CD8+ T-cells suppress antigen-specific and allogeneic CD4+ T-cell responses to herpes simplex virus type 2-infected human dendritic cells

In this study we show that human dendritic cells (DC), productively infected with herpes simplex virus type 2 (HSV-2), activate CD8+ T cells that suppress antigen-specific and alloreactive CD4+ T cell expansion. Addition of CD8+ T cells to cultures of DC and CD4+ T cells blocked CD4+ T-cell proliferation in response to HSV-2-infected but not to uninfected DC. The effect was independent of prior HSV exposure or cognate MHC class I-restricted CD8-DC recognition as it was induced in CD8+ T cells from HSV-2-seronegative individuals and in mixed lymphocyte reactions using allogeneic DC. Both CD8+ CD25+ and CD8+ CD25- cells were shown to have suppressive capacities. The blood-derived CD25+ CD8+ T cells did not express Foxp3 mRNA but had a bona fide antiproliferative capacity in response to both uninfected and HSV-2-infected DC, whereas the CD25-CD8+ T cells were selectively activated to become antiproliferative by HSV-2-infected DC. These data imply that HSV infection of DC could modulate the immune response by activating CD8+ T cells.     

Quantitation of antigen-specific CD8+ T-cell responses

Enumeration of antigen-specific CD8+ T-cells has traditionally been largely dependent on the use of techniques that require a period of expansion in vitro. However the recent emergence of methods that can be used directly ex vivo to quantify antigen-specific CD8+ T cells has prompted a re-evaluation of the magnitude of viral-specific cellular immune responses. The purpose of this review is to describe the commonly used methods currently available to quantify and characterise antigen-specific T-cells and then to focus in more detail on the use of tetrameric complexes of HLA/peptide to study the cellular immune response.     

Memory CD4 T cells enhance primary CD8 T-cell responses

CD4 T-cell help is required for optimal memory CD8 T-cell responses. We have found that engaging preexisting CD4 Th1, but not Th2, memory cells at the time of CD8 T-cell priming results in increased CD8 effector responses to both bacterial and viral pathogens. The enhanced responses are characterized by increased numbers of cytokine-producing, antigen-specific cells. These findings suggest that engaging endogenous memory Th1 cells may increase cellular responses in an immunotherapy or vaccination setting.     

Activation of human CD8+ suppressor T cells by an antigen-specific CD4+ T-cell line in vitro

We generated an antigen (streptococcal cell wall antigen)-specific T-cell line from peripheral blood mononuclear cells of a healthy donor with an intermediate response to streptococcal cell wall antigen. Proliferation of the T-cell line was completely blocked by a monoclonal antibody to HLA-DR. This line activated autologous CD8+ T cells in an antigen-specific manner in the presence of autologous monocytes. This activation was mediated by a factor derived from this line and was blocked by a monoclonal antibody against HLA class I molecules. The resultant CD8+ T blasts showed antigen-nonspecific suppression but no cytolytic activity. This antigen-specific generation of the CD8+ T-cell line in vitro by the antigen-specific CD4+ T-cell line is expected to contribute to analyses of functions of CD8+ T-cell subsets, particularly in the down-regulating system, at both cellular and molecular levels.     

Generation and characterization of antigen-specific CD4+, CD8+, and CD4+CD8+ T-cell clones from patients with carbamazepine hypersensitivity

BACKGROUND: Hypersensitivity is a serious manifestation of anticonvulsant therapy characterized by infiltration of the epidermis and dermis by activated CD8(+) and CD4(+) T-cells, respectively. Attempts to characterize drug-specific CD8(+) T cells have been largely unsuccessful. OBJECTIVES: The aim of these studies was to generate and characterize CD4(+), CD8(+), and CD4(+)CD8(+) T cells in patients with carbamazepine hypersensitivity. METHODS: Carbamazepine-specific T-cell clones were generated from 5 patients by using modified cloning methodologies. Cell surface receptor phenotype, functionality, and mechanisms of antigen presentation were then compared. RESULTS: Ninety CD4(+), 23 CD8(+), and 14 CD4(+)CD8(+) carbamazepine-specific T-cell clones were generated. CD4(+) T-cell clones proliferated vigorously with carbamazepine associated with MHC class II but exhibited little cytotoxic activity. In contrast, most CD8(+) T cells proliferated weakly but effectively killed target cells via an MHC class I or MHC class II restricted, perforin-dependent pathway. CD4(+)CD8(+) T cells displayed characteristics similar to those of CD4(+) T cells; however, drug stimulation was demonstrable in the absence of antigen-presenting cells. Carbamazepine was presented to CD4(+), CD8(+), and CD4(+)CD8(+) T cells in the absence of antigen processing. Drug stimulation resulted in the secretion of IFN-gamma and IL-5. A panel of CD11a(+)CD27(-) clones differentially expressed the receptors CXCR4, CCR4, CCR5, CCR8, CCR9, and CCR10. CONCLUSION: Carbamazepine-specific CD4(+), CD8(+), and CD4(+)CD8(+) T cells exist in the peripheral circulation of hypersensitive patients, often many years after the resolution of clinical manifestations. CLINICAL IMPLICATIONS: Carbamazepine-specific CD4(+), CD8(+), and CD4(+)CD8(+) T cells displaying different effector functions and homing characteristics persist in hypersensitive patients' blood for many years after resolution of clinical symptoms.     

