Functional Characterization Of CD8 Research Articles

Identification and functional characterization of CD8+ T regulatory cells in type 1 diabetes patients.

Type 1 diabetes is an autoimmune disease where autoreactive T lymphocytes destroy pancreatic beta cells. We previously reported a defect in CD4+ Tregs cell proliferation and reduced CD4+ Tregs PD-1 expression in patients. Another ‘memory-like’ regulatory subset, CD8+ Tregs, evaluated as CD8+CD25+FOXP3+, has recently raised interest for their effective suppressive activity. Different CD8+ T cell populations, their proliferation capacity and expression of PD-1 molecule were evaluated by flow-cytometer analysis in newly diagnosed, long-term Type 1 diabetes patients compared to healthy normal donors. Under basal conditions, CD8+ Tregs and CD8+ Teffs were seemingly represented among study groups while there was evidence of diminished expression of PD-1 in Teff subsets of long-term patients. After 3 days of PMA/ionomycin stimulation, patients CD8+ Tregs showed decreased percentage in respect to control group. CD8+ Teffs were instead increased in long-term diabetics versus controls. PD-1+CD8+ Tregs were represented at a much lower percentage in long-term diabetic patients, in respect to controls. Importantly, patients CD8+ Tregs and CD8+ Teffs presented a significant proliferation defect in respect to the control group. In conclusion, our study indicates that a defect of CD8+ Tregs is observed in diabetics. This subset could thus represent a novel target of immunotherapy in patients.

Evaluating the repertoire of immune checkpoint markers expressed on peripheral and ascites CD8+ T cells in ovarian cancer.

5571 Background: Understanding the immune checkpoint marker repertoire can facilitate development of therapeutic strategies to improve efficacy of immune-based therapies. We used a novel high-dimensional flow cytometry panel to determine co-expression patterns of immune checkpoint markers and effector function of CD8+ T cells from peripheral blood and ascites of patients newly diagnosed with ovarian cancer. Methods: Peripheral blood and ascites samples were collected from patients with epithelial ovarian cancer (n=8). Cells isolated from peripheral blood and ascites were used for immune profiling by multiparameter flow cytometry of 5 inhibitory receptors (PD-1, LAG-3, TIM-3, TIGIT, and BTLA) on CD8+ T cells, along with 4 functional parameters (production of each of the following: TNF-α, IFN-γ, IL-2, and upregulation of CD107a). A complementary multiplex analysis on plasma and ascites fluid was performed to quantify 14 soluble checkpoint markers. Results: The concentrations of soluble PD-1, TIM-3, LAG-3, CTLA-4, BTLA, IDO, and CD137 were increased in ascites fluid compared to plasma from patients with ovarian cancer. Ascites CD8+ T cells co-express higher levels of inhibitory receptors than peripheral CD8+ T cells. In total, CD8+ T cells in ascites retained the ability to produce effector functions at levels similar to peripheral blood. However, IFN-γ production was retained in PD-1 only expressing CD8+ T cells and decreased in CD8+ T cells co-expressing multiple receptors. Conclusions: High-dimensional flow cytometry allowed for the phenotypic and functional characterization of CD8+ T cells from ovarian cancer patients. The profile of receptor co-expression was distinct in peripheral blood compared to ascites. Collectively, our study suggests that co-expression of factors beyond PD-1 influences CD8+ T cell activity. Thus blocking PD-1 and PD-L1 alone may not be sufficient for CD8+ T cells expressing multiple inhibitory receptors.

Human leukocyte antigen class 1 genotype distribution and analysis in persons with active tuberculosis and household contacts from Central Uganda.

BackgroundTo determine the distribution of Human leukocyte antigen (HLA) class I genotypes in a Ugandan population of persons with tuberculosis (TB) and establish the relationship between class I HLA types and Mycobacterium tuberculosis (MTB) disease.MethodsBlood samples were drawn from HIV negative individuals with active TB and HIV negative household controls. DNA was extracted from blood samples and HLA typed by the polymerase chain reaction-sequence specific primer method. The allelic frequencies were determined by direct count.ResultsHLA-A*02, B*15, C*07, C*03, B*58, C*04, A*01, A*74, C*02 and A*30 were the dominant genotypes in this Ugandan cohort. There were differences in the distribution of HLA types between the individuals with active TB and the household controls with only HLA-A*03 allele showing a statistically significant difference (p = 0.017 crude; OR = 6.29 and p = 0.016; OR = 11.67 after adjustment for age). However, after applying the Benjamini and Hochberg adjustment for multiple comparisons the difference was no longer statistically significant (p = 0.374 and p = 0.176 respectively).ConclusionsWe identified a number of HLA class I alleles in a population from Central Uganda which will enable us to carry out a functional characterization of CD8+ T-cell mediated immune responses to MTB. Our results do not show a positive association between the HLA class I alleles and TB in this Ugandan population however the study sample was too small to draw any firm conclusions about the role of HLA class I alleles and TB development in Uganda.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1833-3) contains supplementary material, which is available to authorized users.

