Functional re-expression of CCR7 on CMV-specific CD8+ T cells upon antigenic stimulation

During latency circulating human cytomegalovirus (CMV)-specific CD8(+) T cells do not express the chemokine receptor CCR7. We here show that antigen-specific stimulation in vitro with the specific CMV-peptide in combination with CMV-antigen, IL-2 or IL-21 induced re-expression of CCR7 on CMV-specific CD8(+) T cells. Although IL-15 induced strong proliferation of peptide-pulsed CMV-specific CD8(+) T cells, these cells did not re-express CCR7. CMV-specific cells that re-expressed CCR7 also expressed CD62L and were able to react to specific chemokine stimulation with changes in the cytoskeleton. In addition, activated CMV-specific cells specifically migrated towards a chemokine gradient in a transwell assay, with and without an endothelial cell monolayer. We conclude that antigenic stimulation induced functional re-expression of CCR7 which suggests that the migratory properties of virus-primed T cells are flexible and depend on the presence or absence of antigen and cytokines.     

TSLP promotes influenza-specific CD8+ T-cell responses by augmenting local inflammatory dendritic cell function

Thymic stromal lymphopoietin (TSLP) is a mucosal tissue-associated cytokine that has been widely studied in the context of T helper type 2 (Th2)-driven inflammatory disorders. Although TSLP is also produced upon viral infection in vitro, the role of TSLP in antiviral immunity is unknown. In this study we report a novel role for TSLP in promoting viral clearance and virus-specific CD8+ T-cell responses during influenza A infection. Comparing the immune responses of wild-type and TSLP receptor (TSLPR)-deficient mice, we show that TSLP was required for the expansion and activation of virus-specific effector CD8+ T cells in the lung, but not the lymph node. The mechanism involved TSLPR signaling on newly recruited CD11b+ inflammatory dendritic cells (DCs) that acted to enhance interleukin-15 production and expression of the costimulatory molecule CD70. Taken together, these data highlight the pleiotropic activities of TSLP and provide evidence for its beneficial role in antiviral immunity.     

Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2-polarizing conditions

Background Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP. Objective Because TSLPR‐expressing DCs express TLR3, we examined how the relationship between TSLP and TLR3 ligand stimulation influences DC activation. Methods CD11c⁺DCs purified from adult peripheral blood were cultured in TLR ligands containing media with or without TSLP and then co‐cultured with allogeneic naïve CD4⁺T cells. Results CD11c⁺ DCs responded to a combination of TSLP and TLR3 ligand, poly(I : C), to up‐regulate expression of the functional TSLP receptor and TLR3. Although TSLP alone did not induce IL‐23 production by DCs, poly(I : C) alone primed DCs for the production of IL‐23, and a combination of TSLP and poly(I : C) primed DCs for further production of IL‐23. The addition of poly(I : C) did not inhibit TSLP‐activated DCs to prime naïve CD4⁺ T cells to differentiate into inflammatory Th2 cells. Furthermore, DCs activated by a combination of TSLP and poly(I : C) primed more naïve CD4⁺ T cells to differentiate into Th17‐cytokine–producing cells with a central memory T cell phenotype compared with DCs activated by poly(I : C) alone. Conclusions These results suggest that through DC activation, human TSLP and TLR3 ligands promote differentiation of Th17 cells with the central memory T cell phenotype under Th2‐polarizing conditions.     

Activated B cells modified by electroporation of multiple mRNAs encoding immune stimulatory molecules are comparable to mature dendritic cells in inducing in vitro antigen-specific T-cell responses

Ex-vivo-activated B cells are an alternative source of antigen-presenting cells (APCs) and a potential replacement for dendritic cells (DCs) in immunotherapy. However, the ability of ex-vivo-activated B cells to function as potent APCs has been a concern, especially when compared to DCs. Our study investigated whether modification of activated B cells with immune stimulatory molecules could enhance the ability of activated B cells to stimulate T cells. We show that murine splenic B cells, activated with a combination of Toll-like receptor agonist and agonistic anti-CD40, stimulated antigen-specific CD8+ T cells more efficiently than cells activated with Toll-like receptor agonist or anti-CD40 alone, probably by down-regulation of the immune regulatory cytokine interleukin-10 (IL-10). However, the activated B cells were still poor T-cell stimulators compared to mature DCs. Therefore, we modified the activated B cells by simultaneous electroporation of multiple messenger RNAs encoding costimulatory molecules (OX40L and 4-1BBL), cytokines (IL-12p35 and IL-12p40) and antigen. We found that de novo expression or overexpression of OX40L, 4-1BBL and IL-12p70 on activated B cells synergistically enhanced proliferation as well as IL-2 and interferon-gamma production by CD8+ T cells. Furthermore, the RNA-modified activated B cells induced antigen-specific cytotoxic T lymphocyte responses as efficiently as mature DCs in vitro. Unexpectedly, modified activated B cells were inferior to mature DCs at in vivo induction of CD8+ T-cell responses. In summary, activated B cells modified to express immune stimulatory molecules are a potent alternative to DCs in immunotherapy.     

