relevant miHAg allo-HCT model we found 3 waves of T cell migration with peaks at days +6, +11 and +15 after allo-HCT. In the peripheral blood CD4 T cells increased 20-fold, CD8 T cells 50-fold between day+3 and day+6, but more than 40-fold (CD4) and 400-fold (CD8) between day+3 and day+11. After the third peak on day+15 a period followed when we could only detect very few migrating donor T cells in the peripheral blood before aGvHD became clinically apparent on day+21. Next, we asked whether we could identify alloreactive T cells by testing a large panel of surface markers at the defined migration peaks. Indeed, allogeneic T cells upregulated certain homing receptors at these peaks (e.g. at day+11: α4β7 integrin: 27% of CD4 T cells, 3.4×104/ml, 60% of CD8 T cells, 1.6×105/ml; P-selectin ligand: 28% of CD4 T cells, 3.5×104/ml, 35% of CD8 T cells, 9.1×104/ml). In contrast, syngeneic transplanted mice only showed a constant low expression level of those receptors (e.g. at day+11: α4β7 integrin: 20% of CD4 T cells, 9.6×103/ml, 5% of CD8 T cells, 3.1×103/ml; P-selectin ligand: 17% of CD4 T cells, 8.5×103/ml, 10% of CD8 T cells, 6.6×103/ml). However, other markers such as CD44 could be found on more than 80% of all donor T cells in allogeneic or syngeneic recipients. Our results in this clinical relevant mouse model show accelerating waves of T cell migration consistent with an enhancing feedback loop model of aGvHD pathogenesis. The homing receptor expression profile of donor T cells correlated with critical migration waves and clearly differed between mice with or without aGvHD. The assessment of critical time points frame a diagnostic window for a potential predictive test based on the dynamic change of the T cell homing receptor profile after allo-HCT. This preclinical study now awaits to be evaluated in patients undergoing allo-HCT. Disclosures: No relevant conflicts of interest to declare.

Determination of CD4+ and CD8+ T cells in the peripheral blood of dogs with demodicosis

Parasitology ◽  
2010 ◽  
Vol 137 (13) ◽  
pp. 1921-1924 ◽  
Author(s):  
S. K. SINGH ◽  
U. DIMRI ◽  
M. C. SHARMA ◽  
B. SHARMA ◽  
M. SAXENA
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Alteration ◽  
Healthy Controls ◽  
Healthy Dogs

SUMMARYThe aim of this study was to evaluate the CD4+/CD8+ ratio in peripheral blood of dogs with localized and generalized demodicosis. Sixteen dogs were examined, 8 with localized and 8 with generalized demodicosis, while 8 healthy dogs were used as controls. Peripheral blood was obtained and CD4+ and CD8+ T cells were determined by flow cytometry. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were found in dogs with generalized demodicosis compared to dogs with localized demodicosis and healthy controls. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were also found in dogs with localized demodicosis compared to healthy controls. The CD4+/CD8+ ratio was also found to be significantly lower in dogs with generalized demodicosis in comparison with dogs with localized demodicosis and healthy controls. It is concluded that significant alteration in the CD4+/CD8+ ratio may be implicated in the pathogenesis of generalized canine demodicosis.

scholarly journals Decreased Expression of T-Cell Costimulatory Molecule CD28 on CD4 and CD8 T Cells of Mexican Patients with Pulmonary Tuberculosis

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
German Bernal-Fernandez ◽  
Patricia Espinosa-Cueto ◽  
Rosario Leyva-Meza ◽  
Nathalie Mancilla ◽  
Raul Mancilla
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Pulmonary Tuberculosis ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Costimulatory Molecule ◽  
Cell Phenotype ◽  
Mexican Patients

Patients with tuberculosis frequently develop anergy, a state of T-cell hyporesponsiveness in which defective T-cell costimulation could be a factor. To know if the expression of T-cell costimulatory molecules was altered in tuberculosis, we analyzed the peripheral blood T-cell phenotype of 23 Mexican patients with pulmonary tuberculosis. There was severe CD4 (P<.001) and CD8 (P<.01) lymphopenia and upregulation of costimulatory molecule CD30 on CD4 and CD8 T cells (P<.05); this increase was higher in relapsing tuberculosis. The main finding was severe downregulation of the major costimulatory molecule CD28 on both CD8 and CD4 T cells (P<.001). Depletion of the CD4/CD28 subset, a hitherto undescribed finding, is relevant because CD4 T cells constitute the main arm of the cell-mediated antimycobacterial immune response.

