CD8(+) T cells secreting type 2 lymphokines are defective in protection against viral infection

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8(+) T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-gamma secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules.     

Imaging of lytic granule exocytosis in CD8+ cytotoxic T lymphocytes reveals a modified form of full fusion

Here we imaged the exocytosis of lytic granules from human CD8⁺ cytotoxic T lymphocytes using rapid total internal reflection microscopy, Lamp-1 tagged with mGFP to follow the fate of the lytic granule membrane, and granzyme A, granzyme B or serglycin tagged with mRFP to follow the fate of lytic granule cargo. Lytic granules were released by full fusion with the plasma membrane, such that the entire granule content for all three cargos visualized was released on a subsecond time scale. The behavior of GFP-Lamp-1 was, however, more complex. While it entered the plasma membrane in all cases, the extent to which it then diffused away from the site of exocytosis varied from nearly complete to highly restricted. Finally, the diffusion properties upon release of the three cargos examined put an upper limit on the size of the macromolecular complex of granzyme and serglycin that is presented to the target cell.     

Both CD4(+) T cells and CD8(+) T cells are required for iodine accelerated thyroiditis in NOD mice

The nonobese diabetic (NOD) mouse, a spontaneous animal model for insulin-dependent diabetes mellitus, displays a tendency in common with human diabetic populations to develop autoimmune thyroiditis although incidence and severity of thyroid lesions vary widely among different colonies around the world. A congenic strain of NOD mice bearing I-Ak on a NOD background (NOD-H2(h4)) has recently been derived and displays a much greater tendency to develop thyroiditis and autoantibodies to mouse thyroglobulin (MTg) although it is free of diabetes. Both thyroid infiltrates and autoantibody formation are accelerated and enhanced in NOD-H2(h4) mice by increased iodine intake. The effect of increased iodine intake on NOD mice themselves has not been directly investigated although a recent study of these animals given high or low doses of iodine showed no follicular destruction unless the mice were first rendered goitrous by iodine deprivation. We found that dietary iodine increased both the incidence and the severity of thyroid lesions in our NOD mice although autoantibodies to MTg were absent. NOD background genes appear to be essential for the development of these lesions, which were maximal after 4 weeks of iodine administration and showed no significant regression when the iodine was stopped. Furthermore, our studies show for the first time that both CD4(+) and CD8(+) T cells are necessary for the development of this accelerated but essentially spontaneous murine thyroid disease.     

CD4(+) T cells are required for the development of cytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection.

The control of acute and chronic Mycobacterium tuberculosis infection is dependent on CD4(+) T cells. In a variety of systems CD8(+) T cell effector responses are dependent on CD4(+) T cell help. The development of CD8(+) T cell-mediated immune responses in the absence of CD4(+) T cells was investigated in a murine model of acute tuberculosis. In vitro and in vivo, priming of mycobacteria-specific CD8(+) T cells was unaffected by the absence of CD4(+) T cells. Infiltration of CD8(+) T cells into infected lungs of CD4(-/-) or wild-type mice was similar. IFN-gamma production by lung CD8(+) T cells in CD4(-/-) and wild-type mice was also comparable, suggesting that emergence of IFN-gamma-producing mycobacteria-specific CD8(+) T cells in the lungs was independent of CD4(+) T cell help. In contrast, cytotoxic activity of CD8(+) T cells from lungs of M. tuberculosis-infected mice was impaired in CD4(-/-) mice. Expression of mRNA for IL-2 and IL-15, cytokines critical for the development of cytotoxic effector cells, was diminished in the lungs of M. tuberculosis-infected CD4(-/-) mice. As tuberculosis is frequently associated with HIV infection and a subsequent loss of CD4(+) T cells, understanding the interaction between CD4(+) and CD8(+) T cell subsets during the immune response to M. tuberculosis is imperative for the design of successful vaccination strategies.     

