Blockade of Tim-3 signaling restores the virus-specific CD8⁺ T-cell response in patients with chronic hepatitis B

Chronic hepatitis B (CHB) is characterized by functionally impaired virus-specific CD8(+) T-cell responses. However, the mechanism underlying this dysfunction has not been fully clarified. We examined the role of a newly identified protein, T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3), in regulating the antiviral CD8(+) T-cell response in CHB patients. Tim-3 expression on peripheral virus-specific CD8(+) T cells from 20 CHB patients and 20 healthy controls was determined by flow cytometry. The phenotypes and cytokine-producing capacity were compared between Tim-3(+) CD8(+) and Tim-3(-) CD8(+) T cells. The impact of Tim-3 signaling on cellular proliferation and cytokine-producing capacity was also studied. Tim-3 expression on hepatitis B virus (HBV)-specific CD8(+) T cells was higher than expression on cytomegalovirus (CMV)-specific CD8(+) T cells. Tim-3(+) CD8(+) T cells exhibited proliferative senescence phenotypes and decreased cytokine production upon antigen challenge. Finally, blocking the Tim-3 pathway significantly improved proliferation and antiviral cytokine secretion of CD8(+) T cells in response to HBV-specific antigen peptides. Tim-3 negatively regulates antiviral responses of CD8(+) T cells isolated from CHB patients, and this response is reversed by blocking the Tim-3 pathway.     

Vaccinia virus infection of mature dendritic cells results in activation of virus-specific naïve CD8+ T cells: a potential mechanism for direct presentation

Understanding how vaccinia virus (VV) generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of na?ve CD8(+) T cells. In order to optimally activate na?ve CD8(+) T cells, DC must undergo maturation, during which costimulatory molecules are upregulated and cytokines are produced. In this report, we show that VV infection of immature murine bone marrow-derived DC (BMDC) failed to induce maturation. Similar results were obtained when CD8(+) DC were analyzed, a subset shown previously to be important in vivo in the generation of a vaccinia-specific response. The finding that VV infection of DC resulted in APC that were incapable of initiating T-cell activation was surprising given the previously reported role for direct presentation in the generation of anti-VV CD8(+) T-cell responses in mice. To address the potential mechanism responsible for direct presentation, we tested the hypothesis that previously matured DC were susceptible to vaccinia virus infection and could present newly synthesized VV-derived epitopes for CD8(+) T-cell activation. Our results show, that during VV infection of mature DC, threshold levels of viral protein are produced that promote T-cell activation. These results suggest that, even though VV cannot mature DC, previously matured DC exposed to VV can generate a VV-specific CD8(+) T-cell response providing a potential mechanism by which direct infection results in T-cell activation in vivo.     

Detailed analysis of Epstein-Barr virus-specific CD4+ and CD8+ T cell responses during infectious mononucleosis

We studied simultaneously Epstein-Barr virus (EBV)-specific CD4(+) and CD8(+) T cell responses during and after infectious mononucleosis (IM), using a previously described 12-day stimulation protocol with EBNA1 or BZLF1 peptide pools. Effector function of EBV-specific T cells was determined after restimulation by measuring intracellular interferon-gamma production. During IM, BZLF1-specifc CD4(+) T cell responses were dominant compared with CD8(+) T cell responses. EBNA1-specific CD4(+) and CD8(+) T cell responses were low and remained similar for 6 months. However, 6 months after IM, BZLF1-specific CD4(+) T cell responses had declined, but CD8(+) T cell responses had increased. At diagnosis, EBV-specific CD8(+) T cells as studied by human leucocyte antigen class I tetramer staining comprised a tetramer(bright)CD8(bright) population consisting mainly of CD27(+) memory T cells and a tetramer(dim)CD8(dim) population consisting primarily of CD27(-) effector T cells. The remaining EBV-specific CD8(+) T cell population 6 months after the diagnosis of IM consisted mainly of tetramer(bright)CD8(bright) CD27(+) T cells, suggesting preferential preservation of memory T cells after contraction of the EBV-specific T cell pool.     

