David C. Gondek

Cutting Edge: Contact-Mediated Suppression by CD4+CD25+ Regulatory Cells Involves a Granzyme B-Dependent, Perforin-Independent Mechanism

CD4+CD25+ regulatory T cells (Treg) are potent immunosuppressive cells that are pivotal in the regulation of peripheral tolerance. In this report, we identify granzyme B (GZ-B) as one of the key components of Treg-mediated suppression. Induction of regulatory activity is correlated with the up-regulation of GZ-B expression. Proof of a functional involvement of GZ-B in contact-mediated suppression by Treg is shown by the reduced ability of Treg from GZ-B−/− mice to suppress as efficiently as Treg from WT mice. GZ-B-mediated suppression is perforin independent, because suppression by Treg from perforin−/− and WT is indistinguishable. Additionally, suppression mediated by Treg appears to be mediated, in part, by the induction of apoptosis in the CD4+CD25− effector cell. In summary, GZ-B is one of the key mechanisms through which CD4+CD25+ Treg induce cell contact-mediated suppression.

Mast cells are essential intermediaries in regulatory T-cell tolerance

Contrary to the proinflammatory role of mast cells in allergic disorders, the results obtained in this study establish that mast cells are essential in CD4+CD25+Foxp3+ regulatory T (TReg)-cell-dependent peripheral tolerance. Here we confirm that tolerant allografts, which are sustained owing to the immunosuppressive effects of TReg cells, acquire a unique genetic signature dominated by the expression of mast-cell-gene products. We also show that mast cells are crucial for allograft tolerance, through the inability to induce tolerance in mast-cell-deficient mice. High levels of interleukin (IL)-9—a mast cell growth and activation factor—are produced by activated TReg cells, and IL-9 production seems important in mast cell recruitment to, and activation in, tolerant tissue. Our data indicate that IL-9 represents the functional link through which activated TReg cells recruit and activate mast cells to mediate regional immune suppression, because neutralization of IL-9 greatly accelerates allograft rejection in tolerant mice. Finally, immunohistochemical analysis clearly demonstrates the existence of this novel TReg–IL-9–mast cell relationship within tolerant allografts.

VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses

VISTA suppresses T cell proliferation and cytokine production and can influence autoimmunity and antitumor responses in mice.