Antiretroviral cytolytic T-lymphocyte nonresponsiveness: FasL/Fas-mediated inhibition of CD4(+) and CD8(+) antiviral T cells by viral antigen-positive veto cells

C57BL/6 (H-2(b)) mice generate type-specific cytolytic T-lymphocyte (CTL) responses to an immunodominant Kb-restricted epitope, KSPWFTTL located in the membrane-spanning domain of p15TM of AKR/Gross murine leukemia viruses (MuLV). AKR.H-2(b) congenic mice, although carrying the responder H-2(b) major histocompatibility complex (MHC) haplotype, are low responders or nonresponders for AKR/Gross MuLV-specific CTL, apparently due to the presence of inhibitory AKR. H-2(b) cells. Despite their expression of viral antigens and Kb, untreated viable AKR.H-2(b) spleen cells cause dramatic inhibition of the C57BL/6 (B6) antiviral CTL response to in vitro stimulation with AKR/Gross MuLV-induced tumor cells. This inhibition is specific (AKR.H-2(b) modulator spleen cells do not inhibit allogeneic MHC or minor histocompatibility antigen-specific CTL production), dependent on direct contact of AKR.H-2(b) cells in a dose-dependent manner with the responder cell population, and not due to soluble factors. Here, the mechanism of inhibition of the antiviral CTL response is shown to depend on Fas/Fas-ligand interactions, implying an apoptotic effect on B6 responder cells. Although B6.gld (FasL-) responders were as sensitive to inhibition by AKR.H-2(b) modulator cells as were B6 responders, B6.lpr (Fas-) responders were largely insensitive to inhibition, indicating that the responder cells needed to express Fas. A Fas-Ig fusion protein, when added to the in vitro CTL stimulation cultures, relieved the inhibition caused by the AKR.H-2(b) cells if the primed responders were from either B6 or B6.gld mice, indicating that the inhibitory AKR.H-2(b) cells express FasL. Because of the antigen specificity of the inhibition, these results collectively implicate a FasL/Fas interaction mechanism: viral antigen-positive AKR.H-2(b) cells expressing FasL inhibit antiviral T cells ("veto" them) when the AKR.H-2(b) cells are recognized. Consistent with this model, inhibition by AKR.H-2(b) modulator cells was MHC restricted, and resulted in approximately a 10- to 70-fold decrease in the in vitro expansion of pCTL/CTL. Both CD8(+) CTL and CD4(+) Th responder cells were susceptible to inhibition by FasL+ AKR.H-2(b) inhibitory cells as the basis for inhibition. The CTL response in the presence of inhibitory cells could be restored by several cytokines or agents that have been shown by others to interfere with activation-induced cell death (e.g. , interleukin-2 [IL-2], IL-15, transforming growth factor beta, lipopolysaccharide, 9-cis-retinoic acid) but not others (e.g., tumor necrosis factor alpha). These results raise the possibility that this type of inhibitory mechanism is generalized as a common strategy for retrovirus infected cells to evade immune T-cell recognition.     

Vgamma4(+) T cells promote autoimmune CD8(+) cytolytic T-lymphocyte activation in coxsackievirus B3-induced myocarditis in mice: role for CD4(+) Th1 cells

T cells expressing the Vgamma4 T-cell receptor (TCR) promote myocarditis in coxsackievirus B3 (CVB3)-infected BALB/c mice. CD1, a major histocompatibility complex (MHC) class I-like molecule, is required for activation of Vgamma4(+) cells. Once activated, Vgamma4(+) cells initiate myocarditis through gamma interferon (IFN-gamma)-mediated induction of CD4(+) T helper type 1 (Th1) cells in the infected animal. These CD4(+) Th1 cells are required for activation of an autoimmune CD8(+) alphabeta TCR(+) effector, which is the predominant pathogenic agent in this model of CVB3-induced myocarditis. Activated Vgamma4(+) cells can adoptively transfer myocarditis into BALB/c mice infected with a nonmyocarditic variant of CVB3 (H310A1) but cannot transfer myocarditis into either uninfected or CD1(-/-) recipients, demonstrating the need for both infection and CD1 expression for Vgamma4(+) cell function. In contrast, CD8(+) alphabeta TCR(+) cells transfer myocarditis into either infected CD1(-/-) or uninfected recipients, showing that once activated, the CD8(+) alphabeta TCR(+) effectors function independently of both virus and CD1. Vgamma4(+) cells given to mice lacking CD4(+) T cells minimally activate the CD8(+) alphabeta TCR(+) cells. These studies show that Vgamma4(+) cells determine CVB3 pathogenicity by their ability to influence both the CD4(+) and CD8(+) adaptive immune response. Vgamma4(+) cells enhance CD4(+) Th1 (IFN-gamma(+)) cell activation through IFN-gamma- and CD1-dependent mechanisms. CD4(+) Th1 cells promote activation of the autoimmune CD8(+) alphabeta TCR(+) effectors.     