Batf3 transcription factor-dependent DC subsets in murine CMV infection: differential impact on T-cell priming and memory inflation

Priming of CD8(+) T cells specific for viruses that interfere with the MHC class I presentation pathway is a challenge for the immune system and is believed to rely on cross-presentation. Cytomegalovirus (CMV) infection induces vigorous CD8(+) T-cell responses despite its potent immune evasion strategies. Furthermore, CD8(+) T cells specific for a subset of viral epitopes accumulate and are maintained at high levels exhibiting an activated phenotype - referred to as "inflationary T cells". Taking advantage Batf3(-/-) mice in which the development of cross-presenting CD8α(+) and CD103(+) DCs is severely compromised, we analyzed their role in the induction and inflation of murine (M)CMV-specific CD8(+) T-cell responses. We found that priming of MCMV-specific CD8(+) T cells was severely impaired in the absence of cross-presenting DCs. However, inflation of two immuno-dominant MCMV-specific CD8(+) T-cell populations was largely normal in the absence of cross-presenting DCs, indicating that inflation during latency was mainly dependent on direct antigen presentation. These results highlight differential antigen presentation requirements during acute and latent MCMV infection.     

LIGHT-deficiency impairs CD8+ T cell expansion,but not effector function

LIGHT, a newly identified member of the tumor necrosis factor (TNF) family, is expressed on activated T lymphocytes. To evaluate how LIGHT contributes to T cell functions, we generated LIGHT-deficient (LIGHT(-/-)) mice using gene targeting. Disruption of LIGHT significantly reduced CD8(+) T cell-cycle progression, leading to reduced proliferation to anti-CD3, anti-CD3/anti-CD28 or allogeneic stimulation, whereas proliferation of CD4(+) T cells remained unchanged. In contrast to the observed proliferative defects, isolated CD8(+) T cells from LIGHT(-/-) mice displayed normal cytotoxic effector function development when compared to wild-type CD8(+) T cells. Underlying a potential mechanism of reduced CD8(+) T cell proliferation, LIGHT(-/-) CD8(+) T cells displayed reduced surface levels of CD25 and a diminished ability to proliferate in response to exogenous IL-2. Furthermore, addition of IL-12 to LIGHT(-/-) CD8(+) T cell cultures could not ameliorate this proliferative defect. These results reveal a potential mechanism of action for LIGHT as a positive regulator of CD8(+) T cell expansion, but not lytic effector function development.     

Phosphatidylinositol 3-kinase inhibitors prevent mouse cytotoxic T-cell development in vitro

To become competent killer cells, CD8(+) T cells require stimulation through signal transduction pathways associated with the T-cell receptor, costimulatory molecules such as CD28, and cytokine receptors such as the interleukin (IL)-2 receptor. We used wortmannin and LY294002, two inhibitors of phosphatidylinositol 3-kinase (PI3-K), to study the role of PI3-K in mouse cytotoxic T-lymphocyte (CTL) induction in response to mitogenic anti-CD3 antibody. Anti-CD3-induced CD8(+) T-cell proliferation and CTL development were inhibited dose dependently by both PI3-K inhibitors. IL-2 synthesis by anti-CD3-activated CD8(+) T cells was also diminished by PI3-K inhibition. PI3-K inhibition resulted in a modest decrease in anti-CD3-induced CD4(+) T-cell proliferation but failed to affect IL-2 expression by anti-CD3-activated CD4(+) T cells. PI3-K inhibition during CTL induction resulted in decreased levels of mRNAs coding for granzyme B, perforin, and Fas ligand. In addition, CTL induced in the presence of PI3-K inhibitors failed to conjugate normally with P815 target cells. Exogenous IL-2 did not reverse the effects of PI3-K inhibition on CD8(+) T-cell proliferation and CTL induction. These results support the conclusion that PI3-K activation is involved in T-cell receptor, CD28, and IL-2 receptor signaling of CD8(+) T cells. PI3-K is, therefore, an important component of multiple signal transduction pathways involved in CTL generation.     