Dendritic cells generated in the presence of interferon-alpha stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells

Dendritic cells (DCs) generated in the presence of IFN-alpha (IFN-DCs) exhibit high expression of major histocompatibility and co-stimulatory molecules and a potent ability to stimulate CD8(+) T-cell responses. Here, we found that IFN-DCs were more potent stimulators of bulk and purified CD8(+) T-cell proliferation, as compared with IL-4-DCs. In contrast, IFN-DCs were less efficient than IL-4-DCs in stimulating allogeneic CD4(+) T-cell proliferation, due to a weak induction of naive CD4(+)CD45RO(-) T-cell proliferation by these DCs. However, both DC populations induced similar levels of proliferation of memory CD4(+)CD45RO(+) T cells. IFN-DCs and IL-4-DCs exhibited a similar phenotype and production of IL-10 following maturation induced by CD40 ligation. In contrast, IFN-DCs produced higher levels of IL-10 during the first days of differentiation. In addition, neutralization of IL-10 during the differentiation of DCs increased the expression of DC-LAMP and MHC class II by IFN-DCs, and the ability of IFN-DCs to stimulate allogeneic CD4(+) T-cell proliferation at similar levels, than IL-4-DCs. Independently of IL-10 production, IFN-DCs were found to induce higher levels of CD4(+)T-cell apoptosis, this effect being more sticking on naive T cells. Finally, we demonstrated that IFN-DCs induced a differentiation bias of naive CD4(+) T cells towards Th1 and Tr1 cells, compared to IL-4-DCs. Taken together, these results indicate that, despite the induction of Tr1 cells and enhanced apoptosis of naive CD4(+) T cells, IFN-DCs are potent stimulators of CD8(+) and memory CD4(+) T cells, and induce a strong polarization of naive CD4(+) T cells towards Th1 cells, further supporting their use in immune-based therapy.     

Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening

CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-gamma) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-gamma by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-gamma and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.     

Properties of CD4(+) T cells in human cytomegalovirus infection

The correlates of protective immunity to disease-inducing viruses in man remain to be elucidated. We determined the kinetics and properties of cytomegalovirus (CMV)-specific CD4(+) T cells in healthy individuals and renal transplant recipients during different stages of CMV infection. Our data reveal that circulating CMV-specific CD4(+) T cells displayed an effector-memory phenotype, and produced the T helper 1 cytokines interferon-gamma and tumor necrosis factor-alpha. In addition, they lacked molecules for secondary lymphoid organ homing and expressed the cytotoxic molecule granzyme B, inferring a direct role of these cells at target sites of infection. In asymptomatic individuals the CMV-specific CD4(+) T-cell response preceded CMV-specific CD8(+) T-cell responses, whereas in symptomatic individuals the CMV-specific effector memory CD4(+) T-cell response was delayed and only detectable after antiviral therapy. The appearance of disease symptoms in these patients suggests that functional CD8(+) T cell and antibody responses are insufficient to control viral replication and that formation of effector memory CD4(+) T cells is necessary for recovery of infection.     

Heterologous priming-boosting immunization of cattle with Mycobacterium tuberculosis 85A induces antigen-specific T-cell responses

Heterologous priming-boosting vaccination regimens involving priming with plasmid DNA antigen constructs and inoculating (boosting) with the same recombinant antigen expressed in replication-attenuated poxviruses have recently been demonstrated to induce immunity, based on CD4(+)- and CD8(+)-T-cell responses, against several diseases in both rodents and primates. We show that similar priming-boosting vaccination strategies using the 85A antigen of Mycobacterium tuberculosis are effective in inducing antigen-specific gamma interferon-secreting CD4(+) and CD8(+) T cells, detected by a bovine enzyme-linked immunospot assay, in Bos indicus cattle. T-cell responses induced by priming with either plasmid DNA or fowlpox virus 85A constructs were enhanced by boosting with modified vaccinia virus Ankara expressing the same antigen administered intradermally. On the basis of the data, it appears that intradermal priming was more effective than intramuscular delivery of the priming dose for boosting with the modified vaccinia virus Ankara strain in cattle. Using either fowlpox virus or DNA priming, there was a significant bias toward induction of CD4(+)- rather than CD8(+)-T-cell responses. These data illustrate the general applicability of priming-boosting vaccination strategies for induction of antigen-specific T-cell responses and suggest that the method may be useful for development of veterinary vaccines.     

Cytomegalovirus-specific CD8+ T cells targeting different peptide/HLA combinations demonstrate varying T-cell receptor diversity

Cytomegalovirus (CMV) infection and reactivation pose a serious threat for patients after haematopoietic stem cell transplantation. We have previously shown that CD8(+) T cells targeting different CMV epitopes correlate with protection at different threshold frequencies in those patients. To investigate if this may relate to a different quality of these cells here we analyse the T-cell receptor diversity of pp50 (245-253)/HLA-A*0101 specific CD8(+) T cells with that of CD8(+) T cells targeting various pp65 peptides. The results from this pilot study show differences in the breadth of the T-cell receptor usage of the different cell populations. We observe for the first time that the T-cell receptor Vβ CDR3 spectratypes used by CMV pp50 (245-253)/HLA-A*0101-specific CD8(+) T cells can reach higher numbers than those used by CD8(+) T cells targeting various pp65 peptides in our patient cohort. This merits further investigation into the effectiveness of the different CMV-specific T cells and their impact on immunosenescence, which is important to eventually define the most useful source of adoptive therapy and monitoring protocols for cytomegalovirus-specific immune responses.     

