Tracking antigen-specific CD8+ T cells in the rat using MHC class I multimers

Studies of the quantitative and qualitative aspects of anti-microbial, anti-tumoral or autoreactive immune responses have been greatly facilitated by the possibility to stain antigen-specific CD8(+) T cells using fluorescently labeled multimeric major histocompatibility complex (MHC) class I/peptide complexes. So far, this technology has been developed for human and mouse, but not yet in the rat. Here, we describe the generation of the first rat MHC multimer. We produced a rat RT1(l) Pro5 MHC Pentamer combined with the immunodominant peptide for Borna disease virus (BDV), in order to study the characteristics of the antiviral CD8(+) T cell response. BDV is an RNA virus that can cause persistent infections of the central nervous system (CNS), often associated with prominent brain inflammation. In adult Lewis rats, of the RT1(l) MHC haplotype, BDV infection leads to severe immune-mediated neurological symptoms. The pathogenic role of the immune response is due primarily to antiviral CD8(+) T cells, many of them being specific for an immunodominant epitope located in the BDV nucleoprotein (N(230-238)). Ex vivo flow cytometry analyses revealed that 3 to 12% of CD8(+) T cells found in the brains of BDV-infected rats stained positively with the BDV-Pentamer. Interestingly, the frequency of Pentamer-positive cells increased up to 3.3 fold after a short resting period in culture. Virus-specific CD8(+) T cells were mainly detected in the brain and were virtually undetectable in peripheral lymphoid organs. This novel rat Pro5 MHC Pentamer represents an attractive tool for the detection, isolation and characterization of antigen-specific CD8(+) T cell responses in the rat.     

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ T cell responses by cross-presentation of exogenous antigen via MHC class I is well known. In contrast, their role in the activation of CD4+ T cells is less clearly defined, although several recent studies in mice and T cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC class II. In this study we have investigated the potential of antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to the human stress-inducible Hsp70 to enhance activation and proliferation of human memory CD4+ T cells. Hsp70:peptide complexes were found to amplify the proliferation of antigen-specific CD4+ T cells as confirmed by HLA-DR tetramer staining. Complex formation of the antigenic peptide with Hsp70 was absolutely required to elicit an antigen-specific amplification. This effect was most pronounced at low doses of antigen and decreasing APC/CD4+ T cell ratios. Taken together, we show the potential of Hsp70 to enhance antigen-specific CD4+ T cell proliferation and to increase the immunogenicity of presented peptides in human CD4+ T cells.     

Immediate antigen-specific effector functions by TCR-transgenic CD8+ NKT cells

Only recently have natural antigens for CD1d-dependent, invariant Valpha14+ natural killer T (iNKT) cells been identified. Similar data for CD1d-independent and CD8+ NKT cell populations are still missing. Here, we show that the MHC class I-restricted CD8+ TCR-transgenic mouse lines OT-I, P14 and H-Y contain a significant proportion of transgenic CD8+ NK1.1+ T cells. In liver, most of NK1.1+ T cells express CD8alphaalpha homodimers. Transgenic NKT cells did not bind invariant Valpha14-to-Jalpha18 TCR rearrangement (Valpha14i)-specific CD1d/alpha-galactosylceramide tetramers and the frequency of iNKT cells was severely reduced. The activated cell surface phenotype and the distribution of transgenic NKT cells in vivo were similar to that reported for iNKT cells. The OT-I and P14 CD8+ NKT cells recognized their cognate antigen in the context of H2-Kb and produced cytokines shortly after TCR stimulation. Importantly, transgenic NKT cells exerted immediate antigen-specific cytotoxicity in vitro and in vivo. Our results demonstrate the presence of transgenic CD8+ NKT cells in MHC class I-restricted TCR-transgenic animals, which are endowed with rapid antigen-specific effector functions. These data imply that experiments studying naive T cell function in TCR-transgenic animals should be interpreted with caution, and that such animals could be utilized for studying CD8+ NKT cell function in an antigen-specific manner.     