scholarly journals T-Cell Subset Counts in Peripheral Blood Can Be Used as Discriminatory Biomarkers for Diagnosis and Severity Prediction of Coronavirus Disease 2019

2020 ◽  
Vol 222 (2) ◽  
pp. 198-202 ◽  
Author(s):  
Mei Jiang ◽  
Yang Guo ◽  
Qing Luo ◽  
ZiKun Huang ◽  
Rui Zhao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Cell Subset ◽  
Lymphocyte Subset ◽  
T Cell Subset ◽  
Diagnosis And Prognosis ◽  
Severity Prediction

Abstract This study evaluated the significance of lymphocyte subset detection in peripheral blood in the diagnosis and prognosis of coronavirus disease 2019 (COVID-19). Our results revealed that CD3+ T cells, CD4+ T cells, CD8+ T cells, and natural killer cells were significantly decreased in patients with COVID-19. These patients had a relatively slight decrease in CD4+ T cells but a severe decrease in CD8+ T cells. The significantly elevated CD4/CD8 ratio was observed in COVID-19 patients. T-cell subset counts were related to the severity and prognosis of COVID-19, suggesting that the counts of CD8+ T and CD4+ T cells can be used as diagnostic markers of COVID-19 and predictors of disease severity.

scholarly journals Comprehensive Immune Profiling from Peripheral Blood and Bone Marrow in Newly Diagnosed and Relapsed/Refractory Multiple Myeloma Patients Reflects Differences in Immune Subsets and Activation Status

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3161-3161 ◽  
Author(s):  
Greg E. Pietz ◽  
Mark Tometsko ◽  
Wilbert B. Copeland ◽  
Elizabeth Whalen ◽  
Frank Schmitz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Cell Populations ◽  
Employment Equity ◽  
Programmed Death ◽  
Equity Ownership

Abstract BACKGROUND: Loss of immune surveillance is critical in the pathogenesis of multiple myeloma (MM) and the progression from smoldering to symptomatic MM. To date, no clear efficacy signal has been observed with programmed-death 1 and programmed death ligand-1 inhibitors in patients with MM. General immune dysfunction in MM is well documented, but the evolving immune landscape in relapsed/refractory MM (RRMM) vs newly diagnosed MM (NDMM) is less well characterized. This study aimed to characterize immune profiles in peripheral blood and bone marrow from patients with NDMM and RRMM. METHODS: Peripheral blood samples were collected from 35 NDMM and 146 RRMM patients and 36 age-matched healthy volunteers (HVs). Cell surface and intracellular antigen staining using fluorochrome labeled antibodies was performed on a BD FACSCanto II flow cytometer. Bone marrow aspirates were collected from 26 NDMM and 73 RRMM patients, and the transcriptome was assessed by mRNA-Seq. RESULTS: In peripheral blood, T-cell populations differed between HVs and NDMM and RRMM patients. Absolute numbers of lymphocytes were higher in HVs than in NDMM and RRMM, regardless of the MM disease state. Absolute numbers of total CD4+ T cells and naïve CD4+ T cells were lower in RRMM patients, whereas CD4+ effector memory T cells as a proportion of total CD4+ T cells were increased in RRMM patients. Blood from RRMM patients also contained increased levels of proliferating CD4+ T cells, as evidenced by Ki67, ICOS, and HLA-DR, compared with blood from NDMM patients; HVs had values much closer to those from NDMM than from RRMM patients, suggesting a trend influenced by disease state or therapeutic intervention. In bone marrow, immunologic gene expression signatures were elevated in NDMM vs RRMM patients; the differences were similar to those in peripheral blood. Using limma to model the differential expression of all measured genes between NDMM and RRMM, we identified 367 genes that were elevated in NDMM patients vs 52 in RRMM patients. Gene set analyses using Molecular Signatures Database immunologic signatures (C7) applied to those 367 genes showed that naïve T-cell genes were increased in the bone marrow of NDMM vs RRMM patients. Gene set enrichment analysis with limma, using 489 gene sets from xCell representing 64 cell types and controlling for differences in tumor burden, indicated that macrophage, monocyte, and neutrophil genes were upregulated and T cells, particularly naïve CD4+ T cells, were downregulated in RRMM patients. Immunohistochemistry results from bone marrow biopsies showed increased programmed death-ligand 1 expression on tumor and infiltrating immune cells and increased CD8 infiltration into bone marrow in RRMM vs NDMM patients. Multiparameter immunofluorescence is underway to confirm these findings and further understand the tumor immune microenvironment in patient subsets. As expected, baseline RRMM immune cell populations depended on prior lines of therapy. Daratumumab-exposed RRMM patients had elevated total CD8+ T cells in peripheral blood but decreased CD38+, CD4+, and CD8+ T cells, as well as decreased total natural killer cells, compared with the daratumumab-naïve patients. Transcriptome analyses of bone marrow from daratumumab-exposed RRMM patients revealed increased T-cell gene expression signatures relative to marrow from daratumumab-naïve patients. Additionally, pomalidomide-exposed RRMM patients had increased activated CD4+ and CD8+ T cells vs pomalidomide-naïve patients. CONCLUSIONS: These data indicate that RRMM patients have peripheral blood and bone marrow environments with highly differentiated T-cell populations, whereas NDMM patients show elevated T-cell levels with proliferative capacity. Furthermore, the bone marrow of RRMM patients is enriched with neutrophils and macrophages; investigation is ongoing to determine if these cell types contribute to an immunosuppressive tumor microenvironment. Understanding immune system function based on disease progression, patient segments, and prior lines of therapy is imperative as treatment of MM improves, and it may inform the administration and sequence of next generation immunotherapeutics and identify predictive biomarkers for optimal treatment selection. Disclosures Pietz: Celgene Corporation: Employment. Tometsko:Celgene Corporation: Employment, Equity Ownership. Copeland:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Thompson:Celgene Corporation: Employment, Equity Ownership. Agarwal:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Buchholz:Celgene Corporation: Employment. Komashko:Celgene Corporation: Employment. Dell'Aringa:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment.