树突状细胞扩增抗原特异性CD4+ CD25+调节性T细胞研究进展

CD4+CD25+调节性T细胞(Tr)是同时具有免疫低反应性和免疫抑制性功能两大特征的T细胞.研究证实,CD4+ CD25+ Tr在抑制器官特异性自身免疫性疾病及GVHD是抗原特异性的,因此,应用器官特异性而不是多克隆性的Tr将大大促进以Tr为基础的免疫治疗.而具有调节活性的CD4+ CD25+ Tr仅占人类外周血CIM+ T细胞的1%～2%,因此,研究体外大量扩增的方法 对于以Tr基础的治疗至关重要.研究表明,树突状细胞(DC)作为机体强有力的专职抗原递呈细胞可以扩增具有抗原特异性的CD4+ CD25+ Tr且能增加后者的抑制活性,这为治疗自身免疫性疾病及GVHD提供了新的治疗前景.     

Type I interferon supports primary CD8+ T-cell responses to peptide-pulsed dendritic cells in the absence of CD4+ T-cell help

CD8(+) T-cell responses to non-pathogen, cell-associated antigens such as minor alloantigens or peptide-pulsed dendritic cells (DC) are usually strongly dependent on help from CD4(+) T cells. However, some studies have described help-independent primary CD8(+) T-cell responses to cell-associated antigens, using immunization strategies likely to trigger natural killer (NK) cell activation and inflammatory cytokine production. We asked whether NK cell activation by MHC I-deficient cells, or administration of inflammatory cytokines, could support CD4(+) T-cell help-independent primary responses to peptide-pulsed DC. Injection of MHC I-deficient cells cross-primed CD8(+) T-cell responses to the protein antigen ovalbumin (OVA) and the male antigen HY, but did not stimulate CD8(+) T-cell responses in CD4-depleted mice; hence NK cell stimulation by MHC I-deficient cells did not replace CD4(+) T-cell help in our experiments. Dendritic cells cultured with tumour necrosis factor-α (TNF-α) or type I interferon-α (IFN-α) also failed to prime CD8(+) T-cell responses in the absence of help. Injection of TNF-α increased lymph node cellularity, but did not generate help-independent CD8(+) T-cell responses. In contrast, CD4-depleted mice injected with IFN-α made substantial primary CD8(+) T-cell responses to peptide-pulsed DC. Mice deficient for the type I IFN receptor (IFNR1) made CD8(+) T-cell responses to IFNR1-deficient, peptide-pulsed DC; hence IFN-α does not appear to be a downstream mediator of CD4(+) T-cell help. We suggest that primary CD8(+) T-cell responses will become help-independent whenever endogenous IFN-α secretion is stimulated by tissue damage, infection, or autoimmune disease.     

CTLA4-Ig modifies dendritic cells from mice with collagen-induced arthritis to increase the CD4+CD25+Foxp3+ regulatory T cell population

