Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages.
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CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2- to 5-fold between individuals. CCR5 was expressed at much higher levels in CD4+/CD45RO+/CD62L-true memory cells compared with CD4+/CD45RO+/CD62L+ cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitro maturation of PBDC resulted in median 3- and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from approximately 5,000 to approximately 50, 000 ABS) whereas granulocyte-macrophage colony-stimulating factor caused a marked decrease of CXCR4 (from approximately 5,000 ABS to <500) while up-regulating CCR5 expression (from approximately 5,000 to approximately 20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms. (+info)
Regulatory T cells in experimental allergic encephalomyelitis. I. Frequency and specificity analysis in normal and immune rats of a T cell subset that inhibits disease.
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We have shown previously that administration of myelin basic protein (MBP)-reactive T cells to naive Lewis rats induces not only autoimmune encephalomyelitis (EAE) but also a near total resistance to subsequent disease. By isolating the effector cells that are responsible for the resistance, we demonstrated that disease protection paralleled with increased numbers of a CD8+ regulatory T cell (RTC) subset and that co-injection of this RTC subset with encephalitogenic T cells aborted the pathogenic activity of the latter cells. Here, we show that a radio-sensitive splenic population of RTC also exists in naive rats that can be recruited and activated to inhibit the onset of secondary episodes of adoptive EAE. In co-transfer experiments, this protective RTC subpopulation can be isolated to neutralize the pathogenic activity of stimulatory MBP-reactive T cells in vivo. We show that the frequency of RTC with specificity for MBP-reactive T cells in naive rats is two orders of magnitude higher than the frequency of MBP-specific precursors, the activity of RTC increases substantially with age and RTC frequencies increase as a consequence of immunization with MBP-reactive cells lines. In specificity studies, we show that RTC isolated from naive rats and RTC from animals primed with one MBP-reactive cell line show cross-reactive responses to a variety of different MBP-reactive T cell lines. However, following repeated stimulation with a given MBP line, these RTC display a more limited, clonotypic response to the selecting line and assume a uniform CD8 phenotype. Finally, functional studies with RTC indicate that proliferative and lytic specificities do not necessarily correlate and that activated rat RTC are especially lytic for a Fas-sensitive murine cell line. (+info)
Peptide dependency of alloreactive CD4+ T cell responses.
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Alloreactivity, the capacity of a large number of T lymphocytes to react with foreign MHC molecules, represents the cellular basis for the rejection of tissue grafts. Although it was originally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells, studies in the MHC class I system have clearly demonstrated that MHC-bound peptides can influence this interaction. It remains unclear, however, whether peptides play an equally important role for the recognition of MHC class II molecules by alloreactive CD4+ T cells. Another issue that remains unresolved is the overall frequency of peptide-dependent versus peptide-independent alloreactive T cells. We have addressed these questions with antigen-presenting cells (APC) from H2-M mutant mice that predominantly express a single MHC class II-peptide complex, H2-Ab bound by a peptide (CLIP) derived from the class II-associated invariant chain. APC from these mice were used as targets and stimulators for alloreactive CD4+ T cells. Results demonstrated that the vast majority of CD4+ alloreactive T cells recognize MHC class II molecules in a peptide-dependent fashion. (+info)
In vivo elimination of viral superantigen-activated CD4+ T cells: apoptosis occurs at a distance from the activation site.
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Local injection of mouse mammary tumor virus (MMTV) induces a local immune response, with activation of viral superantigen (vSAG)-specific T cell subsets followed by their clonal deletion. We investigated the fate of vSAG-reactive T cells following footpad injection of MMTV(SW) to mice. Activated T cells accumulated in draining lymph nodes. However, we demonstrated that apoptosis did not occur at the activation site, on the contrary of what has been shown after bacterial SAG activation. Although activated T cells were already shown to have the capacity to migrate to the gut, the fate of gut homing cells remains unclear. We demonstrate that the number of vSAG-specific T cells activated in the periphery was increasing in the follicles of gut-associated lymphoid organs, together with the number of apoptotic cell clusters. These results strongly suggested that gut-associated lymphoid tissue was the specific graveyard for apoptotic vSAG-activated CD4 T cells. (+info)
Differential CD4/CD8 subset-specific expression of highly homologous rat Tcrb-V8 family members suggests a role of CDR2 and/or CDR4 (HV4) in MHC class-specific thymic selection.
