ELISPOT assay to measure antigen-specific murine CD8(+) T cell responses

The enzyme-linked immunospot technique (ELISPOT) relies on the visualization of cytokine secretion by individual T cells following in vitro stimulation with antigen. This assay has been developed and standardized for the quantitative detection of antigen-specific CD8(+) T cells in mice subjected to different immunization protocols [J. Immunol. Methods 181 (1995) 45]. We have identified important variables that affect the efficacy of the ELISPOT assay and in this protocol we describe this methodology in detail. As a model, we used the production of interferon-gamma by CD8(+) T cells from peripheral blood, spleen and liver of mice immunized with malaria sporozoites expressing the H-2K(d)-restricted SYVPSAEQI. This protocol has also been used successfully to detect Th1 and Th2 epitope specific CD4(+) T cells.     

Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15--the Amplispot assay

The importance of the enzyme-linked immunosorbent spot (ELISPOT) assay as a tool for studying immune responses in vitro is becoming increasingly apparent. However, there remains a need for enhanced sensitivity for the detection of low frequency antigen-specific T cell responses. We reasoned that the addition of a combination of the cytokines interleukin (IL)-7 and IL-15 would selectively increase interferon-gamma (IFN-gamma) production from antigen-stimulated CD4+ and CD8+ effector memory T cells. Freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC) from four healthy donors were analysed by ELISPOT for the frequency of purified protein derivative (PPD)-specific CD4+ T cells or cytomegalovirus (CMV) peptide-specific CD8+ T cells. Addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC and up to 18-fold in cryopreserved PBMC. The cytokines also increased the number of CMV peptide-specific CD8+ T cells in fresh PBMC up to 7.5-fold. No additional increases were seen when antibodies to co-stimulatory molecules CD28 and CD49d were applied together with the cytokine combination. These data demonstrate that the sensitivity of the ELISPOT assay may be significantly augmented by addition of the cytokines IL-7 and IL-15 to antigen-stimulated cells. This method will be particularly useful for the assessment of antigen-stimulated cytokine production by T cells in cryopreserved biological specimens.     

Direct exosome stimulation of peripheral human T cells detected by ELISPOT

Exosomes from APC are nano-vesicles that can induce antigen-specific T cell responses and are presently explored as therapeutic tools in different clinical settings. Investigations of the capacity of exosomes to stimulate T cells in vitro have mostly been performed on T cell hybridomas, clones or lines. Whether exosomes can stimulate T cells directly or need the presence of dendritic cells (DC) is debated. We could detect exosome-induced antigen-specific CD8(+) T cell responses in peripheral blood from humans. Exosomes from monocyte-derived DC (MDDC) were loaded with a mix of 23 immunogenic peptides from EBV, CMV and influenza virus, and added to autologous peripheral CD8(+) T cells. IFN-gamma-producing cells were detected by enzyme-linked immunospot assay (ELISPOT). MDDC-exosomes induced IFN-gamma production in CD8(+) T cells without addition of DC. The response was exosome dose dependent, and dependent on exosomal MHC class I. Furthermore, we detected an enhanced T cell stimulatory capacity by exosomes from lipopolysaccharide-matured MDDC compared to exosomes from immature MDDC. Exosomes could also induce TNF-alpha production. These results show, for the first time, that exosomes can directly stimulate human peripheral CD8(+) T cells in an antigen-specific manner and that ELISPOT is a suitable method for detecting exosome-induced peripheral T cell responses. This system may provide a useful tool when developing exosomes as therapeutic agents.     

Characterization of HIV-1 Gag-specific T cell responses in chronically infected Indian population

India is at the epicentre of the global HIV/AIDS epidemic in South-east Asia, predominated by subtype C infections. It is important to characterize HIV-1-specific T cell responses in this particular population with the aim of identifying protective correlates of immunity to control HIV-1 infection. In this study, we performed a comprehensive analysis of the breadth and magnitude of T cell responses directed at HIV-1 subtype C Gag, one of the most conserved HIV-1 proteins. The study population consisted of antiretroviral naive, chronic HIV-1 subtype C-infected individuals at various stages of infection. We used recent advanced techniques such as enzyme-linked immunospot (ELISPOT) assay and intracellular cytokine staining to quantify the total CD4(+) and CD8(+) T cell response to HIV-1 gag at single peptide level, regardless of HLA haplotype of the infected individual. The p24-Gag was identified as the most frequently recognized subunit protein with the greatest magnitude of CD4(+) and CD8(+) T cell responses. Stronger and broader CD8 T cell responses were recognized, contrasting with the weaker and narrower CD4 T cell responses with regard to Gag protein subunits. The magnitude of the HIV-specific interferon (IFN)-gamma responses was observed to be higher than the corresponding interleukin (IL)-2 response, indicating the persistence of antigenic load in chronically infected Indian population due to the probable dysfunction of HIV-specific, IFN-gamma-secreting CD8 T cells in absence of IL-2 help.     

Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells

CD8(+) T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan and bacterial pathogens. Activated CD8(+) T cells are able to induce cytolysis of infected cells (perforin and CD95-CD95L mediated pathways) and also elaborate cytokines, including IFN-gamma and TNF after appropriate MHC class I-peptide recognition. New technologies for the detection of antigen-specific CD8(+) T cells, including tetrameric MHC class I-peptide complexes, intracellular IFN-gamma staining and IFN-gamma ELISPOT analysis have revised our understanding of the magnitude of the CD8(+) T cell response to infection. Here, using intracellular cytokine staining, we compare detection of IFN-gamma and TNF in the analysis of pathogen-specific CD8(+) T cell lines and CD8(+) T cells after primary viral infection (LCMV) or secondary bacterial infection (Listeria monocytogenes). Under multiple conditions and with multiple epitopes, we find that staining for intracellular IFN-gamma consistently detects a higher frequency of antigen-specific CD8(+) T cells than detection of intracellular TNF. However, (a) intracellular staining for TNF can be used to detect antigen-specific CD8(+) T cell responses and (b) intracellular staining for cytokines is a useful approach for in vitro characterization of antigen-specific CD8(+) T cell lines.     

The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8(+) T lymphocytes in IFN-gamma ELISPOT assays.

ELISPOT assays are increasingly used for a direct detection and quantification of single antigen-specific T cells in freshly isolated peripheral blood mononuclear cells (PBMC). They are particularly attractive for the monitoring of specific T lymphocyte responses in clinical trials assessing antigen-specific immunizations in patients with cancer or chronic viral infections. However, one major limitation for the broad clinical implementation of ELISPOT assays is the lack of an inexhaustible source of suitable HLA-matched antigen-presenting cells (APC). Currently available allogeneic or xenogeneic APC (such as the human lymphoid hybrid T2 or HLA-transfected insect cells) can either lead to strong background spot production by APC-reactive T lymphocytes or have a low antigen presentation capability. Both phenomena can prevent the detection of low frequency T cell responses in PBMC. In search of alternative APC for ELISPOT assays, the human chronic myelogenous leukemia cell line K562 that per se does not express HLA class I and class II molecules on the cell surface was transfected with the HLA-A*0201 gene. Clonal HLA-A*0201-expressing K562 (K562/A*0201) cells were able to process and present endogenously expressed and exogenously loaded melanoma peptide antigens to HLA-A*0201-restricted cytolytic T lymphocyte clones in cytotoxicity and IFN-gamma ELISPOT assays. K562/A*0201 cells were then used as APC in IFN-gamma spot assays to detect ex vivo CD8(+) T lymphocytes responsive to known HLA-A*0201-binding peptide epitopes derived from cytomegalovirus, Epstein-Barr virus, influenza virus and melanoma in PBMC from HLA-A*0201-positive donors. In the majority of cases, peptide-pulsed K562/A*0201 cells were similarly efficient in the ability to visualize single antigen-specific CD8(+) T lymphocytes when compared to T2 cells. However, in contrast to T2, background reactivity of CD8(+) T cells responsive to unpulsed K562/A*0201 was regularly found to be negligible, thereby enhancing the sensitivity of the ELISPOT assay, particularly in donors with strong anti-T2 reactivity. K562 cells transfected with HLA-A*0201 or other HLA genes can serve as standard APC for monitoring T lymphocyte responses against tumor and viral peptide antigens.     

Cytokine production of CD8+ immune T cells but not of CD4+ T cells from Toxoplasma gondii-infected mice is polarized to a type 1 response following stimulation with tachyzoite-infected macrophages.