Rescue of memory CD8+ T cell reactivity in peptide/TLR9 ligand immunization by codelivery of cytokines or CD40 ligation

The capability of cellular immune components to rapidly recall upon challenge in most situations decides the efficacy of a vaccine. Here, we show that immunization of mice with SSIEFARL peptide (immunodominant epitope in glycoprotein B of herpes simplex virus type 1, aa498-505) combined with TLR9 ligand in the absence of helper CD4(+) T cell activation generates a functionally impaired CD8(+) T cell memory response. Codelivery of IL-12, IL-15, or anti-CD40 together with MHC class-I-restricted peptide combined with TLR9 ligand at inception of immunization resulted in generation of memory CD8(+) T cells that were several fold less compromised than immunization with peptide alone. Furthermore, administration of either plasmid DNA encoding IL-15 or anti-CD40 mAb but not rIL-12 during the memory phase restored the reactivity of memory CD8(+) T cells. Moreover, the rescued CD8(+) T cells preserved their cytotoxic capability and were able to clear a recombinant vaccinia virus encoding glycoprotein B of HSV. Our results indicate that good memory CD8(+) T cell response to peptide immunization can be achieved by using costimulatory procedures at the time of priming or recall immunization.     

CD27 and CD57 expression reveals atypical differentiation of human immunodeficiency virus type 1-specific memory CD8+ T cells

The failure of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells to control chronic HIV-1 infection could be due to the progressive loss of their capacities to undergo normal memory effector differentiation. We characterized and compared the expressions of CD27, CD28, CD57, and CD62L by Epstein-Barr virus (EBV)-, cytomegalovirus (CMV)-, and HIV-1-specific CD8+ T cells by six-color, eight-parameter flow cytometry. In contrast to the maturation of EBV- and CMV-specific memory CD8+ T cells, we found that HIV-1-specific CD8+ T cells did not display coordinated down-regulation of CD27 and up-regulation of CD57 and accumulated in an atypical CD27(high) CD57(low) subset. Moreover, the accumulation of CD27(high) CD57(low) HIV-1-specific CD8+ T cells was positively correlated with HIV-1 plasma viremia. The differentiation of HIV-1-specific CD8+ T cells to an effector subset is therefore impaired during chronic HIV-1 infection. This lack of normal CD8+ T-cell differentiation could contribute to the failure of cellular immune control of HIV-1 infection.     

Frequency of proteinase 3 (PR3)-specific autoreactive T cells determined by cytokine flow cytometry in Wegener's granulomatosis

OBJECTIVE: Previous studies have shown proliferation to Wegener's autoantigen, proteinase 3 (PR3). We tested immunogenicity of PR3-derived peptides and determined frequencies of PR3-specific T cells using cytokine flow cytometry in Wegener's granulomatois (WG). METHODS: Peripheral blood T-cell responses were measured after stimulation with previously described PR3-derived peptides. PBMC were stimulated with PR3-derived peptides or control stimuli for up to 10 days. Cells were stained with antibodies against CD4 or CD8, CD69 and intracellular TNF-alpha and analyzed by flow cytometry. PR3-specific T cells were counted as the percentage of CD69(+)TNF-alpha(+)double positive T cells after stimulation. RESULTS: Frequencies of PR3-specific peripheral blood T cells after short-term stimulation (相似文献   

The use of class-I HLA tetramers for the detection of hepatitis C virus NS3-specific CD8(+) T cells in patients with chronic infection

BACKGROUND/AIMS: New methods to detect virus-specific T-cell responses have recently been developed. Several human leukocyte antigen (HLA)-peptide tetramers for the detection of hepatitis C virus (HCV)-specific CD8(+) T cells are under evaluation. METHODS: Evaluation of one HLA class I-tetramer (HCVNS3-2) for the detection of HCV NS3-specific CD8(+) T cells in a series of 38 HLA-A2(+) chronically infected patients. RESULTS: Almost half (42%) of the patients had detectable NS3-specific CD8(+) T cells. The frequencies of such cells ranged from 0.01% to 0.22% of total CD8(+) T cells. No significant differences in clinical features or mean viral load were detected between patients with or without tetramer + CD8(+) T cells. CONCLUSIONS: The tetramer HCVNS3-2 may be very useful for the study of the HCV-specific CD8(+) immune response. Combination of this reagent with other tetramers based on other HCV peptides may help in the understanding of the immune response to the virus. However, a panel of tetramers based on several parts of the HCV polyprotein may be a mandatory requirement to explore the whole breadth of the CD8(+) T-cell response against HCV and to detect that response in the majority of patients with chronic infection.     

Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8(+) T cells with specificity for a novel mycobacterial epitope

Although CD4(+) T cells are essential for protective immunity against Mycobacterium tuberculosis infection, recent reports indicate that CD8(+) T cells may also play a critical role in the control of this infection. However, the epitope specificity and the mechanisms of activation of mycobacteria-reactive CD8(+) T cells are poorly characterized. In order to study the CD8(+) T cell responses to the model mycobacterial antigen, MPT64, we used recombinant vaccinia virus expressing MPT64 (VVWR-64) and a panel of MPT64-derived peptides to establish that the peptide MPT64(190-198) contains an H-2D(b)-restricted CD8(+) T cell epitope. A cytotoxic T lymphocyte response to this peptide could be demonstrated in M. bovis bacillus Calmette Guerin (BCG)-infected mice following repeated in vitro stimulation. When bone marrow-derived dendritic cells (DC) were infected with BCG, the expression of MHC class I molecules by DC was up-regulated in parallel with MHC class II and B7-2, whereas CD1d expression level was not modified. Moreover, BCG-infected DC activated MPT64(190-198)-specific CD8(+) T cells to secrete IFN-gamma, although with a lower efficacy than VVWR-64-infected DC. The production of IFN-gamma by MPT64(190-198)-specific CD8(+) T cells was inhibited by antibodies to MHC class I, but not to CD1d. These data suggest that mycobacteria-specific CD8(+) T cells are primed during infection. Therefore, anti-mycobacterial vaccine strategies targeting the activation of specific CD8(+) T cells by DC may have improved protective efficacy.     

Vaccinia assay for the rapid detection of functional HIV-specific CD8+ cytotoxic T lymphocytes

Human immunodeficiency virus (HIV)-specific T cells play a critical role in anti-virus immunity. Therefore, during the clinical development of immune-based therapies, it is important to perform a diagnostic test that rapidly quantifies and characterizes cellular immune responses. For detection of functional HIV-specific CD8(+) cytotoxic T cells (CTL), we used a rapid vaccinia assay that employs recombinant vaccinia virus-infected peripheral blood mononuclear cells (PBMC). This assay was compared with the traditional 51Cr-release CTL assay and with the peptide pool-based enzyme-linked immunospot assay (ELISPOT). We demonstrated a close correlation between these assays by using identical antigens in parallel assays. However, the vaccinia assay was the least expensive and time- and labor-consuming test. Regarding sensitivity, the vaccinia assay was similar to both the peptide pool-based ELISPOT and CTL assays. We showed that human histocompatibility leukocyte antigen (HLA)-Dr(+) cells are responsible for presenting recombinant vaccinia antigens to T cells. The recombinant vaccinia-based assay is a rapid, sensitive and quantitative test and is thus suitable for the detection of functional antigen-specific CD8(+) CTL in large-scale clinical studies.     

Immunodominance of HIV-1-specific CD8(+) T-cell responses in acute HIV-1 infection: at the crossroads of viral and host genetics

The development of HIV-1-specific CD8(+) T-cell responses during acute HIV-1 infection is associated with a dramatic decline in HIV-1 replication and the resolution of the acute retroviral syndrome. These HIV-1-specific CD8(+) T cells typically target a small number of viral epitopes in a distinct hierarchical order, and high-level viremia in chronic progressive infection leads to broadly diversified HIV-1-specific CD8(+) T-cell responses with a less clear immunodominance pattern. It is argued here that the specific hierarchical pattern of immune responses in acute HIV-1 infection is the result of a tightly regulated process that, among other factors, is critically impacted by the kinetics of viral protein expression, the HLA class I background of the infected individual and the autologous sequence of the infecting virus.     