A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells

The right combination for protection Despite its prevalence, no vaccine exists to protect against infection with the sexually transmitted bacterium Chlamydia trachomatis. Stary et al. now report on one potential vaccine candidate (see the Perspective by Brunham). Vaccinating with an ultraviolet light-inactivated C. trachomatis linked to adjuvant-containing charged nanoparticles protected female conventional and humanized mice against C. trachomatis infection. The vaccine conferred protection only when delivered through mucosal routes. Protection relied on targeting the bacteria to a particular population of immunogenic dendritic cells and inducing memory T cells that resided in the female genital tract. Science, this issue 10.1126/science.aaa8205; see also p. 1322 A nanoparticle-based vaccine protects mice against infection with Chlamydia trachomatis. [Also see Perspective by Brunham] INTRODUCTION Administering vaccines through nonmucosal routes often leads to poor protection against mucosal pathogens, presumably because such vaccines do not generate memory lymphocytes that migrate to mucosal surfaces. Although mucosal vaccination induces mucosa-tropic memory lymphocytes, few mucosal vaccines are used clinically; live vaccine vectors pose safety risks, whereas killed pathogens or molecular antigens are usually weak immunogens when applied to intact mucosa. Adjuvants can boost immunogenicity; however, most conventional mucosal adjuvants have unfavorable safety profiles. Moreover, the immune mechanisms of protection against many mucosal infections are poorly understood. RATIONALE One case in point is Chlamydia trachomatis (Ct), a sexually transmitted intracellular bacterium that infects >100 million people annually. Mucosal Ct infections can cause female infertility and ectopic pregnancies. Ct is also the leading cause of preventable blindness in developing countries and induces pneumonia in infants. No approved vaccines exist to date. Here, we describe a Ct vaccine composed of ultraviolet light–inactivated Ct (UV-Ct) conjugated to charge-switching synthetic adjuvant nanoparticles (cSAPs). After immunizing mice with live Ct, UV-Ct, or UV-Ct–cSAP conjugates, we characterized mucosal immune responses to uterine Ct rechallenge and dissected the underlying cellular mechanisms. RESULTS In previously uninfected mice, Ct infection induced protective immunity that depended on CD4 T cells producing the cytokine interferon-γ, whereas uterine exposure to UV-Ct generated tolerogenic Ct-specific regulatory T cells, resulting in exacerbated bacterial burden upon Ct rechallenge. In contrast, mucosal immunization with UV-Ct–cSAP elicited long-lived protection. This differential effect of UV-Ct–cSAP versus UV-Ct was because the former was presented by immunogenic CD11b+CD103– dendritic cells (DCs), whereas the latter was presented by tolerogenic CD11b–CD103+ DCs. Intrauterine or intranasal vaccination, but not subcutaneous vaccination, induced genital protection in both conventional and humanized mice. Regardless of vaccination route, UV-Ct–cSAP always evoked a robust systemic memory T cell response. However, only mucosal vaccination induced a wave of effector T cells that seeded the uterine mucosa during the first week after vaccination and established resident memory T cells (TRM cells). Without TRM cells, mice were suboptimally protected, even when circulating memory cells were abundant. Optimal Ct clearance required both early uterine seeding by TRM cells and infection-induced recruitment of a second wave of circulating memory cells. CONCLUSIONS Mucosal exposure to both live Ct and inactivated UV-Ct induces antigen-specific CD4 T cell responses. While immunogenic DCs present the former to promote immunity, the latter is instead targeted to tolerogenic DCs that exacerbate host susceptibility to Ct infection. By combining UV-Ct with cSAP nanocarriers, we have redirected noninfectious UV-Ct to immunogenic DCs and achieved long-lived protection. This protective vaccine effect depended on the synergistic action of two memory T cell subsets with distinct differentiation kinetics and migratory properties. The cSAP technology offers a platform for efficient mucosal immunization that may also be applicable to other mucosal pathogens. Protection against C. trachomatis infection after mucosal UV-Ct–cSAP vaccination. Upon mucosal vaccination, dendritic cells carry UV-Ct–cSAP to lymph nodes and stimulate CD4 T cells. Effector T cells are imprinted to traffic to uterine mucosa (first wave) and establish tissue-resident memory cells (TRM cells). Vaccination also generates circulating memory T cells. Upon genital Ct infection, local reactivation of uterine TRM cells triggers the recruitment of the circulating memory subset (second wave). Optimal pathogen clearance requires both waves of memory cells. Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ–producing CD4 T cells. By contrast, we report that mucosal exposure to ultraviolet light (UV)–inactivated Ct (UV-Ct) generated regulatory T cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAPs) elicited long-lived protection in conventional and humanized mice. UV-Ct–cSAP targeted immunogenic uterine CD11b+CD103– dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b–CD103+ DCs. Regardless of vaccination route, UV-Ct–cSAP induced systemic memory T cells, but only mucosal vaccination induced effector T cells that rapidly seeded uterine mucosa with resident memory T cells (TRM cells). Optimal Ct clearance required both TRM seeding and subsequent infection-induced recruitment of circulating memory T cells. Thus, UV-Ct–cSAP vaccination generated two synergistic memory T cell subsets with distinct migratory properties.

Transplantation survival is maintained by granzyme B+ regulatory cells and adaptive regulatory T cells.

Granzyme B (GZB) has been implicated as an effector mechanism in regulatory T cells (Treg) suppression. In a model of Treg-dependent graft tolerance, it is shown that GZB- deficient mice are unable to establish long-term tolerance. Moreover, mice overexpressing the inhibitor of GZB, serine protease inhibitor 6, are also resistant to tolerization to alloantigen. Graft survival was shorter in bone marrow-mixed chimeras reconstituted with GZB-deficient Treg as compared with wild-type Treg. Whereas there was no difference in graft survival in mixed chimeras reconstituted with wild-type, perforin-deficient, or Fas ligand-deficient Treg. Finally, data also show that if alloreactive effectors cannot express FoxP3 and be induced to convert in the presence of competent Treg, then graft tolerance is lost. Our data are the first in vivo data to implicate GZB expression by Treg in sustaining long-lived graft survival.