B cells can prime naive CD4+ T cells in vivo in the absence of other professional antigen-presenting cells in a CD154-CD40-dependent manner

The role of B cells as APC is well established. However, their ability to prime naive T cells in vivo has been difficult to examine because of the presence of dendritic cells. The current studies were undertaken to examine this issue in a model of adoptive transfer of antigen-specific B cells and T cells into histoincompatible Rag2(-/-) mice. By means of this system, we were able to demonstrate that antigen-specific B cells are competent APC for naive CD4(+) T cells specific for the same antigen. In vivo antigen presentation resulted in expansion of both CD4(+) T cells and B cells. The antigen-presenting function of the transferred B cells was dependent on the CD154-CD40 interaction, as transfer of CD154-deficient antigen-specific CD4(+) T cells or CD40-deficient B cells failed to induce T and B cell expansion in response to immunization. These results indicate that antigen-specific B cells have the capacity to induce primary T cell responses in the absence of other competent APC.     

Transgenic expression of CCL2 in the central nervous system prevents experimental autoimmune encephalomyelitis

CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, a member of the CC chemokine family, is a chemoattractant for monocytes and T cells through interaction with its receptor CCR2. In the present study, we examined a T helper cell type 1 (Th1)-dependent disease, proteolipid protein-induced experimental autoimmune encephalomyelitis, in a transgenic mouse line that constitutively expressed low levels of CCL2 in the central nervous system (CNS) under control of the astrocyte-specific glial fibrillary acidic protein promoter. CCL2 transgenic mice developed significantly milder clinical disease than littermate controls. As determined by flow cytometry, mononuclear cell infiltrates in the CNS tissues of CCL2 transgenic and littermate-control mice contained equal numbers of CD4+ and CD8+ T cells, and the CCL2 transgenic mice showed an enhanced number of CNS-infiltrating monocytes. CNS antigen-specific T cells from CCL2 transgenic mice produced markedly less interferon-gamma. Overexpression of CCL2 in the CNS resulted in decreased interleukin-12 receptor expression by antigen-specific T cells. Collectively, these results indicate that sustained, tissue-specific expression of CCL2 in vivo down-regulates the Th1 autoimmune response, culminating in milder clinical disease.     

Anti-OX40 stimulation in vivo enhances CD8+ memory T cell survival and significantly increases recall responses

There is growing evidence that engagement of OX40 (CD134), a member of the TNF receptor superfamily, can directly stimulate antigen-specific CD8+ T cells. It has been shown that CD8+ T cells express OX40 following activation, but the response of antigen-specific CD8+ T cells to OX40 stimulation has not been fully characterized. We utilized an antigen-specific transgenic CD8+ T cell model (OT-I) to determine if OX40 engagement can boost the generation of antigen-specific CD8+ T cell memory. Our results demonstrate that enhanced OX40 costimulation, via an agonist anti-OX40 antibody, increases CD25 and phospho-Akt expression on the antigen-specific CD8+ T cells and significantly increases the generation of long-lived antigen-specific CD8+ memory T cells. The increased numbers of memory CD8+ T cells generated via anti-OX40 treatment still required the presence of CD4+ T cells for their long-term maintenance in vivo. In addition, anti-OX40 costimulation greatly enhanced antigen-specific CD8+ T cell recall responses. These data show that OX40 engagement in vivo increases the number of antigen-specific CD8+ memory T cells surviving after antigen challenge and has implications for the development of more potent vaccines against pathogens and cancer.     

Soluble, dimeric HLA DR4-peptide chimeras: an approach for detection and immunoregulation of human type-1 diabetes

Still there are no effective methods to predict or cure type 1 diabetes (T1D) in humans. Soluble, dimeric MHC class II-peptide (DEF) chimeras have potential for both early diagnosis and immunospecific therapy. DEF chimeras prevent and reverse diabetes in mice by stimulating antigen-specific type 1 T regulatory cell (Tr1)-like cells. We also showed that diabetes could be predicted by changes in the phenotype of autoreactive CD4 T cells in peripheral blood. Herein, we demonstrated that human DEF (HLA-DR*0401/Fcgamma1) chimeras expressing peptides of beta-cell antigens stimulate Tr1-like cells in blood of patients with T1D, non-diabetic relatives, and controls. Furthermore, the specific and stable binding of DEF chimeras to cognate TCR and CD4 coreceptor allowed quantification and phenotyping of autoreactive CD4 T cells in non-stimulated blood by FACS. Our results indicate that (1) autoreactive CD4 T cells to GAD65 autoantigen are commonly present in humans expressing diabetes-susceptible HLA-DR*0401 molecules; (2) these autoreactive T cells undergo avidity maturation upon encountering the self antigen early in life; (3) the disease is associated with an imbalance between autoreactive CD4+CD25+ and CD4+CD69+ T cells specific for GAD65. Based on this, we propose a model to explain the kinetics of autoreactive CD4 T cells in blood during the natural history of T1D.     