Flow cytometry-based enumeration and functional characterization of cd8 t regulatory cells in patients with multiple myeloma before and after lenalidomide plus dexamethasone treatment

Multiple myeloma (MM) is a malignancy of plasma cells frequently associated with immune abnormalities. Several studies have confirmed that in MM immune deregulation can be mediated by increased numbers of CD4 T regulatory (Treg) cells, and these cells were also associated with poor outcome. In this study, we aimed to study CD8 Treg cells before and after lenalidomide plus dexamethasone (len-dex) treatment in MM patients. Using flow cytometry, we enumerated and assessed suppressive function of CD8 Treg cells in 16 MM patients before and after len-dex treatment. Numbers of CD8 Treg cells (CD8+CD25hi+FoxP3+) (P < 0.01) were significantly increased in MM patients (before treatment) compared to healthy donors. However, no significant changes were observed in CD4 and CD8 T cells. A significant increase in CD8 Treg cells was observed after len-dex treatment compared to pre-treatment but no significant difference was observed in CD4 and CD8 T cells. Proliferation assay data showed that CD8 Treg cells inhibited proliferation of CD4 T cells and IFN-γ secretion in a concentration dependent manner. Suppressive activity of CD8 Treg cells did not differ significantly between healthy donors, untreated and len-dex treated MM patients. A significant abnormal level of IL-10 was observed from proliferation assays of untreated and len-dex treated MM patients compared to healthy donors (P ≤ 0.03). Using flow cytometry, we have shown that suppressive CD8 Treg cells are increased in MM patients and len-dex treatment is unable to control these suppressive CD8 Treg cells.

Flow cytometry based enumeration and functional characterization of CD8 T regulatory cells in patients with multiple myeloma before and after lenalidomide plus dexamethasone treatment.

Multiple myeloma (MM) is a malignancy of plasma cells frequently associated with immune abnormalities. Several studies have confirmed that in MM immune deregulation can be mediated by increased numbers of CD4 T regulatory (Treg) cells, and these cells were also associated with poor outcome. In the present study, we aimed to study CD8 Treg cells before and after lenalidomide plus dexamethasone (len-dex) treatment in MM patients. Using flow cytometry, we enumerated and assessed suppressive function of CD8 Treg cells in 16 MM patients before and after len-dex treatment. Numbers of CD8 Treg cells (CD8+CD25hi+FoxP3+) (P<0.01) were significantly increased in MM patients (before treatment) compared to healthy donors. However, no significant changes were observed in CD4 and CD8 T cells. A significant increase in CD8 Treg cells was observed after len-dex treatment compared to pre-treatment but no significant difference was observed in CD4 and CD8 T cells. Proliferation assay data showed that CD8 Treg cells inhibited proliferation of CD4 T cells and IFN-γ secretion in a concentration dependent manner. Suppressive activity of CD8 Treg cells did not differ significantly between healthy donors, untreated and len-dex treated MM patients. A significant abnormal level of IL-10 was observed from proliferation assays of untreated and len-dex treated MM patients compared to healthy donors (P≤0.03). Using flow cytometry, we have shown that suppressive CD8 Treg cells are increased in MM patients and len-dex treatment is unable to control these suppressive CD8 Treg cells. © 2013 Clinical Cytometry Society.

Murine CD8 + regulatory T lymphocytes: The new era

Regulatory T lymphocytes unequivocally play a major role in the maintenance of immunologic homeostasis. The first descriptions of regulatory T lymphocytes concerned CD8 + cells, but this field was brought into discredit when some of its central tenets turned out to be erroneous. CD4 + regulatory T cells took over and, with the help of newly developed molecular tools, rapidly were phenotypically and functionally characterized. We now know that these cells control a large variety of immune responses. However some observations of in vitro or in vivo immune regulation could not be explained with CD4 + regulatory T cell activity and depended on the action of a variety of CD8 + T cell populations. In recent years, substantial progress has been made in the phenotypic and functional characterization of CD8 + regulatory T cells. These cells play a role in the control of intestinal immunity, immunopathology, and autoimmunity, as well as in immune privilege of the eye, in oral tolerance, and in prevention of graft-versus-host disease and graft-rejection. The suppressor effector mechanisms used by these cells are in part shared with CD4 + regulatory T cells and in part unique to this population. We here review the current literature on naturally occurring and experimentally induced murine CD8 + regulatory T-cell populations.

