Switching from a restricted to an effective CD4 T cell response by activating CD8+ murine dendritic cells with a Toll-like receptor 9 ligand

Freshly isolated quiescent splenic dendritic cell (DC) subtypes differ in their capacity to activate naive CD4 T cells in culture. The CD8+ DC showed a reduced capacity to stimulate T cell proliferation compared to either of the CD8- DC subsets, regardless of antigen and DC dose. In contrast to CD8- DC, the quiescent CD8+ DC did not induce IFN-gamma production from CD4 T cells. The difference between the DC subtypes appeared to be at the level of initial surface molecule interactions, but could not be attributed to differences in expression of MHC class II or B7 family molecules, or to the expression of Fas ligand on DC. However, when activated by inclusion of the Toll-like receptor 9 ligand CpG in culture, CD8+ DC became potent stimulators of both CD4 T cell proliferation and IFN-gamma production. In contrast, similar activation of CD8- DC produced a more modest increase in capacity to stimulate CD4 T cell proliferation and no increase in capacity to stimulate IFN-gamma production. The difference between a quiescent and an activated state is therefore more extreme for CD8+ than for CD8- DC. The especially tight regulation of the activity of CD8+ DC may be essential for the maintenance of self tolerance.     

CD4+ effector T cell distribution in vivo: TGF-beta 1/TGF-beta receptor II interaction during activation mediates accumulation in the target tissue by preferential proliferation

CD4(+) effector T cells generated in mesenteric lymph nodes (mLN) leave into the blood. Although they enter many tissues, mLN effector T cells are later found preferentially in the gut,the drainage area of mLN. We show in the rat that this is not an intrinsic property of mLN T cells. Instead, within the mLN milieu T cells are instructed by cytokines such as transforming growth factor beta 1 (TGF-beta 1) to up-regulate TGF-beta receptor II (TGF-beta RII) during activation. This enables effector T cells to continue proliferation upon subsequent contact with TGF-beta 1 in mLN and gut, and to accumulate in the lymph node draining area, the most likely site of pathogen invasion.     

Characterization of H4: a mouse T lymphocyte activation molecule functionally associated with the CD3/T cell receptor

The monoclonal antibody C398.4A was produced by immunizing Armenian hamsters with the mouse T cell clone D10.G4.1. It recognizes a molecule selectively expressed by activated mouse T cells and was named H4. H4 is expressed on the T cell surface about 24 h after activation and peaks at day 7. By contrast, it is not expressed by resting or activated B cells, macrophages, or fibroblasts. It is also expressed by CD4 or CD8 single-positive mature thymocytes. Immunoprecipitation showed that H4 is a disulfide-linked dimer, precipitating as a broad band at about 50–65 kDa under nonreducing conditions and at 25 and 29 kDa under reducing conditions. Deglycosylation of the reduced H4 by N-glycanase gave rise to a single band of about 21 kDa, suggesting that the two chains may be differentially glycosylated forms of the same protein. The H4 expression pattern and biochemical features, together with cross-blocking, co-capping, co-modulation, and immunoprecipitation preclearing experiments showed that H4 is different from other known co-stimulatory molecules such as CD69, CD2, Ly-6, CD25, OX-40, Mac-1 and LFA-1. By in vitro kinase assay, H4 was found to co-precipitate a tyrosine kinase activity that phosphorylated substrates of about 29 and 25 kDa. Co-modulation and co-capping experiments showed that H4 is physically associated with the CD3/T cell receptor. These data suggest that H4 may function as a T cell-specific co-stimulatory molecule and play a role in the T cell response when the activation stimulus is limited either because the antigen is only available in low concentration or has a low agonistic activity.     

JNK2 negatively regulates CD8+ T cell effector function and anti-tumor immune response

JNK1 and JNK2 have distinct effects on activation, differentiation and function of CD8+ T cells. Our early studies demonstrated that JNK1 is required for CD8+ T cell-mediated tumor immune surveillance. However, the role of JNK2 in CD8+ T cell response and effector functions, especially in anti-tumor immune response, is unknown. To define the role of JNK2 in antigen-specific immune response, we have investigated CD8+ T cells from OT-1 CD8+ transgenic mice in response to either high- or low-affinity peptides. JNK2-/- CD8+ T cells proliferated better in response to both peptides, with more cell division and less cell death. In addition, JNK2-/- CD8+ T cells produced higher levels of IFN-gamma, which is associated with increased expression of T-bet and Eomesodermin (Eomes). Moreover, JNK2-/- CD8+ T cells expresses high levels of granzyme B and show increased CTL activity. Finally, the enhanced expansion and effector function of JNK2-/- CD8+ T cells was further evidenced by their capacity to delay tumor growth in vivo. In summary, our results demonstrate that JNK2 negatively regulates antigen-specific CD8+ T cell expansion and effector function, and thus selectively blocking JNK2 in CD8+ T cells may potentially enhance anti-tumor immune response.     