Chronic ethanol induces inhibition of antigen-specific CD8+ but not CD4+ immunodominant T cell responses following Listeria monocytogenes inoculation

Chronic ethanol consumption results in immunodeficiency. Previous work with chronic ethanol-fed mice has shown reduced splenic weight and cellularity, including reduced numbers of CD8+ T cells. However, antigen-specific CD8+ and CD4+ T cell responses in chronic ethanol-fed mice have been studied relatively little. We have used an attenuated Listeria monocytogenes strain DPL 1942 (LM DeltaactA) to inoculate mice and subsequently used CD4+ and CD8+ immunodominant peptides of LM to measure the CD4+ and CD8+ T cell responses after chronic ethanol exposure. We found no major differences between control and ethanol-fed mice in the kinetics and persistence of antigen-specific CD4+ T cells in response to an immunodominant LM peptide, as measured by intracellular IFN-gamma staining. In contrast to CD4+ responses, three methods of in vitro antigen presentation indicated that the primary response of CD8+ T cells to several different epitopes was reduced significantly in mice chronically fed ethanol. Antigen-specific CD8+ T cells were also reduced in chronic ethanol-fed mice during the contraction phase of the primary response, and memory cells evaluated at 29 and 60 days after inoculation were reduced significantly. BrdU proliferation assays showed that in vivo proliferation of CD8+ T cells was reduced in ethanol-fed mice, and IL-2-dependent in vitro proliferation of naive CD8+ T cells was also reduced. In conclusion, these results suggest that antigen-specific CD4+ T cell responses to LM are affected little by chronic ethanol consumption; however, antigen-specific CD8+ T cell responses are reduced significantly, as are in vivo and in vitro proliferation. The reduction of antigen-specific CD8+ T cells may contribute strongly to the immunodeficiency caused by ethanol abuse.     

Tumor antigen drives a persistent oligoclonal expansion of CD8+ T cells in aged mice

Oligoclonal T cell expansions (TCE) are common in old humans and mice, but it is not known whether the T cell response to a specific antigen is more restricted in old vs. young animals. Herein, we describe an enhanced and prolonged response of tumor antigen-specific CD8 cells in old mice identified by K(d)/peptide tetramers and Vbeta10 staining. At the onset of the response CD8 T cell numbers and Vbeta10+CD8+ cells at the site of tumor injection were lower in old mice, hinting that control of initial tumor growth may not be optimal. As further evidence of a dysregulated response in old mice, antibody titers to the tumor were deficient and the CD8 tumor antigen-specific response was greater and more prolonged in the blood and spleen. Old mice selected a more oligoclonal TCR repertoire based on TCRbeta chain CDR3 length analysis and sequences. Persistent expansions of Vbeta10+CD8+ cells in old mice had memory/activation phenotypes. This induced tumor antigen-specific response may represent a model for the spontaneous TCE observed with aging and demonstrates that the CD8 response to a defined peptide/MHC antigen is indeed more oligoclonal in old mice.     

Antigen-dependent suppression of alloresponses by Foxp3-induced regulatory T cells in transplantation

Adoptive transfer of polyclonal CD4+CD25+ regulatory T cells (Treg) can tolerize transplantation alloresponses. Treg are activated via their specific TCR, but the antigen specificity of wild-type Treg remains elusive, and therefore controlling potency and duration of Treg activity in the transplantation setting is still not feasible. In this study, we used murine graft-versus-host disease (GVHD) as a model system to show that antigen-specific Treg suppress the response of T effector cells to alloantigens in vitro and prevent GVHD in vivo. The suppressive potential of antigen-specific Treg was much greater than that of polyclonal Treg. To acquire large numbers of antigen-specific Treg, we transduced CD4+CD25- cells with foxp3, and found that these foxp3-induced Treg suppress alloresponses in vitro and prevent GVHD in vivo as effectively as naturally derived CD4+CD25+ Treg. Furthermore, we used an antigen-specific CD4 Th1 clone as a source of foxp3-induced Treg after transduction with foxp3, and found those Treg to effectively prevent GVHD in an antigen-dependent manner. The findings of this study provide a basis for the concept that the onset and potency of the suppression by Treg can be regulated, and suggest a novel approach to enhance the feasibility and effectiveness of inducing tolerance by Treg as an adoptive immunotherapy in transplantation.     

