Distinct APC Subtypes Drive Spatially Segregated CD4+ and CD8+ T-Cell Effector Activity during Skin Infection with HSV-1

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4⁺ and CD8⁺ T-cells during skin infection with HSV-1. IFN-γ-producing CD4⁺ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8⁺ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4⁺ T cells, CD8⁺ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8⁺ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4⁺ and CD8⁺ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).     

Involvement of CD244 in Regulating CD4+ T Cell Immunity in Patients with Active Tuberculosis

CD244 (2B4) is a member of the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors and it plays an important role in modulating NK cell and CD8⁺ T cell immunity. In this study, we investigated the expression and function of CD244/2B4 on CD4⁺ T cells from active TB patients and latent infection individuals. Active TB patients had significantly elevated CD244/2B4 expression on M. tuberculosis antigen-specific CD4⁺ T cells compared with latent infection individuals. The frequencies of CD244/2B4-expressing antigen-specific CD4⁺ T cells were significantly higher in retreatment active TB patients than in new active TB patients. Compared with CD244/2B4-dull and -middle CD4⁺ T cells, CD244/2B4-bright CD4⁺ T cell subset had significantly reduced expression of IFN-γ, suggesting that CD244/2B4 expression may modulate IFN-γ production in M. tuberculosis antigen-responsive CD4⁺ T cells. Activation of CD244/2B4 signaling by cross-linking led to significantly decreased production of IFN-γ. Blockage of CD244/2B4 signaling pathway of T cells from patients with active TB resulted in significantly increased production of IFN-γ, compared with isotype antibody control. In conclusion, CD244/2B4 signaling pathway has an inhibitory role on M. tuberculosis antigen-specific CD4⁺ T cell function.     

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8⁺ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8⁺ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8⁺ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8⁺ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8⁺ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8⁺ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.     

Human CD8+ T Cells from TB Pleurisy Respond to Four Immunodominant Epitopes in Mtb CFP10 Restricted by HLA-B Alleles

CD8⁺ T cells are essential for host defense to Mycobacterium tuberculosis (Mtb) infection and identification of CD8⁺ T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10) from Mtb is a potent CD8⁺ T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13–15 amino acid (aa) peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8⁺ T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8⁺ T cells. The epitope-specific CD8⁺ T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8⁺ T cells shared biased usage of T cell receptor (TCR) variable region of β chain (Vβ) 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8⁺ T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8⁺ T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.     

Activation Associated ERK1/2 Signaling Impairments in CD8+ T Cells Co-Localize with Blunted Polyclonal and HIV-1 Specific Effector Functions in Early Untreated HIV-1 Infection

We recently observed that a large proportion of activated (CD38⁺HLA-DR⁺) CD8⁺ T cells from recently HIV-1-infected adults are refractory to phosphorylation of ERK1/2 kinases (p-ERK1/2-refractory). Given that the ERK1/2 pathway mediates intracellular signaling critical for multiple T cell functions, including key effector functions, the loss of ERK1/2 responsiveness may have broad consequences for CD8⁺ T cell function. In the current study, we hypothesized that the p-ERK1/2-refractory population, localized largely within the activated CD38⁺HLA-DR⁺ CD8⁺ T cell population, would display impairments in CD8⁺ T cell effector functions, such as cytokine production and degranulation, compared to CD8⁺ p-ERK1/2-responsive cells. We further hypothesized that the p-ERK1/2-refractory phenotype is persistent over time during untreated infection, and would correlate with poorer virologic control, in a manner independent of CD8⁺ T cell activation level. We performed single-cell resolution, flow cytometric assays of phospho-kinase responses paired to intracellular cytokine staining in one assay to examine IFN-γ, perforin and CD107α responses in CD8⁺ T cells by ERK1/2 signaling profile. On a per cell basis, p-ERK1/2-refractory cells, which fall predominantly within the activated CD8⁺ T cell compartment, produced less IFN-γ in response to polyclonal or HIV-1 antigen-specific stimulation, and expressed lower levels of perforin and CD107α. The p-ERK1/2 refractory cell population displayed minimal overlap with the PD-1 and Tim-3 inhibitory exhaustion markers and predicted high viral load independent of activation, suggesting that ERK1/2 may be a unique marker and point of intervention for improving CD8⁺ T cell function. Blunted effector functions, secondary to ERK1/2 signaling deficits concentrated within activated CD8⁺ T cells, may contribute to immunodeficiency and underlie the predictive capacity of CD8⁺ T cell activation on HIV-1 disease progression. (270/300).     

