CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.     

Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells

We recently identified CD8+CD122+ regulatory T cells that directly control CD8+ and CD4+ cells without intervention of APCs. In this study, we investigated the effector mechanism of CD8+CD122+ regulatory T cells by using an in vitro regulation system. The profile of cytokine expression revealed that IL-10 was predominantly produced by CD8+CD122+ cells, whereas other cytokines were similarly expressed in CD8+CD122+ cells and CD8+CD122- cells. Suppression of both proliferation and IFN-gamma production by CD8+CD122- cells by CD8+CD122+ cells was blocked by adding anti-IL-10 Ab to the culture but not by adding anti-TGF-beta Ab. When IL-10 was removed from the conditioned medium from CD8+CD122+ cells, the conditioned medium no longer showed regulatory activity. Finally, CD8+CD122+ cells from IL-10-deficient mice had no regulatory activity in vitro and reduced regulatory activity in vivo. Our results clearly indicate that IL-10 is produced by CD8+CD122+ cells and mediates the regulatory activity of these cells.     

IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response

Immunization with serological identification of Ags by recombinant expression cloning (SEREX)-defined self-Ags leads to generation/activation of CD4+ CD25+ regulatory T cells with suppressive activities and enhanced expression of Foxp3. This is associated with increased susceptibility to pulmonary metastasis following challenge with syngeneic tumor cells and enhanced development of 3-methylcholanthrene-induced primary tumors. In contrast, coimmunization with the same SEREX-defined self-Ags mixed with a CTL epitope results in augmented CTL activity and heightened resistance to pulmonary metastasis, both of which depend on CD4+ Th cells. These active regulatory T cells and Th cells were derived from two distinct CD4+ T cell subsets, CD4+ CD25+ T cells and CD4+ CD25- T cells, respectively. In the present study, IFN-gamma was found to abrogate the generation/activation of CD4+ CD25+ regulatory T cells by immunization with SEREX-defined self-Ag. CD4+ CD25+ T cells from these IFN-gamma-treated mice failed to exhibit immunosuppressive activity as measured by 1) increased number of pulmonary metastasis, 2) enhanced development of 3-methylcholanthrene-induced primary tumors, 3) suppression of peptide-specific T cell proliferation, and 4) enhanced expression of Foxp3. The important role of IFN-gamma produced by CD8+ T cells was shown in experiments demonstrating that CD4+ CD25+ T cells cotransferred with CD8+ T cells from IFN-gamma(-/-) mice, but not from wild-type BALB/c mice, became immunosuppressive and enhanced pulmonary metastasis when recipient animals were subsequently immunized with a SEREX-defined self-Ag and a CTL epitope. These findings support the idea that IFN-gamma regulates the generation/activation of CD4+ CD25+ regulatory T cells.     

Dynamic regulation of IFN-gamma signaling in antigen-specific CD8+ T cells responding to infection

IFN-gamma plays a critical role in the CD8(+) T cell response to infection, but when and if this cytokine directly signals CD8(+) T cells during an immune response is unknown. We show that naive Ag-specific CD8(+) T cells receive IFN-gamma signals within 12 h after in vivo infection with Listeria monocytogenes and then become unresponsive to IFN-gamma throughout the ensuing Ag-driven expansion phase. Ag-specific CD8(+) T cells regain partial IFN-gamma responsiveness throughout the contraction phase, whereas the memory pool exhibits uniform, but reduced, responsiveness that is also modulated during the secondary response. The responsiveness of Ag-specific CD8(+) T cells to IFN-gamma correlated with modulation in the expression of IFN-gammaR2, but not with IFN-gammaR1 or suppressor of cytokine signaling-1. This dynamic regulation suggests that early IFN-gamma signals participate in regulation of the primary CD8(+) T cell response program, but that evading or minimizing IFN-gamma signals during expansion and the memory phase may contribute to appropriate regulation of the CD8(+) T cell response.     

