Not all effector CD8+ T cells are alike

As naive CD8+ T cells circulate throughout the bloodstream and secondary lymphoid tissues (i.e. spleen and lymph nodes), they sample complexes of peptides and MHC class I molecules expressed on the surface of professional antigen presenting cells (APCs). A proper fit between lymphocyte and APCs sets into motion a complex series of events that result in the generation of activated cytotoxic T lymphocytes (CTLs) that are the principal immune effectors against infected and transformed cells. Owing to the severe immunopathology that can result from the aberrant stimulation of CTLs, the activation of na?ve CD8(+) T cells is a tightly regulated process. A growing body of evidence suggests that the quality of stimulation na?ve CD8+ T cells receive during the induction and maintenance of an immune response dictates the functional competency of the responding antigen-specific CTLs, and that CD8+ T cells and their progeny "effector cells" can exist long-term in vastly different activation states.     

Type I IFN negatively regulates CD8+ T cell responses through IL-10-producing CD4+ T regulatory 1 cells

Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8(+) T cell responses in IFN-beta (IFN-beta(-/-))- or type I IFN receptor (IFNAR(-/-))-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR(-/-) or IFN-beta(-/-) mice have normal numbers of CD4(+) and CD8(+) T cells, and CD25(+)FoxP3(+) T regulatory (T(R)) cells in liver and spleen. Twice as many CD8(+) T cells specific for different class I-restricted epitopes develop in IFNAR(-/-) or IFN-beta(-/-) mice than in normal animals after peptide- or DNA-based vaccination. IFN-gamma and TNF-alpha production and clonal expansion of specific CD8(+) T cells from normal and knockout mice are similar. CD25(+)FoxP3(+) T(R) cells down-modulate vaccine-primed CD8(+) T cell responses in normal, IFNAR(-/-), or IFN-beta(-/-) mice to a comparable extent. Low IFN-alpha or IFN-beta doses (500-10(3) U/mouse) down-modulate CD8(+) T cells priming in vivo. IFNAR- and IFN-beta-deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4(+) T cells after polyclonal or specific stimulation in vitro or in vivo. CD8(+) T cell responses are thus subjected to negative control by both CD25(+)FoxP3(+) T(R) cells and CD4(+)IL-10(+) T(R1) cells, but only development of the latter T(R) cells depends on type I IFN.     

Human bone marrow hosts polyfunctional memory CD4+ and CD8+ T cells with close contact to IL-15-producing cells

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4(+) and CD8(+) T cells have not yet been elucidated. In this study, we demonstrate that CD4(+) and CD8(+) effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4(+) and CD8(+) T cells are in the immediate vicinity of IL-15-producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4(+) and CD8(+) T cell subsets. Moreover, the IL-15-inducible molecules Bcl-x(L), MIP-1α, MIP-1β, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15-producing cells, is important for the maintenance of a polyfunctional memory CD4(+) and CD8(+) T cell pool.     

T cytotoxic-1 CD8+ T cells are effector cells against pneumocystis in mice

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. A hallmark of HIV infection is Pneumocystis carinii (PC) pneumonia. Recently, CD8+ T cells, which are recruited to the lung in large numbers in response to PC infection, have been associated with some level of host defense as well as contributing to lung injury in BALB/c mice. In this study, we show that CD8+ T cells that have a T cytotoxic-1 response to PC in BALB/c mice, as determined by secretion of IFN-gamma, have in vitro killing activity against PC and effect clearance of the organism in adoptive transfer studies. Moreover, non-T cytotoxic-1 CD8+ T cells lacked in vitro effector activity and contributed to lung injury upon adoptive transfer. This dichotomous response in CD8+ T cell response may in part explain the clinical heterogeneity in the severity of PC pneumonia.     

IL-12 augments CD8+ T cell development for contact hypersensitivity responses and circumvents anti-CD154 antibody-mediated inhibition.

During sensitization with dinitrofluorobenzene for contact hypersensitivity (CHS) responses, hapten-specific CD8(+) T cells develop into IFN-gamma-producing cells, and CD4(+) T cells develop into IL-4/IL-5-producing cells. Administration of IL-12 during sensitization skews CD4(+) T cell development to IFN-gamma-producing cells, resulting in exaggerated CHS responses. In the current report we tested the role of IL-12 on CD8(+) T cell development during sensitization and elicitation of CHS to dinitrofluorobenzene. Administration of IL-12 during hapten sensitization induced the expression of IL-12Rbeta2 on both CD4(+) and CD8(+) T cells, augmented IFN-gamma production by these T cell populations, and increased the magnitude and duration of the CHS response to hapten challenge. CHS responses were virtually identical in wild-type and IL-12 p40(-/-) mice. Since engagement of CD40 on APC may stimulate IL-12 production, we also tested the role of CD40-CD154 interactions on the development of IFN-gamma-producing CD4(+) and CD8(+) T cells following hapten sensitization. Development of IFN-gamma-producing CD4(+) T cells during hapten sensitization was absent in wild-type mice treated with anti-CD154 mAb or in CD154(-/-) mice. In contrast, the absence of CD40-CD154 signaling had little or no impact on the development of IFN-gamma-producing CD8(+) T cells. These results demonstrate that the development of hapten-specific Th1 effector CD4(+) T cells in CHS requires both CD40-CD154 interactions and IL-12, whereas the development of IFN-gamma-producing effector CD8(+) T cells can occur independently of these pathways.     