MHC tetramers: tracking specific immunity

In an adaptive immune response, antigen is recognized by two distinct sets of highly variable receptor molecules: (1) immunoglobulins, that serve as antigen receptors on B cells and (2) the antigen-specific receptors on T cells. T cells play important role in the control of infection and in the development of protective immunity. These cells can also mediate anti-tumor effects and, in case of autoimmune syndromes, contribute to the development and pathology of disease. The specificity of T cells is determined by T cell receptors (TCR). Understanding of the success of immune responses requires the direct measurement of antigen-specific T lymphocytes. Cell with major histocompatibility complex (MHC) class I molecules are able to present antigens to antigen-specific CD8+ cytotoxic T lymphocytes. MHC class I molecules present small peptides (epitopes) processed from intracellular antigens such as viruses and intracellular bacteria. MHC class I molecules in humans are designated as human leukocyte antigen (HLA) class I and divided into HLA-A, -B and -C. CD8+ T cells recognize MHC class I molecules and after activation produce proteins that destroy infected cells. MHC class II molecules receive their peptides mainly from extracellular and soluble antigens and present them to the CD4+ T helper cells. A recently described technique that can be used in flow cytometry enables us to quantify ex vivo antigen-specific T cells by binding of soluble tetramer MHC-peptide complexes attached to fluorochrome. Quantitative analyses of antigen-specific T cell populations provide important information on the natural course of immune responses. The interaction of T cell receptors on T lymphocytes with tetrameric MHC-peptide complexes mimics the situation on the cell surface, and allows for reliable binding. Tetramers consist of four biotinylated HLA-peptide epitope complexes bound to streptavidin conjugated with fluorescent dye. Tetramer technology has sensitivity of detection as little as 0.02% of total cytotoxic T cell pool or T helper cell pool (i.e. approximately 1 in 50.000 lymphocytes). The combination of this technology with intracellular cytokine staining methods opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production). MHC tetramers class I have yielded useful insights into in vivo dynamic and function of antigen-specific CD8+ T cells in viral infections, parasitic infections, cancer, autoimmune disease and transplantation. This knowledge is of special interest for immunotherapy, diagnostic monitoring of T cell mediated immunity, and the development of new vaccination strategies. There is some possibility for cell therapy with antigen-specific CD8+ T cells for various diseases including cancer and viral infections. Targeted immunotherapy of selective deletion of auto--or alloreactive T cells with MHC tetramers may be important for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs. The utility of this technique for the immunotherapy in vivo needs to be confirmed and modified in further research. Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of cellular immune system.     

A kinetic window constricts the T cell receptor repertoire in the thymus

To characterize the ligand binding properties of a naive T cell repertoire capable of responding to a foreign antigen, we analyzed T cell populations from T cell receptor (TCR) beta transgenic mice using a novel, single cell peptide/major histocompatibility complex (MHC) tetramer dissociation assay. The largely CD4+CD8(-/low) antigen-specific thymocyte repertoire exhibited a broad, bimodal distribution of tetramer binding half-lives (t(1/2)s), with a significant underrepresentation in the intermediate half-life range in which the majority of the peripheral repertoire lies. Thus, cells with the potential to bind foreign antigen with the lowest and highest stability are likely to be selectively removed from the repertoire prior to their establishment in the periphery. These studies provide direct evidence that thymic selection biases the naive peripheral T cell repertoire toward TCR-ligand interactions that fall within a moderate half-life "window."     

Impact of MHC class I alleles on the M. tuberculosis antigen-specific CD8+ T-cell response in patients with pulmonary tuberculosis

Challenged by scattered understanding of protective immunity to Mycobacterium tuberculosis (MTB), we have mapped peptide epitopes to human leukocyte antigen (HLA)-A*0101, A*0201, A*1101, A*2402, B*0702, B*0801 and B*1501 of the secreted mycobacterial antigen Ag85B, a vaccine candidate that may be associated with immune protection. Affinity (ED(50)) and half-life (t(1/2), off-rate) analysis for individual peptide species on HLA-A and HLA-B molecules revealed binding ranges between 10(-3) and 10(-7) M. After selection of the best matches, major histocompatibility complex class I/peptide tetramer complexes were constructed to measure the CD8+ T-cell responses directly ex vivo in peripheral blood mononuclear cells (PBMC) derived from 57 patients with acute pulmonary tuberculosis. Three patterns of (allele-) specific CD8+ recognition were identified: (a). Focus on one dominant epitope with additional recognition of several subdominant T-cell epitopes (HLA-A*0301, A*2402, B*0801 and B*1501); (b). Co-dominant recognition of two distinct groups of peptides presented by HLA-B*0702; and (c). Diverse and broad recognition of peptides presented by HLA-A*0201. Peptides that bound with slow off-rates to class I alleles, that is HLA-A*0201, were associated with low frequency of CD8+ T cells in PBMCs from patients with tuberculosis. HLA-B alleles showed fast off-rates in peptide binding and restricted high numbers (up to 6%) of antigen-specific CD8+ T cells in patients with pulmonary tuberculosis.     