Cytotoxic T lymphocyte antigen-4 (CTLA4) and IgG fusion protein, CTLA4-Ig, is a therapeutic agent used for rheumatoid arthritis. It binds B7 molecules on dendritic cells (DCs) and thereby blocks B7/CD28 costimulatory interaction and inhibits effective T cell proliferation. However, the effect of CTLA4-Ig on the regulatory T cell (Treg) is still not known. In this study, we investigated the influence of CTLA4-Ig on the CD4+CD25+Foxp3+ Treg population in collagen-induced arthritis (CIA) mouse model. CTLA4-Ig suppressed CIA and increased the CD4+CD25+Foxp3+ Treg population in joint and spleen. When CD11c + DCs and CD4+T cells from CIA mice were cultured with anti-CD3, CTLA4-Ig increased the CD4+CD25 + Foxp3+ Treg population in a TGF-β-dependent manner. When CD11c + DCs from CIA mice were treated with CTLA4-Ig and adoptively transferred into CIA-induced mice, arthritis did not develop in association with the increase in CD4+CD25+Foxp3+ Treg population. However, in CTLA4-Ig-untreated DC-transferred CIA mice, arthritis developed and then rapidly progressed. Our study demonstrated that CTLA4-Ig suppressed CIA by modifying DCs from CIA mice into tolerogenic DCs to increase the CD4+CD25+Foxp3+ Treg population and this seems to be the new immune regulatory mechanism of CTLA4-Ig.     

Influence of CD4 or CD8 deficiency on collagen-induced arthritis

The role of T cells in the mouse collagen-induced arthritis (CIA) model for rheumatoid arthritis is not clarified, and different results have been reported concerning the role of CD4 and CD8 T cells. To address this issue, we have investigated B10.Q mice deficient for CD4 or CD8. The mice lacking CD4 were found to be less susceptible to disease, but not completely resistant, whereas the CD8 deficiency had no significant impact on the disease. No difference in the development of late occurring relapses was noted. Interestingly, the CD4-deficient mice had a severely reduced response to the glycosylated form of the immunodominant type II collagen (CII) 256-270 peptide whereas the response to the non-glycosylated peptide was not significantly different. Furthermore, CD4-deficient mice had lower antibody responses to CII, explaining the lower disease susceptibility. In comparison with previously reported results, it is apparent that the lack of CD4 molecules has a different impact on CIA if present on different genetic backgrounds, findings that could possibly be related to the occurrence of different disease pathways of CIA in different mouse strains.     

A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo

Immune-stimulating complexes (ISCOMS) are adjuvant vectors which are unusual in being able to prime both CD4(+) and CD8(+) T cells by parenteral and mucosal routes. However, their mode of action is unclear and to define better the cellular interactions involved we have studied the ability of ISCOMS containing ovalbumin (OVA) to prime TCR transgenic CD4(+) or CD8(+) T cells in vivo. Immunization with OVA ISCOMS caused activation and clonal expansion of CD4(+) and CD8(+) T cells in the T cell areas of the draining lymph nodes, followed by the migration of both CD4(+) and CD8(+) T cells into the B cell follicle. The T cells were primed to proliferate and secrete IFN-gamma after re-stimulation in vitro with the appropriate OVA peptide and CD8(+) T cell priming occurred in the absence of CD4(+) T cells. Increasing the number of dendritic cells (DC) in vivo with flt3 ligand augmented the expansion and activation of the OVA-specific T cells, particularly CD8(+) T cells. These studies indicate DC play a central role in the priming of both CD4(+) and CD8(+) T cells in vivo, and suggest that an ability to target DC may allow ISCOMS to be powerful vaccine vectors for stimulating protective immunity.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Presentation of exogenous whole inactivated simian immunodeficiency virus by mature dendritic cells induces CD4+ and CD8+ T-cell responses

Interactions between HIV-1 and dendritic cells (DCs) play an important role in the initial establishment and spread of infection and development of antiviral immunity. We used chemically inactivated aldrithiol-2 (AT-2) simian immunodeficiency virus (SIV) with functional envelope glycoproteins to study virus interactions with DCs and developed an in vitro system to evaluate the quality of SIV antigen (Ag) presentation by DCs to T cells. AT-2 SIV interacts authentically with T cells and DCs and thus allows assessment of natural SIV-specific responses. CD4+ and CD8+ T cells from blood or lymph nodes of SIV-infected macaques released interferon-gamma (IFN gamma) and proliferated in response to a variety of AT-2 SIV isolates. Responses did not vary significantly as a function of the quantitative envelope glycoprotein content of the virions. Presentation of Ags derived from AT-2 SIV by DCs was more potent than presentation by comparably Ag-loaded monocytes. Interestingly, SIV-pulsed mature DCs stimulated both CD4+ and CD8+ T-cell responses, whereas immature DCs primarily stimulated CD4+ T cells. Further studies using AT-2 inactivated virus may help to define better the details of the virus-DC interactions critical for infection versus induction of antiviral immune responses.     