Human CD4+ T cell differentiation and effector function

Human CD4+ memory T cells progress through stages of postthymic differentiation that have been characterized by distinct phenotypes. We have investigated the factors regulating cytokine production, and the correlation between phenotype and effector function in normal and autoimmune individuals. These studies suggest that antigen-induced proliferation in the periphery drives CD4+ T cells through successive stages of differentiation that culminate in optimal effector function and resistance to external modulatory influences. Moreover, these studies support the concept that in autoimmune individuals, the chronic accumulation of differentiated proinflammatory T cells perpetuate the inflammatory response resulting in aggressive disease.     

In vitro model for the activation of CD4 and CD8 T cell receptors

Previously, most models that sought to explain the misregulation of immune cell function assumed molecular similarities between the disease-causing pathogens and the host's proteins. In recent time several different models have been proposed and in this study, these concepts are compared to a new hypothesis proposing another explanation for this immune dysregulation: the possibility that the mislocalization of proteins may be responsible for autoimmune activity. Based on this hypothesis, proteins are recognized as self or non-self depending on where they appear in sufficiently high concentrations. To examine this new idea, the intracellular human proteins β-actin, GAPDH, and hemoglobin as well as the extracellular human proteins insulin and albumin, were added to human whole blood samples. After an incubation period, the activation of whole-blood T lymphocytes in the samples was measured. The observed activation pattern of the T lymphocytes fit well with the proposed hypothesis. Therefore, these data suggest that protein mislocalization and/or errors within protein trafficking might be important in the development of autoimmune diseases.     

CD28-mediated induction of proliferation in resting T cells in vitro and in vivo without engagement of the T cell receptor: Evidence for functionally distinct forms of CD28

JJ316 and JJ319 are rat CD28-specific monoclonal antibodies (mAb) of the γ1χ isotype with identical co-stimulatory potency. At a concentration 100–1000-fold higher than that required for co-stimulation, JJ316, but not JJ319 induces massive proliferation of all T cell subsets in vitro without T cell receptor (TCR) triggering. “Direct” stimulation by JJ316 is fully blocked by JJ319, indicating that it is not due to cross-reactivity of JJ316 with the TCR complex or other activating receptors. JJ316 binds much more slowly to primary T cells than JJ319, whereas both antibodies bind with similar kinetics to CD28-transfected L-929 cells, suggesting that JJ316 binding to T cells requires redistribution or a conformational change of CD28. In vivo, JJ316 but not JJ319 induces rapid and transient proliferation of most CD4 T cells and, indirectly, of B cells. These data show that TCR engagement is not an absolute prerequisite either in vitro or in vivo for the induction of T cell proliferation through CD28 and suggest that mAb JJ316 is able to stimulate resting T cells directly by recruiting CD28 molecules from an inactive to an active form.     

Expression and co-stimulatory function of B7-2 on murine CD4+ T cells

Co-stimulatory signals mediated by the interaction of B7-1/B7-2 with CD28 are important for the activation of CD4⁺ T cells stimulated with antigen on antigen-presenting cells. There are controversies about the expression and function of B7-1/B7-2 on CD4⁺ T cells. The aim of this study was to analyze the expression of B7-1/B7-2 on naive and memory CD4⁺ T cells and the co-stimulatory function in the activation of naive CD4⁺ T cells stimulated by TCR ligation. Present results indicate that memory CD4⁺ T cells express B7-2 molecules on their surface, whereas naive CD4⁺ T cells do not. Neither memory nor naive CD4⁺ T cells expressed B7-1 molecule on their surface, although B7-1 mRNA was faintly expressed in memory T cells. B7-2 molecules expressed on memory T cells co-stimulated CD4⁺ naive T cells stimulated with plate-coated anti-CD3 to produce IL-2. Naive CD4⁺ T cells were shown to express B7-2 after co-stimulation with B7-2 and TCR ligation, because the naive T cells stimulated with anti-CD3 and B7-2CHO expressed B7-2 on their surface, although it remained to be studied whether the co-stimulation with B7-2 directly induced B7-2 expression on naive T cells. Our present results indicate that memory CD4⁺ T cells play some role in the activation of naive CD4⁺ T cells through the co-stimulation with B7-2 molecules.     