Memory CD8+ T cells provide an early source of IFN-gamma

During the non-Ag-specific early phase of infection, IFN-gamma is believed to be primarily provided by NK and NKT cells in response to pathogen-derived inflammatory mediators. To test whether other cell types were involved in early IFN-gamma release, IFN-gamma-producing cells were visualized in spleens and lymph nodes of LPS-injected mice. In addition to NK and NKT cells, IFN-gamma was also detected in a significant fraction of CD8(+) T cells. CD8(+) T cells represented the second major population of IFN-gamma-producing cells in the spleen ( approximately 30%) and the majority of IFN-gamma(+) cells in the lymph nodes ( approximately 70%). LPS-induced IFN-gamma production by CD8(+) T cells was MHC class I independent and was restricted to CD44(high) (memory phenotype) cells. Experiments performed with C3H/HeJ (LPS-nonresponder) mice suggested that CD8(+) T cells responded to LPS indirectly through macrophage/dendritic cell-derived IFN-alpha/beta, IL-12, and IL-18. IFN-gamma was also detected in memory CD8(+) T cells from mice injected with type I IFN or with poly(I:C), a synthetic dsRNA that mimics early activation by RNA viruses. Taken together, these results suggest that in response to bacterial and viral products, memory T cells may contribute to innate immunity by providing an early non-Ag-specific source of IFN-gamma.     

Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN-gamma production

Although CD8+ IL-2Rbeta (CD122)+ T cells with intermediate TCR reportedly develop extrathymically, their functions still remain largely unknown. In the present study, we characterized the function of CD8+ CD122+ T cells with intermediate TCR of C57BL/6 mice. The proportion of CD8+ CD122+ T cells in splenocytes gradually increased with age, whereas CD8+ IL-2Rbeta-negative or -low (CD122-) T cells conversely decreased. The IFN-gamma production from splenocytes stimulated with immobilized anti-CD3 Ab in vitro increased with age, whereas the IL-4 production decreased. When sorted CD8+ CD122+ T cells were stimulated in vitro by the anti-CD3 Ab, they promptly produced a much larger amount of IFN-gamma than did CD8+ CD122- T cells or CD4+ T cells, whereas only CD4+ T cells produced IL-4. The depletion of CD8+ CD122+ T cells from whole splenocytes greatly decreased the CD3-stimulated IFN-gamma production and increased the IL-4 production, whereas the addition of sorted CD8+ CD122+ T cells to CD8+ CD122+ T cell-depleted splenocytes restored the IFN-gamma production and partially decreased IL-4 production. It is of interest that CD8+ CD122+ T cells stimulated CD4+ T cells to produce IFN-gamma. The CD3-stimulated IFN-gamma production from each T cell subset was augmented by macrophages. Furthermore, CD3-stimulated CD8+ CD122+ T cells produced an even greater amount of IFN-gamma than did liver NK1.1+ T cells and also showed antitumor cytotoxicity. These results show that CD8+ CD122+ T cells may thus be an important source of early IFN-gamma production and are suggested to be involved in the immunological changes with aging.     

IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.     

Sequential activation of CD8+ T cells in the draining lymph nodes in response to pulmonary virus infection

We have used a TCR-transgenic CD8+ T cell adoptive transfer model to examine the tempo of T cell activation and proliferation in the draining lymph nodes (DLN) in response to respiratory virus infection. The T cell response in the DLN differed for mice infected with different type A influenza strains with the onset of T cell activation/proliferation to the A/JAPAN virus infection preceding the A/PR8 response by 12-24 h. This difference in T cell activation/proliferation correlated with the tempo of accelerated respiratory DC (RDC) migration from the infected lungs to the DLN in response to influenza virus infection, with the migrant RDC responding to the A/JAPAN infection exhibiting a more rapid accumulation in the lymph nodes (i.e., peak migration for A/JAPAN at 18 h, A/PR8 at 24-36 h). Furthermore, in vivo administration of blocking anti-CD62L Ab at various time points before/after infection revealed that the virus-specific CD8+ T cells entered the DLN and activated in a sequential "conveyor belt"-like fashion. These results indicate that the tempo of CD8+ T cell activation/proliferation after viral infection is dependent on the tempo of RDC migration to the DLN and that T cell activation occurs in an ordered sequential fashion.     