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4(+)CD25(+) T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.     

IFN-gamma promotes Fas ligand- and perforin-mediated liver cell destruction by cytotoxic CD8 T cells

To study liver cell damage by CTL, CD8 T cells from P14 TCR transgenic (tg) mice specific for the gp33 epitope of lymphocytic choriomeningitis virus with either deficiency in IFN-gamma (P14.IFN-gamma(null)), functional Fas ligand (P14.gld), or perforin (P14.PKO) were transferred into H8 tg mice ubiquitously expressing gp33 Ag. Treatment of H8 recipient mice with agonistic anti-CD40 Abs induced vigorous expansion of the transferred P14 T cells and led to liver cell destruction determined by increase of glutamate dehydrogenase serum levels and induction of caspase-3 in hepatocytes. Liver injury was mediated by the Fas/Fas ligand (FasL) pathway and by perforin, because P14.gld and P14.PKO T cells failed to induce increased glutamate dehydrogenase levels despite strong in vivo proliferation. In addition, H8 tg mice lacking Fas were resistant to the pathogenic effect of P14 T cells. Besides FasL and perforin, IFN-gamma was also required for liver cell damage, because P14.IFN-gamma(null) T cells adoptively transferred into H8 mice failed to induce disease. Moreover, Fas expression on hepatocytes from H8 recipient mice was increased after transfer of wild-type compared with P14.IFN-gamma(null) T cells, and wild-type P14 T cells expressed higher levels of FasL than P14 T cells lacking IFN-gamma. Thus, our data suggest that IFN-gamma released by activated CD8 T cells upon Ag contact facilitates liver cell destruction.     

Germinal center helper T cells are dual functional regulatory cells with suppressive activity to conventional CD4+ T cells

Germinal center (GC) reaction is a T cell-dependent process in which activated B cells mature to produce high-affinity Abs and differentiate into memory B cells. The GC microenvironment is almost exclusively reserved for the optimal Ag-specific B cell clonal expansion, selection, and maturation, but lack significant conventional CD4(+) T cell responses. The mechanisms that ensure such a focused B cell response in the GC are not known. In this study, we report that human CD4(+)CD57(+) T cells, which are the major helper T cells in GCs, actively suppress the activation of conventional CD4(+) T cells, particularly Th1 cells, via a direct contact-dependent mechanism and soluble mediators. Our findings demonstrate that GC T cells are unique regulatory cells that provide critical help signals for B cell response but suppress conventional effector T cells in the same local environment.     

Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity

NK cells possess both effector and regulatory activities that may be important during the antitumor immune response. In fact, the generation of antitumor immunity by the administration of an agonistic mAb against CD137 is NK cell-dependent. In this study, we report that NK cells could be induced by IL-2 and IL-15 to express CD137 and ligation of CD137-stimulated NK cell proliferation and IFN-gamma secretion, but not their cytolytic activity. Importantly, CD137-stimulated NK cells promoted the expansion of activated T cells in vitro, demonstrating immunoregulatory or "helper" activity for CD8(+)CTL. Furthermore, tumor-specific CTL activity against P815 tumor Ags was abrogated following anti-CD137 treatment in NK-depleted mice. We further demonstrate that CD137-stimulated helper NK cells expressed the high-affinity IL-2R and were hyperresponsive to IL-2. Taken together with previous findings that CD137 is a critical receptor for costimulation of T cells, our findings suggest that CD137 is a stimulatory receptor for NK cells involved in the crosstalk between innate and adaptive immunity.     