Proximal signaling control of human effector CD4 T cell function

The functional coupling of T cell receptor (TCR)-mediated signaling events in primary human T cells remains undefined. We demonstrate here that alterations in the expression of proximal TCR-coupled signaling subunits are associated with distinct effector capacities in differentiated human CD4 T cells. Analysis of proximal signaling profiles using biochemical and single cell approaches reveals decreased CD3zeta and ZAP-70 expression correlating with functional anergy, with increased CD3zeta/ ZAP-70 expression and phosphorylation connoting acquisition of effector capacity. By contrast, the FcRgamma signaling subunit known to be expressed in human effector cells and in T cells from the autoimmune disease SLE is up-regulated upon activation, yet does not correlate with functional capacity in effector cells, and does not alter signaling or function in primary FcRgamma transfectants. Our results have implications for targeting signaling molecules in immunotherapy and evaluating the functional consequence of signaling alterations associated with autoimmunity and chronic diseases.     

Regulation of 2B4 (CD244)-mediated NK cell activation by ligand-induced receptor modulation

Natural killer (NK) cell activity can be stimulated by different surface receptors. 2B4 is a member of the signaling lymphocyte activation molecule (SLAM)-related receptor family and is important for stimulating human NK cell cytotoxicity and cytokine production. Here we show that stimulation of human NK cells by antibody-mediated 2B4 cross-linking or incubation with target cells expressing the 2B4 ligand CD48 results in a strong down-modulation of 2B4 surface expression. This down-modulation is observed in NK cell lines, purified human NK cells and NK cell clones, and is accompanied by an internalization of 2B4. The modulation of 2B4 is dependent on the activity of Src-family kinases, but independent of PI3 K activity or actin polymerization. Inhibitory receptors can interfere with 2B4-mediated signals and NK cell activation. However, co-engagement of inhibitory killer cell Ig-like receptors has no influence on the down-modulation of 2B4. This suggests that the modulation of 2B4 expression is independent of inhibitory receptors. The lower surface expression of 2B4 after ligand-induced down-modulation results in reduced 2B4-mediated NK cell activation and cytotoxicity. The modulation of activating surface receptors may therefore be another mechanism for the fine-tuning of NK cell activity and may lead to the adaptation of NK cell cytotoxicity in tissues with high ligand expression.     

T cell antigen receptor engagement abrogates CD4-mediated T cell deletion in vivo

We have previously shown that the engagement of CD4 by specificantibody in the mouse initiates a T cell apoptosis responsewith the following features: spleen and lymph node CD4⁺ T cellsmigrate into the bloodstream within minutes of anti-CD4 administrationwhere they exhibit the phenotype of null cells. If they arecapable of expressing functional Fas protein on their surfacethey degrade their DNA and disintegrate rapidly. We show herethat the engagement of the T cell antigen receptor blocks theCD4-medlated deletion process in mouse. Anti-CD4-reactive Tcells avoid the exodus into the bloodstream when their TCR Isengaged by anti-CD3 or by a superantigen, do not modulate surfacereceptors and are not deleted. In contrast to the apoptoslsinducingCD4-specific antibody which causes migration of lymphocytesfrom lymphoid organs into the blood stream, the T cell-activatingCD3-specific antibody causes lymphoid cell redistribution inthe opposite direction, from the bloodstream to lymphoid organs.The TCR-mediated protection of T cells against CD4-mediateddeletion lasts for several hours but ceases before the T cellsbecome blasts.     

Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.     

The effector to memory transition of CD4 T cells

The small number of antigen-specific memory CD4 T cells surviving long-term after antigen or pathogen challenge are often characterized by a surprising degree of phenotypic and functional heterogeneity. We here propose that the immune system has evolved to express this diversity in memory T-cell populations, in order to provide flexibility in recall responses, via a rapid transition from heterogeneous effector cells into correspondingly heterogeneous memory cells. Little attention has been paid to another important transition—from resting memory cell to re-activated effector. We would suggest that superior functional attributes of secondary effectors arising from memory CD4 T cells, as compared to primary effectors arising from naïve precursors, play an important and underappreciated role in protective secondary immune responses.     

Mycobacterium tuberculosis lipoproteins directly regulate human memory CD4(+) T cell activation via Toll-like receptors 1 and 2

The success of Mycobacterium tuberculosis as a pathogen relies on its ability to regulate the host immune response. M. tuberculosis can manipulate adaptive T cell responses indirectly by modulating antigen-presenting cell (APC) function or by directly interacting with T cells. Little is known about the role of M. tuberculosis molecules in direct regulation of T cell function. Using a biochemical approach, we identified lipoproteins LprG and LpqH as major molecules in M. tuberculosis lysate responsible for costimulation of primary human CD4(+) T cells. In the absence of APCs, activation of memory CD4(+) T cells with LprG or LpqH in combination with anti-CD3 antibody induces Th1 cytokine secretion and cellular proliferation. Lipoprotein-induced T cell costimulation was inhibited by blocking antibodies to Toll-like receptor 2 (TLR2) and TLR1, indicating that human CD4(+) T cells can use TLR2/TLR1 heterodimers to directly respond to M. tuberculosis products. M. tuberculosis lipoproteins induced NF-κB activation in CD4(+) T cells in the absence of TCR co-engagement. Thus, TLR2/TLR1 engagement alone by M. tuberculosis lipoprotein triggered intracellular signaling, but upregulation of cytokine production and proliferation required co-engagement of the TCR. In conclusion, our results demonstrate that M. tuberculosis lipoproteins LprG and LpqH participate in the regulation of adaptive immunity not only by inducing cytokine secretion and costimulatory molecules in innate immune cells but also through directly regulating the activation of memory T lymphocytes.     