Selection of T Cell Clones Expressing High-Affinity Public TCRs within Human Cytomegalovirus-Specific CD8 T Cell Responses

Assessment of clonal diversity of T cell responses against human CMV (HCMV), a major cause of morbidity in immunodepressed patients, provides important insights into the molecular basis of T cell immunodominance, and has also clinical implications for the immunomonitoring and immunotherapy of HCMV infections. We performed an in-depth molecular and functional characterization of CD8 T cells directed against an immunodominant HLA-A2-restricted epitope derived from HCMV protein pp65 (NLV/A2) in steady state and pathological situations associated with HCMV reactivation. NLV/A2-specific T cells in healthy HCMV-seropositive donors showed limited clonal diversity and usage of a restricted set of TCR Vbeta regions. Although TCRbeta-chain junctional sequences were highly diverse, a large fraction of NLV/A2-specific T cells derived from distinct individuals showed several recurrent (so-called "public") TCR features associated in some cases with full conservation of the TCRalpha chain junctional region. A dramatic clonal focusing of NLV/A2-specific T cells was observed in situations of HCMV reactivation and/or chronic inflammation, which resulted in selection of a single clonotype displaying similar public TCR features in several patients. In most instances the NLV/A2-specific dominant clonotypes showed higher affinity for their Ag than subdominant ones, thus suggesting that TCR affinity/avidity is the primary driving force underlying repertoire focusing along chronic antigenic stimulation.

Cross-staining of cytotoxic T lymphocyte populations with peptide-MHC class I multimers of natural HIV-1 variant antigens.

Multimeric soluble peptide-MHC class I complexes bind to T cell receptors (TCR) on the surface of CD8 cytotoxic T lymphocytes (CTL) with sufficient avidity to enable the labelling of CTL according to their antigen specificity [1]. Visualization of antigen-specific CD8+T lymphocytes with fluorogen-conjugated peptide-MHC class I multimers enables both the physical quantification and the phenotypic characterization of CTL responses directly ex vivo, and these properties have led to the widespread adoption of this technique throughout the field of experimental immunology. However, it is well documented that TCR can exhibit a degree of promiscuity in terms of ligand recognition [2]. We recently demonstrated that the multimerization of peptide-MHC class I can allow binding to TCR with affinities that are too low to generate ligand-induced physiological responses, and result in crossreactive staining of HIV-specific CTL lines and clones by unrecognized natural variant antigens [3]. These results suggest that crossreactivity between peptide-MHC class I multimers and low avidity CTL could be relevant to the interpretation of direct ex-vivo staining. The possibility of such cross-staining is likely to be considerably greater when using peptide-MHC class I multimers to stain CTL specific for epitopes derived from antigenically variable pathogens such as HIV-1 [4]. Here we extend our previous study with the demonstration that the cross-staining of CTL populations with peptide-MHC class I multimers can complicate analyses performed directly ex vivo.

Functional Characterization of CD8+ Antigen‐Specific Cytotoxic T Lymphocytes after Enrichment Based on Cytokine Secretion: Comparison with the MHC‐Tetramer Technology

Cell therapy with antigen‐specific T cells holds promise for various diseases including cancer and viral infections. The powerful enrichment procedure based on major histocompatibility complex (MHC)‐tetramers, however, is of limited applicability so far. Therefore, the recently developed cell surface affinity matrix technology that allows direct identification and enrichment of life antigen‐specific T cells based on cytokine secretion was evaluated in this respect. To this end, CD8+ T cells directed against the HLA‐A*0201‐restricted melanoma‐associated peptide Melan‐A (aa26–35) were generated by combining stimulation of peptide‐pulsed autologous dendritic cells (DC) with antigen‐independent expansion with anti‐CD3/anti‐CD28 monoclonal antibodies (MoAb). Antigen‐specific cytotoxic T lympocyte (CTL) were detected based on stimulation‐induced interferon (IFN)‐γ and interleukin (IL)‐4 secretion and enriched &gt; 100‐fold using the cell surface affinity matrix technology. The resulting IFN‐γ‐ and IL‐4‐secreting CTL lines contained &gt; 80% and &gt; 70% cytokine positive T cells, respectively. They exhibited a cytotoxic activity against Melan‐A expressing target cells that was significantly higher as compared to nonpurified CTL. Direct staining of enriched CTL with HLA‐A2‐Melan‐A‐tetramers revealed a high correlation between the results obtained from the cell surface affinity matrix technology and those obtained from tetrameric complexes. Altogether, our study demonstrates that cytokine‐driven enrichment based on the cell surface affinity matrix technology enables selective isolation of functionally active antigen‐specific CTL that may be used for an adoptive T cell transfer in immunotherapy.