Hierarchy Low CD4+/CD8+ T-Cell Counts and IFN-γ Responses in HIV-1+ Individuals Correlate with Active TB and/or M.tb Co-Infection

Objective

Detailed studies of correlation between HIV-M.tb co-infection and hierarchy declines of CD8+/CD4+ T-cell counts and IFN-γ responses have not been done. We conducted case-control studies to address this issue.

Methods

164 HIV-1-infected individuals comprised of HIV-1⁺ATB, HIV-1⁺LTB and HIV-1⁺TB^- groups were evaluated. Immune phenotyping and complete blood count (CBC) were employed to measure CD4+ and CD8+ T-cell counts; T.SPOT.TB and intracellular cytokine staining (ICS) were utilized to detect ESAT6, CFP10 or PPD-specific IFN-γ responses.

Results

There were significant differences in median CD4+ T-cell counts between HIV-1⁺ATB (164/μL), HIV-1⁺LTB (447/μL) and HIV-1⁺TB^- (329/μL) groups. Hierarchy low CD4+ T-cell counts (<200/μL, 200-500/μL, >500/μL) were correlated significantly with active TB but not M.tb co-infection. Interestingly, hierarchy low CD8+ T-cell counts were not only associated significantly with active TB but also with M.tb co-infection (P<0.001). Immunologically, HIV-1⁺ATB group showed significantly lower numbers of ESAT-6-/CFP-10-specific IFN-γ+ T cells than HIV-1⁺LTB group. Consistently, PPD-specific IFN-γ+CD4+/CD8+ T effector cells in HIV-1⁺ATB group were significantly lower than those in HIV-1⁺LTB group (P<0.001).

Conclusions

Hierarchy low CD8+ T-cell counts and effector function in HIV-1-infected individuals are correlated with both M.tb co-infection and active TB. Hierarchy low CD4+ T-cell counts and Th1 effector function in HIV-1+ individuals are associated with increased frequencies of active TB, but not M.tb co-infection.     

Protection against Mycobacterium tuberculosis Infection Offered by a New Multistage Subunit Vaccine Correlates with Increased Number of IFN-γ+IL-2+ CD4+ and IFN-γ+ CD8+ T Cells

Protein subunit vaccines present a compelling new area of research for control of tuberculosis (TB). Based on the interaction between Mycobacterium tuberculosis and its host, five stage-specific antigens of M. tuberculosis that participate in TB pathogenesis—Rv1813, Rv2660c, Ag85B, Rv2623, and HspX—were selected. These antigens were verified to be recognized by T cells from a total of 42 whole blood samples obtained from active TB patients, patients with latent TB infections (LTBIs), and healthy control donors. The multistage polyprotein A1D4 was developed using the selected five antigens as a potentially more effective novel subunit vaccine. The immunogenicity and protective efficacy of A1D4 emulsified in the adjuvant MTO [monophosphoryl lipid A (MPL), trehalose-6,6′-dibehenate (TDB), components of MF59] was compared with Bacillus Calmette-Guerin (BCG) in C57BL/6 mice. Our results demonstrated that A1D4/MTO could provide more significant protection against M. tuberculosis infection than the PBS control or MTO adjuvant alone judging from the A1D4-specific Th1-type immune response; however, its efficacy was inferior to BCG as demonstrated by the bacterial load in the lung and spleen, and by the pathological changes in the lung. Antigen-specific single IL-2-secreting cells and different combinations with IL-2-secreting CD4⁺ T cells were beneficial and correlated with BCG vaccine-induced protection against TB. Antigen-specific IFN-γ⁺IL-2⁺ CD4⁺ T cells were the only effective biomarker significantly induced by A1D4/MTO. Among all groups, A1D4/MTO immunization also conferred the highest number of antigen-specific single IFN-γ⁺ and IFN-γ⁺TNF-α⁺ CD4⁺ T cells, which might be related to the antigen load in vivo, and single IFN-γ⁺ CD8⁺ T cells by mimicking the immune patterns of LTBIs or curable TB patients. Our strategy seems promising for the development of a TB vaccine based on multistage antigens, and subunit antigen A1D4 suspended in MTO adjuvant warrants preclinical evaluation in animal models of latent infection and may boost BCG vaccination.     