Characterisation of the expression and function of the GM-CSF receptor alpha-chain in mice

The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine able to regulate a variety of cell functions including differentiation of macrophages and granulocytes, dendritic cell development and the maintenance of homeostasis. It binds specifically to its receptor, which is composed of a cytokine-specific alpha-chain (GM-CSF receptor alpha-chain, GMRalpha) and a beta-chain shared with the receptors for interleukin-3 and interleukin-5. In this report, we present a comprehensive study of GMRalpha in the mouse. We have found that the mouse GMRalpha is polymorphic and alternatively spliced. In the absence of specific antibodies, we generated a novel chimeric protein containing the Fc fragment of human IgG1 coupled to mouse GM-CSF, which was able to specifically bind to GMRalpha and induce proliferation of GMRalpha-transduced Ba/F3 cells. We used this reagent to perform the first detailed FACS study of the surface expression of mouse GMRalpha by leucocytes. Highest expression was found on monocytes and granulocytes, and variable expression on tissue macrophages. The GM-CSF receptor in mice is specifically expressed by myeloid cells and is useful for the detection of novel uncharacterised myeloid populations. The ability to detect GM-CSF receptor expression in experimental studies should greatly facilitate the analysis of its role in immune pathologies.     

Lipopolysaccharide enhances in vivo interleukin-2 production and proliferation by naive antigen-specific CD4 T cells via a Toll-like receptor 4-dependent mechanism

Microbial adjuvants are essential for the development of T-cell-dependent antibody production, recall T-cell proliferation and interferon-gamma production following immunization with protein antigens. Using an adoptive transfer approach, we showed that the adjuvant lipopolysaccharide enhanced the frequency of cells producing interleukin-2, enhanced clonal expansion by antigen-specific CD4 T cells and increased CD86 and interleukin-1alpha production by antigen-presenting cells. All of these effects were dependent on Toll-like receptor-4 (TLR4) expression by cells other than the antigen-specific CD4 T cells. The ability of lipopolysaccharides to increase the number of antigen-specific CD4 T cells that survive after immunization probably explains the previous finding that antigen-specific proliferation by T cells from normal mice depends on previous exposure to antigen and adjuvant.     

CD4-mediated functional activation of human CD4+CD25+ regulatory T cells

Naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells (CD25(+) Tregs) constitute a specialized population of T cells that is essential for the maintenance of peripheral self-tolerance. The immune regulatory function of CD25(+) Tregs depends upon their activation. We found that anti-CD4 antibodies activate the suppressive function of human CD25(+) Tregs in a dose-dependent manner. We demonstrate that CD4-activated CD25(+) Tregs suppress the proliferation of CD4(+) and CD8(+) T cells, their IL-2 and IFN-gamma production as well as the capacity of CD8(+) T cells to re-express CD25. By contrast, anti-CD4 stimulation did not induce suppressive activity in conventional CD4(+) T cells. These results identify CD4 as a trigger for the suppressive function of CD25(+) Tregs and suggest a possible CD4-mediated exploitation of these cells.     

Functional analysis of CD82 in the early phase of T cell activation: roles in cell adhesion and signal transduction

To define T cell co-stimulatory molecules that work in the early phase of T cell activation, we established monoclonal antibodies (mAb) that inhibit or enhance T cell activation by the histiocytic leukemia cell line U937. One of the mAb, 53H5, which recognized both T cells and U937, was identified to bind to CD82 by expression cloning. Functional analyses of CD82 revealed that 1) CD82 needs to exist on both T cells and U937 for the full activation of T cells; 2) CD82 expression is up-regulated on both T cells and U937 by stimulation such as CD3 ligation or treatment with phorbol 12-myristate 13-acetate; 3) overexpression of CD82 enhances both homotypic and heterotypic cell adhesion betweenT cells and U937; 4) CD82 signal co-stimulates T cells and the signal works synergistically with the CD28-mediated T cell co-stimulation signal; 5) in mixed leukocyte reactions using U937 as stimulator cells, CD82 overexpression on U937 correlates with the higher allogeneicity of U937 cells. These results indicate that CD82 co-stimulates T cells not only by sending intra-T cell signals that work synergistically with CD28 signals but also by inducing enhanced T cell-antigen-presenting cell interaction.