Type-I IFN drives the differentiation of short-lived effector CD8+ T cells in vivo

Two subsets of CD8(+) T cells are generated early during an immune response; one of these subsets forms the memory pool, known as memory precursor effector cells (MPECs), identified by high expression of CD127 and low expression of KLRG1, whereas the other subset forms short-lived effector cells (SLECs) identified by low expression of CD127 and high expression of KLRG1. Here, we studied in vivo the role of type-I IFN in this fate decision. We found that under priming conditions dominated by type-I IFN, as observed in lymphocytic choriomeningitis virus (LCMV) infection, type-I IFN signaling directly impacted the regulation of T-bet and thus the early fate decision of CD8(+) T cells. In the absence of type-I IFN signaling, CD8(+) T cells failed to form SLECs but could form MPECs that give rise to functional memory CD8(+) T cells. Together, these findings identify type-I IFN as an important factor driving SLEC differentiation and thus instructing the early division between the effector and memory precursor CD8(+) T-cell pool.     

Loss of antiviral cytotoxic T-lymphocyte activity during high-level antigen stimulation

High levels of antigenic stimulation can result in deactivation of CD8+ T cells through a variety of mechanisms, including insufficient T-cell help. In the present study, an adoptive transfer system was established in which ovalbumin (OVA)-specific CD8+ T cells were transferred to irradiated mice infected with a recombinant vaccinia virus encoding OVA (VV-OVA). Prolonged activation of OVA-specific CD8+ T cells resulted in a proliferative block in these cells, although cytotoxic function was maintained. Unlike naive and recently activated OVA-specific T cells, these nonproliferative cytotoxic CD8+ T cells did not have antiviral activity following further transfer to mice infected with VV-OVA. Provision of interleukin-2 (IL-2) at the site of virus infection using a recombinant virus encoding antigen and IL-2, as well as the addition of helper T cells, had no effect on the generation of these dysfunctional T cells. Thus, there was no evidence that lack of T-cell help was responsible for CD8+ T-cell deactivation in this model.     

HCV-specific CD27- CD28- memory T cells are depleted in hepatitis C virus and Schistosoma mansoni co-infection

Factors that influence the generation and maintenance of memory CD8+ T cells are not fully understood. The homeostasis of memory T cells is highly dynamic and tightly regulated by various stimuli, including cytokines and antigen-major histocompatibility complex ligands. We characterized the hepatitis C virus (HCV)-specific CD8+ T-cell responses in a cohort of HCV-infected individuals with or without Schistosoma mansoni co-infection from Egypt. We observed a significantly decreased CD27- CD28- (late differentiated) memory T-cell population in the HCV co-infected individuals compared to those with HCV infection alone. In contrast, there was no significant difference in the CD27+ CD28+ (early differentiated) memory T cells between the two groups. Analysis of human cytomegalovirus-specific CD8+ T-cell responses in the same individuals failed to reveal a similar pattern of altered memory T-cell differentiation. Thus, S. mansoni co-infection targets a specific subset of memory CD8+ T cells in HCV infection.     

Human plasmacytoid dendritic cells induce CD8⁺ LAG-3⁺ Foxp3⁺ CTLA-4⁺ regulatory T cells that suppress allo-reactive memory T cells

Allo-reactive memory T cells are a major barrier for induction of immunological tolerance to allografts in humans. Here, we report that stimulation of unfractionated human T cells with TLR-stimulated allogeneic plasmacytoid dendritic cells (pDCs) induces CD8(+) regulatory T cells (Tregs) that inhibit T-cell allo-responses, including those of memory T cells. CD3(+) T cells were primed for 7 days with allogeneic pDCs that had been pre-stimulated with TLR-7 or TLR-9 ligands. While the T cells proliferated and produced cytokines during the priming culture, they were profoundly hypo-responsive to re-stimulation with the same allo-antigen in a second culture. Moreover, T cells primed by pDCs exerted donor-specific suppression on allo-responses of both unfractionated and memory CD3(+) T cells. The regulatory capacity of pDC-primed T cells was confined to CD8(+) LAG-3(+) Foxp3(+) CTLA-4(+) T cells, which suppressed allogeneic T-cell responses through a CTLA-4-dependent mechanism. Induction of CD8(+) Tregs by pDCs could be partially prevented by 1-methyl tryptophan, an inhibitor of indoleamine 2,3-dioxygenase. In conclusion, stimulation of human T cells by TLR-stimulated allogeneic pDCs induces CD8(+) Tregs that inhibit allogeneic T-cell responses, including memory T cells. Donor-derived pDCs may be considered as an immunotherapeutic tool to prevent activation of the recipient allo-reactive (memory) T-cell repertoire after allogeneic transplantation.