PD-1/PD-L Pathway Potentially Involved in ITP Immunopathogenesis

2019 ◽  
Vol 119 (05) ◽  
pp. 758-765 ◽  
Author(s):  
Mu Nie ◽  
Yang Liu ◽  
Xiu-xiu Li ◽  
Ya-nan Min ◽  
Dan-dan Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Apoptosis ◽  
Cd4 T Cells ◽  
Peripheral Tolerance ◽  
Healthy Controls ◽  
T Cell Apoptosis ◽  
Ifn Γ

AbstractThe binding of programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 on antigen-presenting cells turns off autoreactive T cells and induces peripheral tolerance. Aberrant PD-1/PD-L signalling could result in a breakdown of peripheral tolerance and lead to autoimmune diseases. In this study, we detected PD-1 and PD-L expression on T cells and dendritic cells (DCs) in immune thrombocytopenia (ITP) patients with active disease by flow cytometry. The effects of PD-L1-Fc fusion protein (PD-L1-Fc) on T cells and on secretion of interferon-γ (IFN-γ) and interleukin-2 (IL-2) were detected by flow cytometry and enzyme-linked immunosorbent assay, respectively. Compared with healthy controls, PD-1 expression was significantly increased in CD4+ T cells and CD8+ T cells from patients with active ITP. However, PD-L1 expression on monocyte-derived DCs was lower in patients with active ITP than in healthy controls. In vitro assays revealed that PD-L1-Fc increased T cell apoptosis, inhibited activation and proliferation of CD4+ T cells and CD8+ T cells and decreased IFN-γ and IL-2 secretion in patients with active ITP. These results suggest that the aberrant PD-1/PD-L negative co-stimulatory pathway may play a role in ITP. Enhancing PD-1/PD-L signalling might be a promising therapeutic approach for ITP patients by enhancing T cell apoptosis, inhibiting T cell activation and proliferation and reducing secretion of inflammatory factors.

Abundant Tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1386-1392 ◽  
Author(s):  
Emmanuel Hanon ◽  
Sarah Hall ◽  
Graham P. Taylor ◽  
Mineki Saito ◽  
Ricardo Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Virus Type ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Type I ◽  
Ctl Response ◽  
Specific Ctls ◽  
Human T Cell