IFN-gamma production by Th1 cells generated from naive CD4+ T cells exposed to norepinephrine

During activation in vivo, naive CD4(+) T cells are exposed to various endogenous ligands, such as cytokines and the neurotransmitter norepinephrine (NE). To determine whether NE affects naive T cell differentiation, we used naive CD4(+) T cells sort-purified from either BALB/c or DO11.10 TCR-transgenic mouse spleens and activated these cells with either anti-CD3/anti-CD28 mAbs or APC and OVA(323-329) peptide, respectively, under Th1-promoting conditions. RT-PCR and functional assays using selective adrenergic receptor (AR) subtype antagonists showed that naive CD4(+) T cells expressed only the beta 2AR subtype to bind NE and that stimulation of this receptor generated Th1 cells that produced 2- to 4-fold more IFN-gamma. This increase was due to more IFN-gamma produced per cell upon restimulation instead of more IFN-gamma-secreting cells, as determined by IFN-gamma-specific immunofluorescence and enzyme-linked immunospot. In contrast, Th1 cell differentiation was unaffected when naive T cells were exposed to NE and activated either in the presence of a neutralizing anti-IL-12 mAb or by APC from IL-12-deficient mice. Moreover, the addition of IL-12 to the IL-12-deficient APC cultures restored the ability of NE to increase Th1 differentiation. Taken together, these results indicate that a possible link may exist between the signaling pathways used by NE and IL-12 to increase naive CD4(+) T cell differentiation to a Th1 cell.     

Bystander central memory but not effector memory CD8+ T cells suppress allograft rejection

Memory T cells respond faster and more vigorously than their naive counterparts and are critical for adaptive immunity. However, it is unknown whether and how memory T cells react in the face of irrelevant Ags. It is generally accepted that bystander memory T cells are neutral in immune responsiveness. In this study, we present the first evidence that bystander central memory (TCM), but not effector memory (TEM), CD8+ T cells suppress allograft rejection as well as T cell proliferation in the draining lymph nodes (DLN) of recipient mice. Both bystander TCM and naive T cells, but fewer TEM cells, migrated to DLN, whereas TCM cells exhibited faster turnover than their naive counterparts, suggesting that bystander TCM cells have an advantage over their naive counterparts in suppression. However, bystander TEM cells migrated to inflammatory graft sites, but not DLN, and yet failed to exert their suppression. These findings indicate that bystander memory T cells need to migrate to lymph nodes to exert their suppression by inhibiting responder T cell activation or homeostatic proliferation. Moreover, the suppression mediated by bystander TCM cells was largely dependent on IL-15, as IL-15 was required for their homeostatic proliferation and TCM-mediated suppression of allograft rejection. This suppression also required the presence of TGFbeta1, as TCM cells expressed TGFbeta1 while neutralizing TGFbeta1 abolished their suppression. Thus, bystander TCM, but not TEM, CD8+ T cells are potent suppressors rather than bystanders. This new finding will have an impact on cellular immunology and may have clinic implications for tolerance induction.     

Status of activation of circulating vaccine-elicited CD8+ T cells

Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209-217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-gamma expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-gamma expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.     

Antigen-specific primary activation of CD8+ T cells within the liver

It is generally accepted that naive T cells recirculate via the blood and lymph, but do not enter nonlymphoid tissues without prior activation and differentiation. In this study, we demonstrate that the liver is an exception to this rule. Naive Des-TCR transgenic CD8(+) T cells specific for H-2K(b) were selectively retained in the liver within a few minutes of adoptive transfer into transgenic Met-K(b) mice expressing H-2K(b) in the liver. Activated CD8(+) cells were found in the liver, but not the blood, as soon as 2 h after transfer and underwent cell division and started to recirculate within 24 h of transfer. In contrast, CD8(+) cells activated in the lymph nodes remained sequestered at that site for 2 days before entering the blood. Our results therefore suggest that, in addition to its previously described role as a non Ag-specific activated T cell graveyard, the liver is involved in Ag-specific activation of naive recirculating CD8(+) T cells. This particular property of the liver, combined with the previously demonstrated ability of hepatocytes to induce tolerance by means of premature CD8(+) T cell death, may be a major mechanism contributing to the acceptance of liver allografts and the chronicity of viral hepatitis.     