Expansions of clonal and oligoclonal T cells in B-cell chronic lymphocytic leukemia are primarily restricted to the CD3(+)CD8(+) T-cell population

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature-appearing clonal B cells exhibiting coexpression of CD5 and CD23. In addition to the accumulation of neoplastic B cells, numerous T-cell abnormalities also occur in B-CLL patients. In this study, the presence, and distribution within the T-cell subsets, of clonal/oligoclonal T cells was studied. Multicolor flow cytometric techniques were employed using combinations of anti-CD3, anti-CD4, and anti-CD8 antibodies coupled with antibodies specific for V(alpha) and V(beta) T-cell receptor (TCR) epitopes. Molecular studies of TCR gene sequences were done to confirm the presence of clonal/oligoclonal T-cell populations. In the flow cytometric studies, examination of V(alpha)/V(beta)expression found evidence of clonal/oligoclonal expansion in 9 of 19 patients studied. In eight of the nine patients, the expansions were restricted to the CD3(+)CD8(+) cell population. Molecular analyses were performed in 16 patients, 12 of whom showed a clonal or oligoclonal pattern. Of the four patients who were negative in the molecular analyses, all demonstrated flow cytometric evidence of clonal/oligoclonal expansions. Thus, when the flow cytometric and molecular analyses were considered together, all 16 patients for whom parallel analyses were done showed evidence of clonal/oligoclonal expansions. These results confirm previous work demonstrating that the majority of B-CLL patients harbor clonal/oligoclonal expansions within the T-cell population. Additionally, based on the relative numbers of cells expressing specific V(alpha) or V(beta)epitopes, these results show that these expansions occur primarily within the CD3(+)CD8(+) T-cell population.     

IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells

The glycoprotein CD86 is an important costimulatory molecule that has been shown to be predominantly expressed on APCs, such as dendritic cells, macrophages, and B cells. More recently, CD86 was also detected on T cells in specific pathological conditions. The mechanisms of how CD86 might be induced and its functional role in T cells are not well understood. In the present study, we showed that treatment with IL-2 markedly upregulated CD86, but not CD80, in human CD4(+) and CD8(+) T cells. This upregulation occurred in the absence of bystander cells, and isolated naive CD4(+) or CD8(+) T cells exhibited different time-dependent CD86-expression patterns in response to IL-2. Upregulation of CD86 on activated T cells was reduced by Abs that block IL-2 and IL-2Rα (CD25), indicating a receptor-mediated mechanism. IL-2-dependent CD86 upregulation was blocked by pharmacological inhibitors of the NFAT and mammalian target of rapamycin pathways and was largely reduced by simultaneous exposure to IFN-α. Importantly, a marked increase in CD86 on T cells was also observed in vivo in IL-2-treated patients. In conclusion, IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells via a receptor-dependent mechanism that involves the NFAT and mammalian target of rapamycin pathways.     

Pathogenesis of murine gammaherpesvirus infection in mice deficient in CD4 and CD8 T cells.

Murine gammaherpesvirus is a natural pathogen of wild mice. The virus infects alveolar cells and spleen cells during the primary infection and establishes a latent/persistent infection in B lymphocytes. Little is known about the immunological response to gammaherpesviruses during a primary infection. To address this issue, we investigated the pathogenesis of murine herpesvirus 68 (MHV-68) infection in mice deficient in CD4 or CD8 T-cell populations. Infection of the lung and spleen were greatly exacerbated in CD8-deficient mice, reflected by elevated virus titers in the lung and an increase in the number of infected splenocytes located around germinal centers. This finding contrasts with clearance of virus from the lung and spleen by day 12 postinfection in CD4-depleted animals. These data clearly indicate a major role for CD8 T cells in recovery from an acute MHV-68 infection. Whereas CD4 T cells fail to influence the course of infection in the lung, they do contribute to lymphoproliferation seen in the spleen (splenomegaly) during the primary infection. The significance of these results are discussed in relation to the immune response to other herpesviruses, in particular Epstein-Barr virus, with which MHV-68 shares similar molecular and biological properties.     

Role of CD8(+)T cells in the host response to Chlamydia

Chlamydia infections constitute a major public health problem. Although multiple arms of the immune system participate in the control of Chlamydia in infected hosts, T lymphocytes are essential. This review focuses on the roles that CD8(+)T cells may play in immunoprotection and immunopathology following recognition of Chlamydia-infected cells.     