To examine whether cytokine production of CD4(+)immune T cells and CD8(+)immune T cells in Toxoplasma gondii-infected mice differ in their responses to infected cells and to soluble antigens of the parasite, we compared the production of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-10 by the immune T cell populations following in vitro stimulation with tachyzoite-infected macrophages and tachyzoite lysate antigens (TLA). Both CD4(+)and CD8(+)immune T cells produced large amounts of IFN-gamma in response to either infected macrophages or TLA, but the CD4(+)T cells produced greater amounts of the cytokine than did the CD8(+)T cells with both stimulations. Both T cell populations also produced IL-2 after stimulation with infected macrophages, whereas only CD4(+)T cells did when stimulated with TLA. CD4(+)immune T cells also produced large amounts of IL-4 and IL-10 after stimulation with infected macrophages, but CD8(+)T cells did not. These results indicate that CD4(+)immune T cells produce IFN-gamma, IL-2, IL-4, and IL-10 in response to infected macrophages, whereas CD8(+)immune T cells produce predominantly IFN-gamma and IL-2. Since IL-4 and IL-10 could suppress IFN-gamma-mediated protective mechanisms against the parasite, the production of these cytokines by CD4(+)immune T cells in response to infected cells could negatively affect their protective activity in vivo.     

A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-gamma HIV-specific immune responses

The single color IFN-γ ELISPOT assay has become a standard for assessing HIV-specific immune responses in HIV-infected subjects. However, recent data suggests that single cytokine detection for immune monitoring of HIV-infected individuals may not be sufficient to fully describe virus-specific immune responses. Here, we have designed and validated a dual color ELISPOT assay capable of detecting both IL-2 and IFN-γ secreting cells simultaneously in response to HIV antigens. We found that a cell input number of 200,000 cells/well provided a good balance between limited availability of cells due to blood volume restrictions and ability to detect all cytokine secretion patterns. The simultaneous detection of IL-2 and IFN-γ resulted in a decreased magnitude of IFN-γ but not IL-2 responses. Measures of intra- and inter-assay variability for the dual color ELISPOT assay were comparable to that seen for single cytokine ELISPOT assay with coefficients of variation below 20% for IL-2, IFN-γ and dual secretion. Although CD8+ T cells mediated most HIV-specific responses in infected subjects, CD4+ T cells mediated responses to HIV were also detected. Features of this assay such as high throughput, cell number requirement and cytokine choice should make this assay a valuable tool for screening for HIV-specific immune responses in several clinically relevant settings.     

The secretory IFN-gamma response of single CD4 memory cells after activation on different antigen presenting cell types

It is unknown to what extent the heterogeneity of antigen presenting cells (APC) influences the IFN-gamma response of CD4 memory cells. We re-stimulated DO11.10 T cell receptor (TCR)-transgenic cells and wild-type CD4 memory cells with OVA-peptide 323-339 presented on purified dendritic cells (DC), macrophages, and B cells. Using IFN-gamma ELISPOT assays, we measured the number of cytokine producing T cells and the amount of cytokine produced by individual T cells at different time points after antigen encounter. The data showed that, when CD4 cells recognized antigen on DC, the induction of cytokine production was accelerated compared to macrophages and B cells. In contrast, the per-cell cytokine productivity was independent of the type of APC by which the T cells were re-stimulated. Moreover, the peptide concentration required for CD4 cell activation was comparable for the different APC. The data suggest that DC induce cytokine production in memory cells with accelerated activation kinetics, whereas 24 h of antigen stimulation on DC, macrophages, and B cells results in comparable levels of T cell activation. These data have implications for the understanding of T cell memory responses when T cells re-encounter antigen on different APC as well as for the monitoring of memory T cell responses ex vivo.     

Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4(+) and CD8(+) T cells secreting INFgamma, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFgamma-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4(+) and CD8(+) T cell subsets, IFNgamma-secreting RV-specific CD8(+) but not CD4(+) T cells were detected in recently infected children. Using the same approach, both CD4(+) and CD8(+) RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFgamma-secreting CD4(+) T cells from adult volunteers preferentially express the intestinal homing receptor alpha4beta7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFgamma-secreting CD4(+) T cells preferentially express L-selectin but not alpha4beta7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFgamma is independent of the expression of these homing receptors.     