Generation and differentiation of IL-17-producing CD4+ T cells in malignant pleural effusion

IL-17-producing CD4(+) T (Th17) cells have been found to be increased in some human cancers; however, the possible implication of Th17 cells in regulating antitumor responses in malignant pleural effusion (MPE) remains to be elucidated. In the current study, distribution and phenotypic features of Th17 cells in both MPE and peripheral blood from patients with lung cancer were determined by flow cytometry or double immunofluorescence staining. The impacts of cytokines on Th17 cell generation and differentiation were explored. The chemoattractant activity of chemokines CCL20 and CCL22 for Th17 cells in vitro was also observed. It was found that the increased Th17 cells could be found in MPE compared with blood. The in vitro experiments showed that IL-1β, IL-6, IL-23, or their various combinations could promote Th17 cell generation and differentiation from naive CD4(+) T cells. MPE was chemotactic for Th17 cells, and this activity was partly blocked by anti-CCL20 and/or CCL22 Abs. Our data also showed that the accumulation of Th17 cells in MPE predicted improved patient survival. It could be concluded that the overrepresentation of Th17 cells in MPE might be due to Th17 cell differentiation and expansion stimulated by pleural proinflammatory cytokines and to recruitment of Th17 cells from peripheral blood induced by pleural chemokines CCL20 and CCL22. Furthermore, the accumulation of Th17 cells in MPE predicted improved patient survival. These data provide the basis for developing immune-boosting strategies based on ridding the cancer patient of this cell population.     

Expression and regulation of IL-22 in the IL-17-producing CD4＋ T lymphocytes

IL-22 is a novel cytokine in the IL-10 family that functions to promote innate immunity of tissues against infection. Although CD4＋ helper T lymphocytes （TH） were found as a source of IL-22, the regulation of this cytokine has been poorly understood. Here, we show that IL-22 is expressed at both mRNA and protein levels by a novel subset of TH cells that also makes IL-17. IL-22 and IL-17 were found to be coordinately regulated by TGFI3 and IL-6 during TH differentiation by real-time PCR as well as ELISA analysis. However, IL-22 does not regulate TH differentiation; exogenous IL-22 or an IL-22 antagonist had no effect on TH differentiation. These data demonstrate a novel cytokine expressed by IL-17-producing T cells, and suggest interaction and synergy of IL-22 and IL-l 7 signaling pathways in tissue inflammation and autoimmune diseases.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.     

IL-2/anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific CD8+ T cell responses and increases the ratio of effector/memory CD8+ T cells to regulatory T cells

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8(+) T cells to T(REG) cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.     

Cutting edge: Lipopolysaccharide induces IL-10-producing regulatory CD4+ T cells that suppress the CD8+ T cell response

TLR ligands are potent activators of dendritic cells and therefore function as adjuvants for the induction of immune responses. We analyzed the capacity of TLR ligands to enhance CD8+ T cell responses toward soluble protein Ag. Immunization with OVA together with LPS or poly(I:C) elicited weak CD8+ T cell responses in wild-type C57BL/6 mice. Surprisingly, these responses were greatly increased in mice lacking CD4+ T cells indicating the induction of regulatory CD4+ T cells. In vivo, neutralization of IL-10 completely restored CD8+ T cell responses in wild-type mice and OVA-specific IL-10 producing CD4+ T cells were detected after immunization with OVA plus LPS. Our study shows that TLR ligands not only activate the immune system but simultaneously induce Ag specific, IL-10-producing regulatory Tr1 cells that strongly suppress CD8+ T cell responses. In this way, excessive activation of the immune system may be prevented.     

Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-gamma after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-gamma production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-gamma production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.     

Lectin-mediated induction of IL-4-producing CD4+ T cells.