Regulation of IFN-gamma signaling is essential for the cytotoxic activity of CD8(+) T cells.

Previous studies have demonstrated that, as naive murine CD4(+) cells differentiate into Th1 cells, they lose expression of the second chain of IFN-gammaR (IFN-gammaR2). Hence, the IFN-gamma-producing subset of Th cells is unresponsive to IFN-gamma. Analysis of IFN-gamma-producing CD8(+) T cells demonstrates that, like Th1 cells, these cells do not express IFN-gammaR2. To define the importance of IFN-gamma signaling for the development of functional CD8(+) T cells, mice either lacking IFN-gammaR2 or overexpressing this protein were examined. While CD8(+) T cell development and function appear normal in IFN-gammaR2(-/-) mice, CD8(+) T cell function in IFN-gammaR2 transgenic is altered. IFN-gammaR2 transgenic CD8(+) T cells are unable to lyse target cells in vitro. However, these cells produce Fas ligand, perforin, and granzyme B, the effector molecules required for killing. Interestingly, TG CD8(+) T cells proliferate normally and produce cytokines, such as IFN-gamma in response to antigenic stimulation. Therefore, although IFN-gamma signaling is not required for the generation of normal cytotoxic T cells, constitutive IFN-gamma signaling can selectively impair the cytotoxic function of CD8(+) T cells.     

Double-negative (CD4- CD8-) T cells with an alpha/beta T cell receptor. Non-MHC-restricted cytolytic activity and lymphokine production

T cell lines with a novel phenotype (CD3+ TCR-alpha/beta+ CD4- CD8-) were developed from the peripheral blood of a patient with a combined immunodeficiency and tissue injury resembling graft-vs-host disease. One of these IL-2-dependent T cell lines demonstrated non-MHC-restricted cytolytic function against tumor targets, syngeneic and allogeneic fibroblasts, and PHA blasts from allogeneic donors. The other cell line only became cytotoxic in the presence of lectin or anti-CD3 antibody. The two cell lines also differed in their expression of the T-200 gene products CD45RO (gp180) and CD45RA (gp220). Both cell lines produced tumor necrosis factor-alpha and -beta and IFN-gamma activity when activated with mitogens or PMA and IL-1. The in vitro functions of these T-cell lines suggest a potential role for alpha/beta double-negative T lymphocytes in tissue injury resembling graft-vs-host disease.     

CD4(+) T regulatory cells are more resistant to DNA damage compared to CD4(+) T effector cells as revealed by flow cytometric analysis

A number of apoptotic stimuli produce a different response by CD4(+) regulatory and effector lymphocytes. So far, little is known concerning the sensitivity of CD4(+) regulatory T cells (Treg) to genotoxic agents. Observations from a mouse model suggest that Treg are more resistant to DNA damage compared to CD4(+) T effector cells (Teff). By flow cytometry we analysed the apoptotic response to genotoxic stimuli in culture, comparing Treg and Teff. CD4(+) regulatory lymphocytes appeared to be more resistant than CD4(+) effector lymphocytes. Results of costaining experiments for CD45RA suggest that this dissimilarity is not related to the differentiation to a CD45RA negative phenotype. Further, neither the antiapoptotic protein Bcl-2 nor Bcl-xL were found to be expressed in greater amounts by Treg compared to Teff. The differential sensitivity of Treg and Teff to DNA-damage inducing agents may be of clinical relevance in cancer therapy. ? 2011 International Society for Advancement of Cytometry.     

Dendritic cells are sufficient to cross-present self-antigens to CD8 T cells in vivo

The mechanism of cross-presentation enables professional APCs to induce CD8 T cell-mediated immune responses against exogenous Ags. Through this mechanism, APCs can induce either immunity against infectious pathogens or tolerance against self-Ag residing in extralymphatic locations. An unanswered question in this field concerns the identity of the cross-presenting APC. All major classes of professional APCs, particularly dendritic cells, macrophages, and B cells, have previously been shown to be able to cross-present Ags in vitro. In the present study, we have created transgenic mice where MHC class I expression is driven selectively in dendritic cells and provide direct in vivo evidence that dendritic cells are sufficient to cross-present exogenous self-Ags and induce Ag-specific cell division of CD8-positive T cells.