Analysis of the immune response of Hantaan virus nucleocapsid protein-specific CD8+ T cells in mice

The major histocompatibility complex (MHC) class-I restricted epitope of Hantaan virus nucleocapsid protein (N) was identified using overlapping peptides and BALB/c mice. Using the MHC tetramer derived from the epitope, we found that the level of N-specific CD8(+) T cells increased to approximately 20% of all antigen-specific CD8(+) T cells in a mouse model of transient infection. However, N-specific CD8(+) T cells were undetectable in a mouse model of persistent infection, both in the persistently infected phase and in the convalescent phase. Levels of CD8(+) T cells producing interferon-gamma were weak in both the acute and convalescent phases in the persistently infected model. These results indicate that hantavirus strongly suppresses the production of N-specific CD8(+) T cells throughout the course of infection in persistently infected mice. Moreover, N-specific CD8(+) T cells were not effective in recovering persistently infected mice, despite the existence of abundant N antigen in vivo.     

Selection of aberrant class II restricted CD8+ T cells in NOD mice expressing a glutamic acid decarboxylase (GAD)65-specific T cell receptor transgene

We previously described the generation of non-obese diabetic (NOD) mice expressing a transgenic T cell receptor (TCR) specific for peptide epitope 286-300 of the diabetes related self antigen, glutamic acid decarboxylase (GAD)65 in the context of I-A(g7) class II MHC, that are paradoxically protected from diabetes. In this report, we examine the atypical CD8+ cells in these mice. Unlike typical class II restricted TCR transgenic mice, GAD286 mice have normal numbers of CD8+ cells, half of which express high levels of the transgenic TCR. These MHC mismatched CD8+ cells persist in the periphery and proliferate to GAD286-300 peptide in vitro and in vivo in a class II restricted fashion. Interestingly, the CD8+ tetramer(-) T cells that are expressing endogenous TCR can delay diabetes induction in a transfer model, as we previously showed for CD4+ tetramer+ T cells in these mice. The MHC mismatched CD8+ cells appear to be positively selected in an atypical fashion, in that they do not upregulate CD69 or reexpress CD44, and they escape negative selection. We find that production of these CD8+ cells is not dependent on NOD thymus or high affinity of the TCR, but is dependent on the atypical TCR transgenic thymic environment.     

抗原特异性初始CD4+T细胞的体内分化及特性

为了探讨抗原特异性CD4+T细胞在体内的分裂、表型、Th1细胞因子的产生和组织器官的分布。将CFSE标记的抗原特异性初始CD4+T细胞静脉被动输给小鼠后,进行免疫,3d后处死小鼠取其脾脏、淋巴结和肺组织,分离单个核细胞,利用流式细胞计数仪在单个细胞水平上,观察细胞的分裂、表型、Th1细胞因子的产生和组织分布。结果显示在没有抗原刺激的情况下,未见初始CD4+T细胞分裂,其主要分布于淋巴结和脾脏。当受到抗原刺激后,CD4+T细胞分裂1～5次,主要分布于脾脏和肺组织,CD25的表达增加,CD62L的表达随着细胞分裂次数的增加而减少。IL-12促进CD25的表达和细胞的分裂。促进Th1细胞的分化和IFN-γ的表达。研究的结果提示,在体内,当CD4+T细胞活化后,主要分布于脾和非淋巴组织发挥其免疫效应。     

Correlation between CD8+ T cells specific for prostate-specific antigen and level of disease in patients with prostate cancer