Cultured murine CD4+ T cells have been shown to differentiate into IL-2 or IL-4-producing subsets. The factors responsible for the development of CD4+ T cells which produce IL-2 but not IL-4 and cells capable of producing IL-4 but not IL-2 are unknown. Here we describe a system that allows the controlled induction of IL-2- or IL-4-producing T cells after one single round of activation. Freshly isolated CD8-depleted T cells were activated with various polyclonal T cell activators for 48 h, washed, and then expanded under different conditions. IL-2 and IL-4 production were induced by restimulation of T cells and were measured with CTLL cells that respond to both cytokines and mAb to IL-2 and IL-4. T cells produced mainly IL-2 and small amounts of IL-4 when restimulated after expansion culture for 12 days with rIL-2 alone. However, after expansion for 12 days in the presence of rIL-2 plus Con A, we observed a 30- to 100-fold up-regulation of IL-4 activity and a 100-fold down-regulation of IL-2 when assessed by responses of CTLL cells incubated with the supernatant of restimulated T cells and by responses of CTLL cells cocultured with restimulated cells. An increase of IL-4 and decrease of IL-2 was also observed when the results were based on the cell numbers at the beginning of the expansion culture. The induction of IL-4 and the down-regulation of IL-2 1) were not reproduced with alpha-methyl-mannoside-treated supernatant of Con A-stimulated spleen cells, 2) were not dependent on the presence of large numbers of APC, 3) did not result from differential consumption of lymphokines after restimulation, 4) were not due to a difference in the time course of IL-2 or IL-4 release in either T cell population, and 5) were obtained regardless of the agents used to activate or to restimulate the T cells. Because Con A remained detectable on the T cell surface and because expansion of activated T cells with IL-2 plus Con A for several days was necessary, our results indicate that mainly IL-4-producing CD4+ T cells can be induced by prolonged engagement of T cell surface molecules.     

Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.     

IL-4-secreting CD4+ T cells are crucial to the development of CD8+ T-cell responses against malaria liver stages

CD4+ T cells are crucial to the development of CD8+ T cell responses against hepatocytes infected with malaria parasites. In the absence of CD4+ T cells, CD8+ T cells initiate a seemingly normal differentiation and proliferation during the first few days after immunization. However, this response fails to develop further and is reduced by more than 90%, compared to that observed in the presence of CD4+ T cells. We report here that interleukin-4 (IL-4) secreted by CD4+ T cells is essential to the full development of this CD8+ T cell response. This is the first demonstration that IL-4 is a mediator of CD4/CD8 cross-talk leading to the development of immunity against an infectious pathogen.     

Regulatory T cells selectively control CD8+ T cell effector pool size via IL-2 restriction

Regulatory T cells (Treg) are key players in maintaining immune homeostasis but have also been shown to regulate immune responses against infectious pathogens. Therefore, Treg are a promising target for modulating immune responses to vaccines to improve their efficacy. Using a viral vector system, we found that Treg act on the developing immune response early postinfection by reducing the extent of dendritic cell costimulatory molecule expression. Due to this change and the lower IL-2 production that results, a substantial fraction of CD8(+) effector T cells lose CD25 expression several days after activation. Surprisingly, such Treg-dependent limitations in IL-2 signaling by Ag-activated CD8(+) T cells prevent effector differentiation without interfering with memory cell formation. In this way, Treg fine-tune the numbers of effector T cells generated while preserving the capacity for a rapid recall response upon pathogen re-exposure. This selective effect of Treg on a subpopulation of CD8(+) T cells indicates that although manipulation of the Treg compartment might not be optimal for prophylactic vaccinations, it can be potentially exploited to optimize vaccine efficacy for therapeutic interventions.     

Increased IL-17-producing CD4(+) T cells in patients with esophageal cancer

Increased interleukin-17 (IL-17)-producing Th (Th17) cells have been described in a variety of human carcinoma cases, however, the mechanism of Th17 cells' accumulation in a tumor microenvironment remains elusive. This study was designed to investigate whether Th17 cells were involved in the development of esophageal cancer. We found that the proportion of Th17 cells increased within the peripheral blood and tumor tissues of esophageal cancer patients. Furthermore, the proportion of circulating Th17 cells was higher in advanced esophageal cancer patients than that in early esophageal cancer patients. In addition, the Th17 cells differentiation-related cytokines (IL-23, IL-1β, and IL-6) and accumulation-related chemokines (CCL22 and CCL20) were present in a tumor microenvironment. Therefore, the findings may partly explain the cause for the increased proportion of Th17 cells and indicate a potential prognostic marker of Th17 cells in esophageal cancer.     

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.     

Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

CD4+ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-gamma or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-gamma producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-gamma alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-gamma- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-gamma and IL-4 producers.     

Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

CD4⁺ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-γ or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-γ producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-γ alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-γ- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-γ and IL-4 producers.