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Different rat Tcrb haplotypes express either TCR beta variable segment (Tcrb-V) 8.2l or 8.4a. Both V segments bind the mAb R78 but differ by one conservative substitution (L14V) and clusters of two and four substitutions in the complementarity-determining region (CDR) 2 and CDR4 [hypervariable loop 4 (HV4)]. Independently of MHC alleles numbers of R78+ CD4+ cells are lower in Tcrb-V8.2l-expressing than in Tcrb-V8.4a-expressing strains. Expression of R78+ TCR during T cell development, analysis of backcross populations and generation of a Tcrb congenic strain [LEW.TCRB(AS)] define two mechanisms how Tcrb haplotypes affect the frequency of R78+ cells, one acting prior to thymic selection leading to up to 2-fold higher frequency of Tcrb-V8.4a versus Tcrb-V8.2l in unselected thymocytes and another occurring between the TCRlow and the CD4/CD8 single-positive stage. The latter leads to a 50% reduction of frequency of Tcrb-V8.4a CD8+ cells but not CD4+ cells and does not affect either subset of Tcrb-V8.2l cells. A comparison of rat classical class I MHC (RT1.A) sequences and current models of TCR-MHC-peptide interaction suggests that this reduction in frequency of Tcrb-V8.4a CD8 cells may be a consequence of differential selection of Tcrb-V8.2l versus Tcrb-V8.4a TCR by differential binding of CDR2beta to highly conserved areas of C-terminal parts of the alpha helices of class I MHC molecules. (+info)
Bovine gammadelta T-cell responses to the intracellular protozoan parasite Theileria parva.
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T cells bearing the gammadelta antigen receptor (gammadelta T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that gammadelta T cells are involved in immune responses against Theileria parva. gammadelta T cells isolated from peripheral blood mononuclear cells (PBMC) of T. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologous T. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of gammadelta T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the gammadelta T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the gammadelta T-cell lines and clones. These gammadelta T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of gammadelta T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine gammadelta T cells are activated by and lyse T. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion. (+info)
Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes.
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Gamma delta intraepithelial lymphocytes are thought to coordinate responses to pathogens that penetrate the epithelial barrier. To directly test this, mice were inoculated with Nocardia asteroides. At doses that were nonlethal for control mice, gamma delta-deficient mice became severely ill and died within 14 days. Histologic examination of these lungs demonstrated the presence of severe tissue damage and unimpeded bacterial growth in the gamma delta-deficient mice compared with neutrophilic lesions and clearance of the organism in control mice. Interestingly, ozone exposure that targets a comparable lung region also resulted in diffuse epithelial necrosis associated with a similar lack of neutrophil recruitment in gamma delta-deficient mice. These data demonstrate that gamma delta intraepithelial lymphocytes can protect the host from pathogenic and nonpathogenic insults by targeting the inflammatory response to epithelial necrosis. (+info)
Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal.
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After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonally expand and then delete to very low levels. Previously, it was shown that deletion can be prevented by coinjection of cytokines or proinflammatory agents such as adjuvants. Here, we demonstrate that ligation of 4-1BB blocks deletion of superantigen-activated T cells in the absence of adjuvant or additional cytokine treatment. Nearly 10 times as many staphylococcal enterotoxin A-specific T cells were detected in the spleens of mice injected 21 days previously with staphylococcal enterotoxin A and an agonist anti-4-1BB Ab compared with mice given staphylococcal enterotoxin A and a control IgG. Even though both CD4- and CD8-activated T cells expressed 4-1BB, a higher proportion of CD8 T cells were rescued compared CD4 T cells. These data suggest that although 4-1BB provides costimulation, it may also promote long-term T cell survival. (+info)