Age-related changes in the occurrence and characteristics of thymic CD4(+) CD25(+) T cells in mice

Natural regulatory CD4(+) CD25(+) T cells play an important role in preventing autoimmunity by maintaining self-tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T-cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age-associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4(+) CD25(+) thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA-4, CD122, FOXP3), usually used to characterize the phenotype of CD4(+) CD25(+) T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus-deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus-deriving CD4(+) CD25(+) T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4(+) CD25(+) thymocytes between young and old animals are discussed in relation to the expression of these surface markers.     

Human thymic stromal lymphopoietin promotes dendritic cell-mediated CD4+ T cell homeostatic expansion

T cell homeostasis is a self-regulating process for maintaining the size of the peripheral T cell pool. Although dendritic cells (DCs) seem to be important in T cell homeostasis, the molecular regulation of DC-mediated T cell homeostasis is unknown. We show that human DCs activated by thymic stromal lymphopoietin (TSLP) induced a robust expansion of autologous CD4(+) T cell populations, which depended on self peptide-major histocompatibility complex. The proliferating T cells adopted and maintained a central memory polyclonal phenotype and could differentiate into T helper type 1 or type 2 effector cells. These results, together with findings of TSLP expression in epithelial cells of mucosal lymphoid tissues and thymus, indicate that TSLP is involved in DC-mediated CD4(+) T cell homeostasis.     

The development and functions of CD4(+) T cells expressing a transgenic TCR specific for an MHC-I-restricted tumor antigenic epitope

It has been reported that the ratio of CD4(+) to CD8(+) T cells has no bias in a few class I major histocompatibility complex (MHC-I)-restricted T-cell receptor (TCR)-transgenic mice specific for alloantigens or autoantigens, in which most CD4(+) T cells express an MHC-I-restricted TCR. In this study, we further showed that more than 50% of CD4(+) T cells in MHC-I-restricted P1A tumor antigen-specific TCR (P1ATCR)-transgenic mice could specifically bind to MHC-I/P1A peptide complex. P1A peptide could stimulate the transgenic CD4(+) T cells to proliferate and secrete both type 1 helper T cell and type 2 helper T cell cytokines. The activated CD4(+) T cells also showed cytotoxicity against P1A-expressing tumor cells. The analysis of TCR α-chains showed that these CD4(+) T cells were selected by co-expressing endogenous TCRs. Our results show that CD4(+) T cells from P1ATCR transgenic mice co-expressed an MHC-I-restricted transgenic TCR and another rearranged endogenous TCRs, both of which were functional.     

TSLP-induced placental DC activation and IL-10(+) NK cell expansion: comparative study based on BALB/c x C57BL/6 and NOD/SCID x C57BL/6 pregnant models

Thymic stromal lymphopoietin (TSLP)-TSLP receptor (TSLP-R) interactions activate CD11c(+) dendritic cells (DCs) and increase epithelial cell Th2-type cytokine production. We detected intracellular TSLP expression on CK7(+) trophoblast cells and TSLP-R expression on placental DCs from pregnant BALB/cxC57BL/6 and NOD/SCIDxC57BL/6 mice on gestational day 12.5. Murine recombinant TSLP activated DCs from BALB/c mice, with increased CD80 and CD83 expressions; TSLP-activated DCs induced IL-10-producing NK cell expansion. This was abrogated by anti-TSLP Ab or by culturing CD49b(+) NK cells alone. No TSLP-DC-induced IL-10(+)CD49b(+) cell expansion occurred when DCs and CD49b(+) cells were cultured separately. Although TSLP-induced DC activation occurred in NOD/SCID mice, the IL-10(+) NK cell percentage was unchanged. CK7(+) trophoblast cells may activate placental DCs via a TSLP-TSLP-R interaction and induce DC-dependent placental NK cell IL-10 production. TSLP-DC and NK cell contact appears necessary for IL-10(+)CD49b(+) cell expansion. Placental NK cells from NOD/SCIDxC57BL/6 mice appear less sensitive to TSLP-DC induction.     