IRF4-dependent dendritic cells regulate CD8+ T-cell differentiation and memory responses in influenza infection

Acute respiratory disease caused by influenza viruses is imperfectly mitigated by annual vaccination to select strains. Development of vaccines that elicit lung-resident memory CD8⁺ T cells (T_RM) would offer more universal protection to seasonal and emerging pandemic viruses. Understanding how lung-resident dendritic cells (DCs) regulate T_RM differentiation would be an important step in this process. Here, we used CD11c-cre-Irf4^f/f (KO) mice, which lack lung-resident IRF4-dependent CD11b⁺CD24^hi DCs and show IRF4 deficiency in other lung cDC subsets, to determine if IRF4-expressing DCs regulate CD8⁺ memory precursor cells and T_RM during influenza A virus (IAV) infection. KO mice showed defective CD8⁺ T-cell memory, stemming from a deficit of T regulatory cells and memory precursor cells with decreased Foxo1 expression. Transfer of wild-type CD11b⁺CD24^hi DCs into KO mice restored CD8⁺ memory precursor cell numbers to wild-type levels. KO mice recovered from a primary infection harbored reduced numbers of CD8⁺ T_RM and showed deficient expansion of IFNγ⁺CD8⁺ T cells and increased lung pathology upon challenge with heterosubtypic IAV. Thus, vaccination strategies that harness the function of IRF4-dependent DCs could promote the differentiation of CD8⁺ T_RM during IAV infection.     

The CD4+ T-cell response to protein immunization is independent of accompanying IFN-gamma-producing CD8+ T cells.

By virtue of their strong bias towards production of interferon-gamma (IFN-gamma), CD8+ T cells have the potential to promote the development of type 1 immune responses. We have previously shown that the CD4+ T-cell response to immunization with the protein antigen keyhole limpet haemocyanin (KLH) has a mixed interleukin-4 (IL-4)/IFN-gamma production profile. Here we show that this immunization regimen also stimulates accumulation in the draining lymph nodes of CD8+ T cells, which preferentially contain IFN-gamma mRNA ex vivo and secrete IFN-gamma protein in vitro. This provides a model to test whether CD8+ cell-derived IFN-gamma participates in the normal control of the immune response to a non-viable exogenous antigen. To investigate regulation of the anti-KLH response by the CD8+ population or IFN-gamma produced by this or other cell types, mice were administered depleting antibodies. Depletion of CD8+ cells had no effect on the frequency of clonogenic KLH-specific CD4+ T cells, the IL-4/IFN-gamma profiles of their progeny, or the isotype profiles of the serum antibody response to KLH. In contrast, IFN-gamma neutralization diminished cell accumulation in the lymph nodes and reduced both the frequency of KLH-specific CD4+ T cells that gave rise to IFN-gamma-producing clones and serum titres of KLH-specific IgG2a and IgG3. Therefore, despite the potential for cross-regulation, the CD4+ T-cell response to this immunogen is independent of the IFN-gamma-skewed CD8+ response.     

Mouse lung CD103+ and CD11bhigh dendritic cells preferentially induce distinct CD4+ T-cell responses

Mouse lung dendritic cells (LDCs) have been recently shown to contain two major subpopulations: CD103(+) CD11b(low or negative) (CD103(+) LDCs) and CD103(-) CD11b(high) LDCs (CD11b(high) LDCs). Although several studies have demonstrated functional differences between them, it is unclear whether the subpopulations induce distinct T helper (Th) cell responses. The present study was conducted to examine whether CD103(+) and CD11b(high) LDCs preferentially generate different Th responses. Naive DO11.10 CD4(+) T cells were primed with CD103(+) or CD11b(high) LDCs obtained from normal BALB/c mice. The primed CD4(+) T cells were restimulated, and their cytokine secretions were assessed. The expression of intracellular cytokines and the mRNA levels of chemokine receptors were also measured. We found that the CD4(+) T cells primed with CD103(+) LDCs secreted significantly larger amounts of IFN-γ and IL-17A, whereas those primed with CD11b(high) LDCs released significantly higher levels of IL-4, IL-6, and IL-10. Intracellular cytokine assay showed that CD103(+) LDCs induced greater frequencies of CD4(+) T cells producing IFN-γ and IL-17A, whereas CD11b(high) LDCs were more efficient at inducing CD4(+) T cells producing IL-4 and IL-10. The mRNA levels of CXCR3 and CCR5, which are expressed preferentially in Th1 cells, were significantly higher in CD4(+) T cells primed with CD103(+) LDCs. The mRNA levels of CXCR4 and CCR4, which are expressed primarily in Th2 cells, were significantly greater in those primed with CD11b(high) LDCs. These data suggest that mouse CD103(+) LDCs predominantly elicit Th1 and Th17 responses, whereas CD11b(high) LDCs primarily provoke a Th2 response under the steady state.     