CD8+ T细胞对HIV-1合成表位的免疫主导应答研究

目的研究CD8^＋ T细胞对人免疫缺陷病毒1型（HIV-1）表位的免疫主导应答。方法分别采用酶联免疫斑点技术（ELtSPOT）和羧乙基锗倍半氧化物（CFSE）标记流式分析技术，以覆盖HIV-1 Env、Pol、Gag、Vif、Nef、Tat区的701个重叠肽段组成的34个肽段库及其部分单肽段作为刺激表位，对一例感染HIV-1的长期不进展者（LTNP）的外周血单个核细胞（PBMC）中CD8^＋ T细胞的了γ-干扰素（IFN-γ）分泌细胞频率和细胞增殖率进行了测定研究。结果HIV-1 Gag区域肽段诱导产生的CD8^＋ T细胞的IFN-γ分泌细胞频率最高，Nef、Tat、Vif区域依次顺减，Env和Pol区域不能诱导产生显著性应答；在IFN-γ ELISPOT实验中，肽段和相应肽段库刺激产生的结果一致；CD8^＋ T细胞在单肽段刺激下，用ELISPOT技术测定的IFN-γ分泌细胞频率和CFSE标记流式分析技术测定的细胞增殖率显示出较好的相关性。结论CD8^＋ T细胞能特异性识别某些HIV-1抗原表位，诱导出免疫主导应答；当进行HIV-1特异性CD8^＋ T细胞反应增殖测定和免疫主导应答研究时，ELISPOT是值得称道的标准实验，同时，推荐一种新颖的CFSE标记流式分析技术。     

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.     

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.     

Early Emergence of CD8+ T Cells Primed for Production of Type 1 Cytokines in the Lungs of Mycobacterium tuberculosis-Infected Mice

Several lines of evidence suggest that CD8 T cells are important in protection against tuberculosis. To understand the function of this cell population in the immune response against Mycobacterium tuberculosis, T cells from lungs of M. tuberculosis-infected mice were examined by flow cytometry. The kinetics of the appearance of CD8 T cells in lungs of infected mice closely paralleled that of CD4 T cells. Both CD4(+) and CD8(+) T cells displaying an activated phenotype were found in the lungs as early as 1 week postinfection. By 2 weeks, total cell numbers in the lungs had tripled and percentages of T cells were increased two- to threefold; the percentages of CD4(+) T cells were ca. twofold higher than those of CD8(+) T cells. Short-term stimulation with M. tuberculosis-infected antigen-presenting cells induced cytokine production by primed CD4(+) and CD8(+) T cells. Intracellular cytokine staining revealed that 30% +/- 5% of CD4(+) and 23% +/- 4% of CD8(+) T cells were primed for production of gamma interferon (IFN-gamma). However, a difference in in vivo IFN-gamma production by T cells was observed with approximately 12% of CD4(+) T cells and approximately 5% of CD8(+) T cells secreting cytokine in the lungs at any given time during infection. The data presented indicate that although early in infection the majority of IFN-gamma is produced by CD4(+) T cells, cytokine-producing CD8(+) T cells are readily available when triggered by the appropriate stimuli.     

Determinants of HIV-specific CD8 T-cell responses in HIV-infected pediatric patients and enhancement of HIV-gag-specific responses with exogenous IL-15

Cellular immune responses play a central role in controlling HIV-1 infection. HIV-specific IFN-gamma production by CD8 T cells was evaluated in 17 HLA-A2+ HIV-infected pediatric patients (age range 1 month to 16 years) in an ELISPOT assay. Most patients (15/17) exhibited responses to HIV-gag, followed by responses to envelope gp120, gp41, and V3 loop. Only 7 patients responded to all four antigenic peptides. Treatment-related immune reconstitution of CD4 T cells was associated with increase in gag-specific responses, but these declined with prolonged viral suppression. Exogenous IL-15 resulted in augmentation of HIV-gag-specific response in 71% of patients, while IL-2 and IL-7 had variable effects, augmenting responses in 25% patients. Thus, HIV-specific CD8 T-cell responses are dependent on both CD4 T-cell help and antigenic stimulation. The cytokine IL-15 may be a useful modality as adjunctive therapy to augment HIV-specific memory CD8 T cells.     

CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens

CD4(+)CD8(dim) T cells represent a minor subset of the total CD3(+) T cell population in peripheral blood. Although transient and persistent expansions of these cells have been reported in both healthy and diseased individuals, the functional properties of the CD4(+)CD8(dim) population are largely unknown. In this study, we examined antigen-specific cytokine and proliferative responses of the CD4(+)CD8(dim) subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV-1, a significant fraction of the CD4(+)CD8(dim) subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4(+)CD8(dim) population contained two- to eightfold higher frequencies of antigen-specific cytokine producing cells than the CD4(+)CD8(-) population. Phenotypic analysis of the cytokine expressing CD4(+)CD8(dim) population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4(+)CD8(dim) cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4(+)CD8(dim) T cells were found to possess higher CMV-specific cytotoxic activity than purified CD4(+)CD8(-) T cells in a standard (51)Cr-release CTL assay. Thus, CD4(+)CD8(dim) T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4(+) cell populations specific for HCMV and HIV-1.     

Detection of low-frequency antigen-specific IL-10-producing CD4(+) T cells via ELISPOT in PBMC: cognate vs. nonspecific production of the cytokine

Single-cell resolution cytokine ELISPOT assays are increasingly used to gain insights into clonal sizes of type 1 and type 2 effector T cell populations in vivo. However, ELISPOT assays permitting monitoring of regulatory IL-10-producing T cells have so far not been established. Unlike IFN-gamma, IL-2, IL-4, and IL-5 assays performed on PBMC in which the recall antigen-induced cytokine spots are T cell-derived, we show here that in such assays IL-10 is primarily monocyte-derived. T cell-derived IL-10 spots were 80 x 10(3) microm(2) in size, seven times larger than spots produced by monocytes, and B cells produced even smaller spots. Based on spot size gating and the use of B cells as APC, we have established test conditions that permit measurement of cognate IL-10 production by low-frequency antigen-specific T cells. IL-10-producing PPD-specific CD4(+) T cells were detected in frequencies comparable to IFN-gamma-secreting CD4(+) T cells in tuberculosis patients, but not in uninfected healthy control individuals. In contrast, IL-10-secreting CD4(+) T cells specific for a panel of recall antigens could not be detected in frequencies >1/100,000 in healthy individuals whose CD4(+) cells responded to these antigens with type 1 or type 2 cytokine production in the 1:100,000-1:1000 frequency range. Therefore, the induction of IL-10-producing T cells seems to be under tighter control than that of Th1/Th2 cells, apparently confined to states of chronic immune stimulation. Access to low-frequency immune monitoring of IL-10-producing T cells will provide new insights into the role of regulatory T cells in health and disease.     

Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+ T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

Immature dendritic cells (DC) infected with an endotheliotropic (Huv(+)) and leukotropic (Leuk(+)) human cytomegalovirus (HCMV) strain were used as a stimulus to determine functional HCMV-specific CD4(+) and CD8(+) T cells. Infected DC were co-cultured with autologous peripheral blood mononuclear cells and both arms of T cell activation were determined by intracellular flow cytometry analysis of IFN-gamma production. Efficient stimulation of HCMV-specific CD4(+) and CD8(+) T cell responses was achieved using DC productively infected with Huv(+) Leuk(+) VR1814 strain. On the contrary, a negligible CD8(+) T cell response was obtained when HCMV strains unable to infect DC, or DC pulsed with inactivated viral antigen, were used. HCMV specificity of the T cell response was confirmed in 46 HCMV-seropositive and 8 HCMV-seronegative healthy subjects. A cut-off was established to discriminate between immune and nonimmune subjects. The novel ex vivo assay enables the simultaneous evaluation of HCMV-specific CD4(+) and CD8(+) T cell responses and may be a useful tool for monitoring HCMV-specific T cell activity in immunocompromised transplanted patients.     

Generation of highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) regulatory T cells by continuous antigen stimulation

Continuous antigen stimulation of CD4(+)CD25(-) T cells leads to generation of adaptive CD4(+)CD25(+)FOXP3(+) regulatory T (T(R)) cells. Here, we show that highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14(+) monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up-regulation and expression of cytotoxic molecules. The CD8(+) T(R) cells inhibit CD4(+) and CD8(+) T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8(+) effector T cells. Although, the CD8(+) T(R) cells express prostaglandin E(2), IL-10 and TGF-beta, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4(+) T(R) cells, the CD8(+) T(R) cells suppress mainly by a contact-dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA-4, CD80 nor CD86 could reverse CD8(+) T(R)-mediated suppression, indicating that other mechanism(s) must be employed by these cells.