The role of the cellular immune response in human T-cell leukemia virus type I (HTLV-I) infection is not fully understood. A persistently activated cytotoxic T lymphocyte (CTL) response to HTLV-I is found in the majority of infected individuals. However, it remains unclear whether this CTL response is protective or causes tissue damage. In addition, several observations paradoxically suggest that HTLV-I is transcriptionally silent in most infected cells and, therefore, not detectable by virus-specific CTLs. With the use of a new flow cytometric procedure, we show here that a high proportion of naturally infected CD4+ peripheral blood mononuclear cells (PBMC) (between 10% and 80%) are capable of expressing Tax, the immunodominant target antigen recognized by virus-specific CTLs. Furthermore, we provide direct evidence that autologous CD8+ T cells rapidly kill CD4+ cells naturally infected with HTLV-I and expressing Tax in vitro by a perforin-dependent mechanism. Consistent with these observations, we observed a significant negative correlation between the frequency of Tax11-19-specific CD8+ T cells and the percentage of CD4+ T cells in peripheral blood of patients infected with HTLV-I. Those results are in accordance with the view that virus-specific CTLs participate in a highly efficient immune surveillance mechanism that persistently destroys Tax-expressing HTLV-I-infected CD4+ T cells in vivo.

scholarly journals Somatic Mutations in CD8+ T Cells in Patients with Chronic Immune Thrombocytopenia Are Associated with Increased Clonality and Cytotoxic Phenotype of CD8+ T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 131-131
Author(s):  
Hanna Rajala ◽  
Paula Savola ◽  
Sofie Lundgren ◽  
Samuli Eldfors ◽  
Tiina Kelkka ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Deep Sequencing ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Somatic Mutations ◽  
Healthy Controls ◽  
Variant Allele Frequency

Abstract Background The role of self-antigen-targeting T cells is well established in multiple autoimmune disorders. In immune thrombocytopenia (ITP), CD8+ cytotoxic T cells target both peripheral blood platelets and bone marrow megakaryocytes. In addition, CD4+ helper T cell imbalance and cytokine secretion leads to the release of autoantibodies by B cells favoring the destruction of platelets. In order to understand if somatic mutations play a role and drive the aberrant immune responses in ITP, we analyzed sorted CD4+ and CD8+ T cells with deep sequencing panel. Methods The study population consisted of 13 adult patients diagnosed with chronic ITP (median age 47 years) at least one year before the first sampling. The median number of lines of therapy was 4, (range 0-8). In addition, 11 healthy control samples were collected for T cell phenotyping and TCR analysis (median age 63 years). After separation of peripheral blood mononuclear cells, immunophenotyping by flow cytometry was performed for CD4+ and CD8+ T cells and both fractions were sorted using magnetic beads. The CD4+ and CD8+ clonality was analyzed by deep sequencing of TCRB CDR3 using Adaptive platform. Custom-made deep sequencing gene panel covering 2433 genes related to immunity and cancer was used to analyze somatic mutations from 11 ITP patients. Mean target coverage was 567X (range 405-757X). Somatic variants with over 2% variant allele frequency (VAF) were called using Varscan2-based bioinformatics tool and CD4+ and CD8+ fractions were used as each other's germline control. Candidate somatic variants were visually validated with Integrative Genomics Viewer. Results ITP patients harbored clonal TCR rearrangements both in CD8+ and CD4+ fractions. The median percentage of the largest T cell clone of all TCR rearrangements in an individual sample was 9.5% (range 1.8%-31.9%) in CD8+ cells and 7.1% (range 0.9-13.6%) in CD4+. The median clone size or clonality index did not differ between ITP patients and healthy controls. Age correlated with the largest rearrangement and clonality index both in CD4+ and CD8+ fractions in ITP patients (r=0.70, p=0.015 and r=0.60, p=0.043 for CD4+ and r=0.63, p=0.033 and r=0.77, p=0.0050 for CD8+), but no correlation was observed in healthy controls. The size of the maximum CD8+ TCR rearrangement and clonality index correlated positively with the percentage of highly cytotoxic CD8+CD57+ (r=0.80, p=0.0029 and r=0.90, p=0.0002) and terminally differentiated CD8+ effector memory with CD45RA (TEMRA) T cells (r=0.66, p=0.022 and r=0.78, p=0.0038). In healthy controls positive correlation was observed only between CD8+ clonality index and CD8+CD57+ T cells, but not with TEMRAs. Somatic mutations in CD8+ T cells were detected in 7/11 (64%) of ITP patients. Somatic variants were distinct between individuals and their mean variant allele frequency (VAF) was 4.5% (range 2.0-13.6%). The mutated genes included those related to IFNg-signaling pathway (STAT1, NLRP4, CARD6, and EBF1), apoptosis (CASP6), and inflammation and cancer (ATM, EGR1, NOD1). ITP patients with CD8+ mutations had larger immunodominant CD8+ TCR clone (Figure 1A) and higher CD8+ clonality index (Figure 1B). The patients with CD8+ mutations had more cytotoxic CD8+CD57+ T cells than mutation-negative cases, but CD8+TEMRAs did not differ between the two groups (Figure 1C). The mutation profile of the CD4+ samples was different: 3/11 (27%) of the ITP patients had mutations in the genes connected to clonal hematopoiesis of indeterminate potential (CHIP). One patient with a maximum of 8% clone in CD4+ T cells harbored mutations both in TET2 (VAF 13.5%) and PPM1D (VAF 12.8%): the VAFs suggest that the mutation was not restricted to the largest CD4+ clone. The second patient had a different mutation in PPM1D gene (VAF 2.8%) and the third one had a PPM1B-mutation both in CD4+ (VAF 18.0%) and CD8+ (VAF 11.1%) T cells. Conclusions To our knowledge, this is the first report of somatic variants in T cells in patients with ITP. Discovered somatic mutations in CD8+ T cells were associated with increased clonality and highly cytotoxic phenotype of CD8+ T cells. Interestingly, three patients harbored mutations in CHIP-associated genes in CD4+ and also in CD8+ fraction, with recurrent PPM1D-mutations. Further studies and screening of a larger ITP patient cohort is warranted to understand the meaning and functional consequence of somatic mutations in the pathogenesis of chronic ITP. Disclosures Ebeling: Boehringer Ingelheim: Consultancy; Celgene: Speakers Bureau; Otsuka Pharma Scandinavia AB: Consultancy. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria; Pfizer: Honoraria, Research Funding.