Detection and analysis of antigen-specific CD8+ T cells

Based on recent advances in techniques that can detect and enumerate antigen-specific CD8+ T cells, it is evident that these cells can differentially regulate CD8+ T cell effector mechanisms at the single-cell level. Interplay between effector mechanisms that are employed by antigen-specific CD8+ T cells during the immune response in vivo can be addressed with different techniques that "count" cells either directly (T cell receptor (TCR) expression) or indirectly (antigen-specific cytokine production).     

Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses

Whereas the definition of B-cell memory is based on well-known cellular properties and differentiation steps, the process of T-cell memory generation was, until recently, less well understood. A series of recent reports, however, have drastically modified our notion of CD8(+) memory T cells. They show that, in addition to division, the generation of efficient memory cells requires a previously unknown differentiation process. As a whole, the generation of CD8(+) memory T cells appears to mimic the generation of memory B cells. Both processes depend on the help of CD4(+) T cells, they are irreversible, they have the same mechanism, and they occur progressively during the late expansion phase of the primary immune response.     

The proliferative response of CD4 T cells to steady-state CD8+ dendritic cells is restricted by post-activation death

CD8(+) splenic dendritic cells (DCs) from steady-state mice are less effective than the CD8(-) DC subset in their capacity to stimulate CD4 T cell proliferation in culture. However, we found that the two DC subtypes were equally potent at activating CD4 T cells, based on up-regulation of CD69 and CD25 expression. Also, we found no difference in the rate of T cell death prior to entry into the first division. We then tracked carboxyfluorescein diacetate succinimidyl ester-labeled T cells and employed a quantitative model to assess in detail the CD4 T cell expansion process in response to stimulation with CD8(+) or with CD8(-) DCs. The time required for most T cells to replicate their DNA prior to the first division was similar in both DC cultures. However, progression of the CD4 T cell population through subsequent divisions was reduced in CD8(+) DCs compared with CD8(-) DC culture. This was associated with an increased loss of viable T cells at each division. Post-activation, division-associated T cell death is therefore a major factor in the reduced response of CD4 T cells to CD8(+) DCs.     

Copulsing tumor antigen-pulsed dendritic cells with zoledronate efficiently enhance the expansion of tumor antigen-specific CD8+ T cells via Vgamma9gammadelta T cell activation

We demonstrate that Vgamma9gammadelta T cells activated by zoledronate can link innate and acquired immunity through crosstalk with dendritic cells (DCs) in a way that can amplify activation and proliferation of tumor antigen-specific CD8+ T cells. DCs pulsed with antigen alone or antigen plus zoledronate were used to stimulate the in vitro expansion of antigen-specific CD8+ T cells. MART-1-modified peptide (A27L peptide) and apoptotic HLA-A*0201-positive, MART-1-positive JCOCB tumor cell lines were used as tumor antigen sources. The percentage of A27L-specific CD8+ T cells within the responding lymphocytes on Day 7 when immature DCs (imDCs) were cultured in the presence of A27L peptide and 0.01 microM zoledronate was significantly higher (P=0.002, n=11) than that observed when imDCs were cultured with the lymphocytes in the presence of the A27L peptide alone. This enhancing effect of zoledronate was significantly reduced when gammadelta T cells were depleted from responding lymphocytes (P=0.030, n=5), indicating that the effect is mediated mainly through Vgamma9gammadelta T cells activated by zoledronate-pulsed imDCs. When imDCs copulsed with zoledronate and apoptotic JCOCB tumor cell lines were used, the percentage of A27L-specific CD8+ T cells was higher than that observed using imDCs with the apoptotic JCOCB lines alone, suggesting that zoledronate treatment of imDCs enhances the cross-presentation ability of DCs. These findings suggest a potentially valuable role for Vgamma9gammadelta T cell activation for expanding antigen-specific CD8+T cells using DCs copulsed with tumor antigen and zoledronate in the design of vaccine therapies for malignancy.