PD-1 and Tim-3 Pathways Regulate CD8+ T Cells Function in Atherosclerosis

T cell-mediated immunity plays a significant role in the development of atherosclerosis (AS). There is increasing evidence that CD8⁺ T cells are also involved in AS but their exact roles remain unclear. The inhibitory receptors programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) are well known inhibitory molecules that play a crucial role in regulating CD8⁺ T cell activation or tolerance. Here, we demonstrate that the co-expression of PD-1 and Tim-3 on CD8⁺ T cells is up-regulated in AS patients. PD-1⁺ Tim-3⁺ CD8⁺ T cells are enriched for within the central T (T_CM) cell subset, with high proliferative activity and CD127 expression. Co-expression of PD-1 and Tim-3 on CD8⁺ T cells is associated with increased anti-atherogenic cytokine production as well as decreased pro-atherogenic cytokine production. Blockade of PD-1 and Tim-3 results in a decrease of anti-atherogenic cytokine production by PD-1⁺ Tim-3⁺ CD8⁺ T cells and in an augmentation of TNF-α and IFN-γ production. These findings highlight the important role of the PD-1 and Tim-3 pathways in regulating CD8⁺ T cells function in human AS.     

Loss of the orphan nuclear receptor NR2F6 enhances CD8+ T-cell memory via IFN-γ

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127⁻KLRG1⁺) or memory precursors cells (MPECs, CD127⁺KLRG1⁻) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8⁺ memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6^−/− OT-I T-cells showed that the augmented memory formation is CD8⁺ T-cell intrinsic. Although the relative difference between the Nr2f6^+/+ and Nr2f6^−/− OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8⁺ T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8⁺ T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8⁺ T cells in vivo.Subject terms: Interferons, Bacterial infection     

Patients with Tuberculosis Disease Have Mycobacterium tuberculosis-Specific CD8 T Cells with a Pro-Apoptotic Phenotype and Impaired Proliferative Capacity,Which Is Not Restored following Treatment

CD8 T cells play a critical role in control of chronic viral infections; however, the role of these cells in containing persistent bacterial infections, such as those caused by Mycobacterium tuberculosis (Mtb), is less clear. We assessed the phenotype and functional capacity of CD8 T cells specific for the immunodominant Mtb antigens CFP-10 and ESAT-6, in patients with pulmonary tuberculosis (TB) disease, before and after treatment, and in healthy persons with latent Mtb infection (LTBI). In patients with TB disease, CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells had an activated, pro-apoptotic phenotype, with lower Bcl-2 and CD127 expression, and higher Ki67, CD57, and CD95 expression, than in LTBI. When CFP-10/ESAT-6-specific IFN-γ⁺ CD8 T cells were detectable, expression of distinct combinations of these markers was highly sensitive and specific for differentiating TB disease from LTBI. Successful treatment of disease resulted in changes of these markers, but not in restoration of CFP-10/ESAT-6-specific CD8 or CD4 memory T cell proliferative capacity. These data suggest that high mycobacterial load in active TB disease is associated with activated, short-lived CFP-10/ESAT-6-specific CD8 T cells with impaired functional capacity that is not restored following treatment. By contrast, LTBI is associated with preservation of long-lived CFP-10/ESAT-6-specific memory CD8 T cells that maintain high Bcl-2 expression and which may readily proliferate.     

Mycobacterium tuberculosis Specific CD8+ T Cells Rapidly Decline with Antituberculosis Treatment

Rationale

Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8⁺ T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment.Objectives: We sought to determine the relationship of Mtb specific CD4⁺ T cells and CD8⁺ T cells with duration of antituberculosis treatment.

Materials and Methods

We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n = 50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4⁺ and CD8⁺ T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24.

Results

There was a significant difference in the Mtb specific CD8⁺ T response, but not the CD4⁺ T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p = 0.023). At 24 weeks, the estimated Mtb specific CD8⁺ T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4⁺ T cell during the treatment. The Mtb specific CD4⁺ T cell response, but not the CD8⁺ response, was negatively impacted by the body mass index.

Conclusions

Our data provide evidence that the Mtb specific CD8⁺ T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8⁺ T cell response can detect early treatment failure, relapse, or to predict disease progression.     

Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8⁺ and CD4⁺ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8⁺ and CD4⁺ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4⁺ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4⁺ T cells was significantly correlated with that of CD8⁺ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4⁺ T cell responses showed more significant changes than CD8⁺ T cell responses. CD8⁺ and CD4⁺ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4⁺ T cells secreted significantly higher cytokine levels than did CD8⁺ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.     