Rapid induction of OX40 ligand on primary T cells activated under DNA-damaging conditions

We have previously demonstrated that normal human T cells either long-term repeatedly stimulated or freshly activated in vitro in the presence of TGF-beta express the cell surface T-cell costimulating molecule OX40 ligand (OX40L). To further elucidate the kinetics of OX40L expression by human T cells, we have examined whether cell proliferation was required for the expression of OX40L. Thus, normal fresh peripheral blood mononuclear cells were stimulated with immobilized anti-CD3 antibody in the presence of the DNA synthesis-blocking agents such as mitomycin C, 5-fluorouracil, or X-ray irradiation. Flow cytometric analyses demonstrated that a significant frequency of these DNA-damaged activated primary CD4(+) and CD8(+) T cells became OX40L(+) as early as 1 hour after treatment. The OX40L induction on the DNA-damaged activated T cells was inhibited by treatment with either RNA or protein synthesis inhibitors, actinomycin D, or cycloheximide, respectively. Induced OX40L on T cells was functional because it bound recombinant OX40. These data indicate that human primary T cells are programmed to rapidly express functional OX40L molecules after stimulation under DNA-damaging conditions, demonstrating that the induction of OX40L by T cells is independent of cell proliferation. The clinical implications of these new findings are discussed.     

Skin-derived TSLP stimulates skin migratory dendritic cells to promote the expansion of regulatory T cells

Therapeutic strategies that enhance regulatory T (Treg) cell proliferation or suppressive function hold promise for the treatment of autoimmune and inflammatory diseases. We previously reported that the topical application of the vitamin D3 analog MC903 systemically expands Treg cells by stimulating the production of thymic stromal lymphopoietin (TSLP) from the skin. Using mice lacking TSLP receptor expression by dendritic cells (DCs), we hereby show that TSLP receptor signaling in DCs is required for this Treg expansion in vivo. Topical MC903 treatment of ear skin selectively increased the number of migratory DCs in skin-draining lymph nodes (LNs) and upregulated their expression of co-stimulatory molecules. Accordingly, DCs isolated from skin-draining LNs but not mesenteric LNs or spleen of MC903-treated mice showed an enhanced ability to promote Treg proliferation, which was driven by co-stimulatory signals through CD80/CD86 and OX40 ligand. Among the DC subsets in the skin-draining LNs of MC903-treated mice, migratory XCR1⁻CD11b⁺ type 2 and XCR1⁻CD11b⁻ double negative conventional DCs promoted Treg expansion. Together, these data demonstrate that vitamin D3 stimulation of skin induces TSLP expression, which stimulates skin migratory DCs to expand Treg cells. Thus, topical MC903 treatment could represent a convenient strategy to treat inflammatory disorders by engaging this pathway.     

Critical role of TSLP-responsive mucosal dendritic cells in the induction of nasal antigen-specific IgA response

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine involved in T helper 2 type immune responses. The primary target of TSLP is myeloid dendritic cells (DCs), however, little is known about the mechanism by which TSLP elicits respiratory IgA immune responses upon mucosal immunization. Here, we found that the levels of TSLP and TSLPR were upregulated in the mucosal DCs of mice nasally immunized with pneumococcal surface protein A (PspA) plus cholera toxin (CT) compared with those immunized with PspA alone. PspA-specific IgA responses, but not IgG Ab responses were significantly reduced in both serum and mucosal secretions of TSLPR knockout mice compared with wild-type mice after nasal immunization with PspA plus CT. Furthermore, CD11c⁺ mucosal DCs isolated from TSLPR knockout mice nasally immunized with PspA plus CT were less activated and exhibited markedly reduced expression of IgA-enhancing cytokines (e.g., APRIL, BAFF, and IL-6) compared with those from equivalently immunized wild-type mice. Finally, exogenous TSLP promoted production of IgAs in an in vitro DC–B cell co-culture system as exhibited by enhanced IL-6 production. These results suggest that TSLP–TSLPR signaling is pivotal in the induction of nasal respiratory immunity against pathogenic pneumococcal infection.     

Thymic stromal lymphopoietin plays an adjuvant role in BCG-mediated CD8 cytotoxic T cell responses through dendritic cell activation

Although Bacillus Calmette–Guérin (BCG) has historically emerged as a potent adjuvant in cancer immunization through dendritic cell (DC) activation, the efficacy of its antitumor effect has been limited. Therefore, the strategy of adjuvant therapy using BCG needs to be improved by adding enhancers. Here we found that thymic stromal lymphopoietin (TSLP) acts as an enhancer for the BCG-mediated antitumor effect. While BCG-stimulated DCs induced CD8⁺ T cell production of IFN-γ without strong cell expansion, TSLP-stimulated DCs induced robust CD8⁺ T cell expansion without high quantities of IFN-γ production. Notably, DCs stimulated with both BCG and TSLP induced robust expansion of CD8⁺ T cells that produced a large amount of IFN-γ with a potent cytolytic activity related to granzyme B expression. Our data suggest that TSLP is a good adjuvant to enhance the BCG-mediated cytotoxic T cell effect through DC activation, and provide a functional basis for a novel strategy for antitumor immune-based therapy.     