Human Bone Marrow as a Source of Multifunctional CMV-Specific CD4+ T Cells for Adoptive Cell Therapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2973-2973
Author(s):  
Il-Kang Na ◽  
Anne Letsch ◽  
Ines Noack ◽  
Sandra Bauer ◽  
Jens Geginat ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Human Bone ◽  
Ifn Γ

Abstract Introduction Adoptive cell transfer of ex vivo primed and expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising approach to treat both infectious and malignant diseases in humans. First clinical studies have shown that transfer of Cytomegalovirus (CMV)-specific CD8+ T cells is safe and effective in reconstitution of cellular immunity against CMV disease. Efficacy of adoptive T cell therapy is limited by the numbers of CTLs in vitro and the survival and function after infusion. CD4+ T cells may enhance activity via direct or indirect effector functions (Matloubian 1994 [1]). In this study we have analysed whether bone marrow is superior to peripheral blood for expansion of CMV-specific T cells. Experimental design Paired peripheral blood and bone marrow samples were obtained from patients who underwent total hip arthroplasty. By using two different protocols T cells were expanded in the presence of IL-2 and IL-7 either from bulk culture with exposure of two different peptide pools (IE1 and pp65) or after selection via IFN-γ secretion by stimulation with pp65. CMV specific immune responses were assessed by using multiparameter flow cytometry staining cells for CD3, CD4, CD8, CCR7 and CD45RA and for the cytokines IFN-γ IL-2 and TNF at day 0 and after 10 days of in vitro expansion. Results Similar frequencies of cytokine-producing pp65– and IE1-specific CD4+ and CD8+ T cells were found in unmanipulated paired PB and BM samples. Expansion of CMV-specific T cells from BM resulted in significantly higher frequencies of specific CD4+ T cells than from PB, whereas no difference in frequencies of CMV-specific CD8+ T cells was observed. Interestingly, significantly higher frequencies of BM pp65 and IE1-specific CD4+ T cells were multifunctional, characterized by producing simultaneously IFN-γ, TNF and IL-2 (IE1: BM mean 0.44% ± 0.16; PB mean 0.09% ± 0.05, p=0.031; pp65: BM mean 3.87% ± 2.46; PB mean 1.24% ± 0.90, p=0.031). Expansion of multi-functional CD4+ T cells from BM was observed with both the bulk and selection assay protocol. Both PB and BM CMV-specific CD4+ and CD8+ T cell lines had a predominant CD45RA-CCR7- effector memory phenotype. Conclusions This study implicates the use of human bone marrow as a source for expansion of multifunctional CMV-specific CD4+ T cells. Recent studies in HIV and Leishmania support the crucial role of multifunctional T cells in disease control (Darrah 2007 [2]).

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