Mycobacterium tuberculosis-Specific IL-21+IFN-γ+CD4+ T Cells Are Regulated by IL-12

In the current study of Mycobacterium tuberculosis (MTB)-specific T and B cells, we found that MTB-specific peptides from early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) induced the expression of IL-21 predominantly in CD4⁺ T cells. A fraction of IL-21-expressing CD4⁺ T cells simultaneously expressed Th1 cytokines but did not secrete Th2 or Th17 cytokines, suggesting that MTB-specific IL-21-expressing CD4⁺ T cells were different from Th1, Th2 and Th17 subpopulations. The majority of MTB-specific IL-21-expressing CD4⁺ T cells co-expressed IFN-γ and IL-21⁺IFN-γ⁺CD4⁺ T cells exhibited obviously polyfunctionality. In addition, MTB-specific IL-21-expressing CD4⁺ T cells displayed a CD45RO⁺CD62L^lowCCR7^lowCD40L^highICOS^high phenotype. Bcl-6-expression was significantly higher in IL-21-expressing CD4⁺ T cells than IL-21^-CD4⁺ T cells. Moreover, IL-12 could up-regulate MTB-specific IL-21 expression, especially the frequency of IL-21⁺IFN-γ⁺CD4⁺ T cells. Taken together, our results demonstrated that MTB-specific IL-21⁺IFN-γ⁺CD4⁺ T cells from local sites of tuberculosis (TB) infection could be enhanced by IL-12, which have the features of both Tfh and Th1 cells and may have an important role in local immune responses against TB infection.     

Helminth Infections Coincident with Active Pulmonary Tuberculosis Inhibit Mono- and Multifunctional CD4+ and CD8+ T Cell Responses in a Process Dependent on IL-10

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4⁺ and CD8⁺ T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections—Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4⁺ and CD8⁺ T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial–specific frequencies of mono- and multi–functional CD4⁺ Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4⁺ and CD8⁺ T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4⁺ T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4⁺ Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4⁺ T cell responses as well as protective systemic cytokine responses in active pulmonary TB.     

Tim-3 Expression Defines Regulatory T Cells in Human Tumors

Tim-3, a member of the novel Tim (T cell immunoglobulin and mucin domain) family, has been reported to negatively regulate the immune responses against viral infection and had implications for autoimmune disease. However, the nature and role of Tim-3⁺ CD4 T cells in human tumors remain largely unknown. In the present study, we characterized Tim-3⁺ CD4 T cells in 100 specimens from human hepatocellular, cervical, colorectal and ovarian carcinoma patients. Compared with peripheral blood and nontumor-infiltrating lymphocytes, the lymphocytes isolated from the corresponding tumor tissues of hepatocellular, cervical, colorectal and ovarian carcinoma patients contained significantly greater proportion of Tim-3⁺ CD4 T cells. The majority of tumor-derived Tim-3⁺ CD4 T cells exhibited an impaired capacity to produce IFN-γ and IL-2, but expressed higher levels of CD25, Foxp3, CTLA-4 and GITR than their Tim-3⁻ CD4 T cell counterparts. In contrast, most Tim-3⁺ CD4 T cells isolated from the paired nontumor tissues and peripheral blood did not express these molecules. Moreover, tumor-derived Tim-3⁺ CD4 T cells, but not tumor-derived Tim-3⁻ CD4 T cells, significantly suppressed the proliferation of autologous CD8⁺ T cells in vitro. Notably, multi-color immunofluorescence and confocal microscopy demonstrated that Tim-3⁺Foxp3⁺CD4⁺ cells were preferentially distributed in the tumor nest rather than the peritumoral stroma of hepatocellular carcinoma. Together, our data indicate that Tim-3-expressing CD4 T cells in human tumors could represent the functional regulatory T cells which contribute to the formation of the immune-suppressive tumor micromilieu.     

CD160Ig Fusion Protein Targets a Novel Costimulatory Pathway and Prolongs Allograft Survival

CD160 is a cell surface molecule expressed by most NK cells and approximately 50% of CD8⁺ cytotoxic T lymphocytes. Engagement of CD160 by MHC class-I directly triggers a costimulatory signal to TCR-induced proliferation, cytokine production and cytotoxic effector functions. The role of CD160 in alloimmunity is unknown. Using a newly generated CD160 fusion protein (CD160Ig) we examined the role of the novel costimulatory molecule CD160 in mediating CD4⁺ or CD8⁺ T cell driven allograft rejection. CD160Ig inhibits alloreactive CD8⁺ T cell proliferation and IFN-γ production in vitro, in particular in the absence of CD28 costimulation. Consequently CD160Ig prolongs fully mismatched cardiac allograft survival in CD4^−/−, CD28^−/− knockout and CTLA4Ig treated WT recipients, but not in WT or CD8^−/− knockout recipients. The prolonged cardiac allograft survival is associated with reduced alloreactive CD8⁺ T cell proliferation, effector/memory responses and alloreactive IFN-γ production. Thus, CD160 signaling is particularly important in CD28-independent effector/memory CD8⁺ alloreactive T cell activation in vivo and therefore may serve as a novel target for prevention of allograft rejection.     

Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8⁺ T cells. However, their independent functional contributions in distinct CD8⁺ T cell subsets are not well understood. In this study, CD8⁺ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA⁺CD27⁺) and memory (CD45RA⁻CD27⁺) CD8⁺ T cells (CD28^Hi), while its expression was significantly lower in effector (CD45RA⁺CD27⁻) CD8⁺ T cells (CD28^Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8⁺ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8⁺ T cells. Co-stimulation of CD28^Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28^Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28^Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8⁺ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8⁺ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8⁺ T cells. However, boosting a recall immune response via memory CD8⁺ T cells or vaccination to stimulate naïve CD8⁺ T cells would require CD28-mediated co-stimulation.     

Differential Expression of CD96 Surface Molecule Represents CD8+ T Cells with Dissimilar Effector Function during HIV-1 Infection

During HIV-1 infection, immune dysregulation and aberrant lymphocyte functions are well-established characteristics. Cell surface molecules are important for immunological functions and changes in expression can affect lymphocyte effector functions, thereby contributing to pathogenesis and disease progression. In this study we have focused on CD96, a member of the IgG superfamily receptors that have generated increasing recent interest due to their adhesive and co-stimulatory functions in addition to immunoregulatory capacity. CD96 is expressed by both T and NK cells. Although the function of CD96 is not completely elucidated, it has been shown to have adhesive functions and enhance cytotoxicity. Interestingly, CD96 may also have inhibitory functions due to its immunoreceptor tyrosine-based inhibitory motif (ITIM). The clinical significance of CD96 is still comparatively limited although it has been associated with chronic Hepatitis B infection and disease progression. CD96 has not previously been studied in the context of HIV-1 infection, but due to its potential importance in immune regulation and relevance to chronic disease, we examined CD96 expression in relation to HIV-1 pathogenesis. In a cross-sectional analysis, we investigated the CD8⁺ T cell expression of CD96 in cohorts of untreated HIV-1 infected adults with high viral loads (non-controllers) and low viral loads (“elite” controllers). We demonstrated that elite controllers have significantly higher CD96 mean fluorescence intensity on CD8⁺ T cells compared to HIV-1 non-controllers and CD96 expression was positively associated with CD4⁺ T cell counts. Functional assessment showed that CD8⁺ T cells lacking CD96 expression represented a population that produced both perforin and IFN-γ following stimulation. Furthermore, CD96 expression on CD8⁺ T cells was decreased in presence of lipopolysaccharide in vitro. Overall, these findings indicate that down-regulation of CD96 is an important aspect of HIV-1 pathogenesis and differential expression is related to cell effector functions and HIV-1 disease course.     

Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

The exact role of CD8⁺ T cells during Mycobacterium tuberculosis (Mtb) infection has been heavily debated, yet it is generally accepted that CD8⁺ T cells contribute to protection against Mtb. In this study, however, we show that the Mtb-susceptible CBA/J mouse strain accumulates large numbers of CD8⁺ T cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (PD-1⁺, Tim-3⁺, CD122⁺). CD8⁺ T cell expansions from the lungs of Mtb-infected CBA/J mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (IL-10), although in vivo CD8⁺ T cell depletion did not significantly alter Mtb burden. Further analysis revealed that pulmonary CD8⁺ T cells from Mtb-infected CBA/J mice were clonally expanded, preferentially expressing T cell receptor (TcR) Vβ chain 8 (8.2, 8.3) or Vβ 14. Although Vβ8⁺ CD8⁺ T cells were responsible for the majority of IL-10 production, in vivo depletion of Vβ8⁺ did not significantly change the outcome of Mtb infection, which we hypothesize was a consequence of their dual IL-10/IFN-γ secreting profiles. Our data demonstrate that IL-10-secreting CD8⁺ T cells can arise during chronic Mtb infection, although the significance of this T cell population in tuberculosis pathogenesis remains unclear.