Phenotypic and Functional Characterization of CD8+T Cell Clones Specific for a Mouse Cytomegalovirus Epitope

A series of CD8+T cell clones, specific for the IE1 epitope YPHFMPTNL, of the immediate-early protein 1 of the murine cytomegalovirus (MCMV) were generated in order to determine their protective activity against this infection and correlate their phenotypic markers with antiviral activity. We found that the adoptive transfer of three of these anti-MCMV CD8+T cell clones into irradiated naive mice resulted in protection against challenge, while another CD8+T cell clone, of the same specificity, failed to confer protection. The clones that conferred protection against lethal challenge reduced greatly viral replication in the lung and other organs of the mice. Using one of the protective anti-MCMV CD8+T cell clones we found that in order to be fully protective the cells had to be transferred to recipient mice no later than 1 day after MCMV challenge. The adoptive transfer of these CD8+T cell clones also protected CD4+T-cell-depleted mice. Phenotypic characterization of the anti-MCMV clones revealed that the nonprotective clone expressed very low levels of CD8 molecules and produced only small amounts of TNF-α upon antigenic stimulation. Most importantly, our current study demonstrates that this MHC class I-restricted IE1 epitope of MCMV is efficiently presented to CD8+T cell clonesin vivoand further strengthens the possibility of the potential use of CD8+T cell clones as immunotherapeutic tools against cytomegalovirus-induced disease.

Phenotypic and functional characterization of CD8+ T lymphocytes from the central nervous system of rats with coronavirus JHM induced demyelinating encephalomyelitis.

Intracerebral infection of Lewis (LEW) inbred rats with the neurotropic strain of the murine coronavirus JHM (JHMV) frequently results in a monophasic paralytic disease. In contrast, infection of Brown Norway (BN) inbred rats does not lead to clinical disease. Previous findings indicated that in both rat strains brain-infiltrating leukocytes consisted mainly of CD8+ T lymphocytes. Here, we phenotypically as well as functionally characterised this T cell subset after isolation from the central nervous system (CNS). Using JHMV-infected target cells, MHC class I restricted, cytotoxic T lymphocytes were demonstrated to be present in the leukocyte fraction from the CNS of both, susceptible LEW and disease-resistant BN rats. However, compared to infected, but healthy BN rats, diseased LEW rats generated an enhanced cytotoxic immune response which became most prominent at the maximum of neurological disease. Recently published observations from our laboratory demonstrated a strong virus-specific antibody response in the CNS of BN rats. In LEW rats, however, the response was delayed and of low magnitude. This suggests, that consequences of cytotoxic T lymphocyte action in JHMV-infected CNS tissue largely depend on the efficacy of an accompanying virus-specific humoral immune response.

An immunological role for the CD8 beta-chain.

Mature T cells can be functionally divided into two categories distinguished by surface expression of either CD4 or CD8, which in turn corresponds to restriction by and binding to class II or class I major histocompatibility complex proteins, respectively. CD8 can be expressed as a homodimer of the alpha-chain, or as a heterodimer of alpha- and beta-chains on human and mouse T cells, although most peripheral T cells seem to express CD8 alpha beta heterodimers exclusively (reviewed in ref. 9). Functional characterization of CD8 has focused primarily on the effect of the alpha-chain, which enhances or reconstitutes T-cell responses in homodimeric form and may play a specific role in thymic selection. In contrast, no role has been ascribed to CD8 beta or alpha beta heterodimers specifically. Here we show that CD8 alpha beta transfectants produce more interleukin-2 than CD8 alpha transfectants in response to specific stimuli. Increased interleukin-2 is also observed in cells expressing hybrid CD8 beta-alpha molecules (extracellular CD8 beta plus CD8 alpha transmembrane and cytoplasmic regions) on their surface. These results indicate that external portions of CD8 beta could be critical and that they may act independently of CD8 alpha in mediating their augmentation effect.