Expression of intracellular IFN-gamma in HSV-1-specific CD8+ T cells identifies distinct responding subpopulations during the primary response to infection

Cutaneous infection in the footpads of C57BL/6 mice with HSV-1 results in an accumulation of activated (CD44high CD25+) CD8+ T cells within the draining popliteal lymph node (PLN). These studies were undertaken to evaluate the frequency and phenotype of the CD8+ T cell population within the PLN, recognizing the single immunodominant HSV-1 epitope derived from the viral envelope glycoprotein, glycoprotein B (gB), using an intracellular IFN-gamma-staining assay. It revealed that approximately 6% of the CD8+ T cells were specific for the gB epitope. Phenotypic analysis of the IFN-gamma-producing gB-specific CD8+ T cells generated in the PLN during the course of the acute infection expressed the CD44high CD25+ phenotype on days 3-5 postinfection. Surprisingly, IFN-gamma-producing CD8+ T cells expressed the CD44high CD25- phenotype on days 5-8 postinfection, in contrast to expectations for a CD8+ effector T cell. IFN-gamma-producing CD25- CD8+ T cells were detected in the PLN on day 21 postinfection, long after infectious virus had been cleared. Throughout the response, the spleen was found to be the major reservoir of gB-specific CD8+ T cells, even during the peak of the response. In contrast to the gB-specific CD8+ T cell population within the PLN, the entire gB-specific CD8+ T cell population within the spleen was CD25-. Collectively, these results suggest the generation of subpopulations of virus-specific CD8+ T cells, distinguished by the expression of CD25, during the acute phase of the primary response to a localized viral infection.     

HIV-specific IL-10-positive CD8+ T cells suppress cytolysis and IL-2 production by CD8+ T cells

IL-10 producing T cells inhibit Ag-specific CD8+ T cell responses and may play a role in the immune dysregulation observed in HIV infection. We have previously observed the presence of HIV-specific IL-10-positive CD8+ T cells in advanced HIV disease. In this study, we examined the suppressive function of the Gag-specific IL-10-positive CD8+ T cells. Removal of these IL-10-positive CD8+ T cells resulted in increased cytolysis and IL-2, but not IFN-gamma, production by both HIV- and human CMV-specific CD8+ T cells. In addition, these IL-10-positive CD8+ T cells mediated suppression through direct cell-cell contact, and had a distinct immunophenotypic profile compared with other regulatory T cells. We describe a new suppressor CD8+ T cell population in advanced HIV infection that may contribute to the immune dysfunction observed in HIV infection.     

CD44 contributes to T cell activation

We demonstrate here that the CD44 molecule, which mediates lymphocyte adhesion to high endothelial venules (HEV), is also involved in the delivery of an activation signal to the T cell. We have produced a CD44 mAb (H90) which is able to block the binding of lymphocytes to high endothelial venules. H90 had no effect on [3H]TdR incorporation of whole PBL stimulated by lectins, allogeneic cells, or CD3 mAb in the soluble phase; in contrast, it strongly increased [3H]TdR incorporation of PBL stimulated by CD2 pairs of mAb or by CD3 mAb linked to the plastic culture plates, when purified T cells were used, H90 mAb could efficiently induce them to proliferate after a primary signal of activation delivered via cross-linked CD3 or via CD2, an effect mediated by Il-2 synthesis and Il-2R expression. Thus, the effect of H90 mAb resembles the mitogenic effect of CD28 "9.3" mAb. However, several results show that CD28 and CD44 mediate different signals to the T cells: i) in contrast to CD28 mAb, CD44 mAb cannot complement the signal delivered by a soluble CD3 mAb, lectins, or PMA; ii) CD44 mAb, at the difference of CD28 mAb, cannot induce CD3+ thymocytes to proliferate in conjunction with a first signal provided via cross-linked CD3 or via CD2; iii) F(ab) fragments of H90 were efficient, whereas divalent fragments of CD29 9.3 mAb are required to produce activation signals; and iv) CD44 and CD28 mAb produce a very strong synergistic effect on T cell proliferation. These results fit with previous ones showing that endothelial cells can play the role of accessory cell in T cell activation and that a hierarchy of signaling can be delivered to T cells via CD3 and CD2.