Modest work has been performed to improve the sensitivity of residual disease detection or investigate the contribution that the immune system makes in controlling metastatic tumor growth, in particular, the frequency and biological actions of peptide-specific CD8+ T lymphocytes in limiting metastatic disease and/or maintaining remission. Fifty-three peripheral blood samples from 32 prostate cancer (PC) patients were investigated for the presence of circulating prostate-specific antigen (PSA)-expressing cells (CPECs) using a highly sensitive and specific assay combining immunomagnetic epithelial cell enrichment with nested RT-PCR of PSA mRNA. Using HLA-A2 tetramer complexes, frequency of CD8+ T cells specific for PSA-derived peptides was determined. Additionally, serum concentrations of PSA and testosterone were measured. CPECs were detected in 26% of peripheral blood samples from PC patients. CD8+ T cells specific for PSA-derived peptides were detected at low frequency in HLA-A2-positive PC patients. The correlation between these PSA-specific CD8+ T cells and residual prostate tumor cells and clinical measures was investigated. Our data suggest that frequency of PSA-specific CD8+ T cells is correlated to CPECs, but not to serum PSA level.     

Class II major histocompatibility complex tetramer staining: progress, problems, and prospects

The use of major histocompatibility complex (MHC) tetramers in the detection and analysis of antigen-specific T cells has become more widespread since its introduction 11 years ago. Early challenges in the application of tetramer staining to CD4+ T cells centred around difficulties in the expression of various class II MHC allelic variants and the detection of low-frequency T cells in mixed populations. As many of the technical obstacles to class II MHC tetramer staining have been overcome, the focus has returned to uncertainties concerning how oligomer valency and T-cell receptor/MHC affinity affect tetramer binding. Such issues have become more important with an increase in the number of studies relying on direct ex vivo analysis of antigen-specific CD4+ T cells. In this review we discuss which problems in class II MHC tetramer staining have been solved to date, and which matters remain to be considered.     

38 000 MW antigen-specific major histocompatibility complex class I restricted interferon-gamma-secreting CD8+ T cells in healthy contacts of tuberculosis.

CD8+ T lymphocytes are required to protect mice against Mycobacterium tuberculosis, although in early infection the mechanism appears not to be via perforin or granzyme-mediated lysis of the infected target, and may be via interferon-gamma (IFN-gamma) production. We therefore investigated whether CD8+ T cells specific for the immunoprotective 38 000 MW antigen of M. tuberculosis could be detected in infected humans. Using a recombinant vaccinia virus expressing the 38 000 MW antigen of M. tuberculosis (rV38) and a control vaccinia virus (rVras) we demonstrated that both viruses stimulated IFN-gamma production from freshly isolated peripheral blood mononuclear cells (PBMC) in a 36-hr enzyme-linked immunospot assay. Cell depletion and antibody blockade established that the bulk of the 38 000 MW antigen-specific IFN-gamma response was mediated by CD8+, major histocompatibility complex class I-restricted T cells, whereas the anti-vaccinia virus response was predominantly mediated by CD4+ T cells. In further evaluations PBMC from all seven healthy tuberculosis-exposed contacts had a 38 000 MW antigen-specific IFN-gamma response, whereas seven patients with untreated sputum-positive pulmonary tuberculosis had very low levels of 38 000 antigen-specific IFN-gamma-producing cells. These preliminary observations demonstrate the utility of recombinant vaccinia viruses in restimulating freshly isolated CD4+ and CD8+ T cells. The bias towards a higher frequency of IFN-gamma-producing CD8+ T cells in contacts rather than patients may indicate a protective role for CD8+ cells in human tuberculosis.     

A model of suppression of the antigen-specific CD4 T cell response by regulatory CD25+CD4 T cells in vivo

Despite intense recent interest, the suppressive mechanisms of regulatory CD25+CD4 T cells remain poorly understood. One deficiency in the field is the lack of in vivo models where the effects of regulatory CD25+CD4 T cells on antigen-specific responder T cells can be measured quantitatively. We describe one such model here. We compared responses of adoptively transferred naive wild-type antigen-specific CD4 T cells in syngeneic CD28-/- and wild-type recipient mice toward a nominal antigen. The cells exhibited a greater degree of proliferation and differentiation in CD28-/- mice and could not be rendered functionally hyporesponsive by systemic exposure to adjuvant-free antigen. The only reason we were able to find to explain this difference was the deficiency of regulatory CD25+CD4 T cells in the CD28-/- mice. Use of CD28-/- mice as adoptive transfer recipients provides a simple model that reveals the contribution of regulatory CD25+CD4 T cells in controlling antigen-driven responses in vivo.