Lactobacillus gasseri OLL2809 and its RNA suppress proliferation of CD4(+) T cells through a MyD88-dependent signalling pathway

Recent studies have shown that probiotics are beneficial in prevention and improvement of inflammatory diseases. Accumulating evidence indicates that probiotics can modulate immune cell responses, although the specific molecular mechanism by which probiotics work remains elusive. Because T cells express receptors for microbial components, we examined whether the probiotic strain Lactobacillus gasseri OLL2809 (LG2809) and its components regulate murine CD4(+) T-cell activation. LG2809, as well as two other Lactobacillus strains, inhibited proliferation of CD4(+) T cells; LG2809 had the strongest suppressive activity among them. RNA isolated from LG2809 was also shown to have suppressive activity. We observed this suppressive effect in the culture of CD4(+) T cells stimulated with anti-CD3/CD28 treatment, suggesting a direct effect on CD4(+) T cells. In contrast, the suppressive effect was not observed for CD4(+) T cells from myeloid differentiation primary response gene 88 (MyD88) protein-deficient mice, and was abrogated in the presence of an anti-oxidant reagent, N-acetyl-cysteine (NAC). These results demonstrate that the suppressive effect of LG2809 and its RNA occurred through a MyD88-dependent signalling pathway and suggest involvement of a reactive oxygen species-dependent mechanism. LG2809 RNA injected subcutaneously suppressed delayed-type-hypersensitivity response in DO11.10 mice, and the suppression was abrogated by treatment with NAC. Collectively, these results suggest that suppression of T-cell proliferation by RNA may be one of the mechanisms when a probiotic bacterial strain exerts suppressive effects on inflammatory responses.     

Induction of CD56+ T cells after prolonged activation of T cells in vitro: a possible mechanism for CD4+ T-cell depletion in acquired immune deficiency syndrome patients

The pathogenic mechanisms responsible for depletion of CD4(+) T cells in aquired immune deficiency syndrome (AIDS) are not fully understood. Systemic immune activation mediated by persistent infection of human immunodeficiency virus (HIV) seems to be one of the predictors of disease progression. We predicted that certain lymphocytes responsible for CD4(+) T-cell depletion could be induced in patients during prolonged activation of lymphocytes. Therefore, we have established an in vitro long-term culture system for peripheral blood mononuclear cells with PHA-P stimulation and Herpesvirus saimiri infection, and examined what types of cells having strong cytotoxic activity to be emerged under the activated conditions. We observed that percentage of CD56(+) T cells was gradually increased in cultures from 30 days after stimulation and exhibited a cytotoxic activity against both autologous and allogeneic targets. Interestingly, HIV-1 infection enhanced the susceptibility of CD4(+) T cells to their cytotoxic effectors, and CD4(+) T cells from HIV-1-infected individuals showed decreased survival rate in the presence of autologous CD56(+) T cells. These findings raised the possibility that induction of autoreactive CD56(+) T cells in consequence of immune activation might be contributed to the depletion of CD4(+) T cells in HIV-1-infected patients.     

The suppression of delayed-type hypersensitivity by CD8+ regulatory T cells requires interferon-gamma

CD8(+) regulatory (suppressor) T cells are induced by complex cellular pathways in the spleens of mice that have received an injection of antigen into the anterior chamber (AC) of an eye, an immune-privileged site. Although these CD8(+) regulatory T cells perform an antigen-specific regulatory function for an immune response to self and non-self antigens, the mechanisms of the activation or function of these regulatory cells are not clear. Here, we describe a novel mechanism for the activation of splenic CD8(+) regulatory T cells induced by injection of antigen into the AC. Immunization of mice with trinitrophenyl and bovine serum albumin (TNP-BSA) amplified AC-induced splenic CD8(+) regulatory T cells that suppressed the initiation of contact sensitivity when transferred to immunized, challenged mice. These CD8(+) regulatory T cells were produced independently of perforin, indicating that they are not canonical cytotoxic T cells. Fas ligand (FasL)-deficient CD8(+) regulatory T-cell function was rescued by inclusion of exogenous interferon-gamma (IFN-gamma), demonstrating that the expression of FasL by CD8(+) regulatory T cells was dispensable, but IFN-gamma was not. Ultimately, we demonstrated that the generation of these CD8(+) regulatory T cells occurred independently of IFN-gamma, but their suppressor function required IFN-gamma receptor stimulation.