CD4+ T Cells Are Necessary and Sufficient To Confer Protection against Chlamydia trachomatis Infection in the Murine Upper Genital Tract

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Chlamydia infections that ascend to the upper genital tract can persist, trigger inflammation, and result in serious sequelae such as infertility. However, mouse models in which the vaginal vault is inoculated with C. trachomatis do not recapitulate the course of human disease. These intravaginal infections of the mouse do not ascend efficiently to the upper genital tract, do not cause persistent infection, do not induce significant inflammation, and do not induce significant CD4+ T cell infiltration. In this article, we describe a noninvasive transcervical infection model in which we bypass the cervix and directly inoculate C. trachomatis into the uterus. We show that direct C. trachomatis infection of the murine upper genital tract stimulates a robust Chlamydia-specific CD4+ T cell response that is both necessary and sufficient to clear infection and provide protection against reinfection.

T Cell Responses in the Absence of IFN-γ Exacerbate Uterine Infection with Chlamydia trachomatis

Infection with the obligate intracellular bacterium Chlamydia trachomatis is controlled primarily by IFN-γ and Th1 immunity. In this study, we used cells from a Chlamydia-specific CD4+ TCR-transgenic mouse to assess the role of IFN-γ in development of Th1 immunity. We show that secretion of host IFN-γ or the ability of host cells to respond to secreted IFN-γ is not required to initiate a Th1 immune response. Additionally, we found that Ag-specific CD4+ cells that were preskewed toward Th1 confer protection, whereas cells preskewed toward Th2 cause a previously unreported exacerbation of disease leading to higher bacterial load. Chlamydia-specific Th1 cells transferred into an IFN-γ−/− recipient mouse demonstrate protective effects, but the same cells exacerbate bacterial burden when transferred into IFN-γR−/− mice. Thus, we demonstrate that the secretion of IFN-γ is necessary for protection against C. trachomatis and that in the absence of host cell IFN-γR expression, both Th1 and Th2 cells lead to increased burden of C. trachomatis.

Different Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine Gammaherpesvirus Infection

Gammaherpesviruses can persist in the host in the face of an aggressive immune response. T cells recognize Ags expressed in both the productive and latent phases of the virus life cycle, however little is known about their relative roles in the long-term control of the infection. In this study we used the murine gammaherpesvirus 68 model system to investigate the relative properties of CD8 T cells recognizing lytic and latent viral Ags. We report that the CD8 T cell response to lytic phase epitopes is maximal in the lungs of infected mice at ∼10 days postinfection, and is of progressively lesser magnitude in the mediastinal lymph nodes and spleen. In contrast, the CD8 T cell response to the latent M2 protein is maximal at ∼19 days postinfection and is most prominent in the spleen, then progressively less in the mediastinal lymph node and the lung. Latent and lytic Ag-specific CD8 T cells had markedly different cell surface phenotypes during chronic infection, with latent Ag-specific cells being predominantly CD62Lhigh or CD43 (1B11)high. Lytic Ag-specific T cells had significantly lower expression of these markers. Importantly, latent but not lytic Ag-specific T cells could kill target cells rapidly in vivo during the chronic infection. These two different sets of CD8 T cells also responded differentially to IL-7, a cytokine involved in T cell homeostasis and the maintenance of T cell memory. These data have important implications for our understanding of immunological control during chronic gammaherpesvirus infections.

NFκB-Inducing Kinase Deficiency Results in the Development of a Subset of Regulatory T Cells, which Shows a Hyperproliferative Activity upon Glucocorticoid-Induced TNF Receptor Family-Related Gene Stimulation

CD4+CD25+ regulatory T cells (Treg) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on Treg has been shown to be a key player of regulating Treg-mediated suppression. A recent study reports that NF-κB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of Treg but not for its suppression capacity. In this report, we have shown that Treg from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive Treg proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type Treg. Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+CD25+CD62Llow Treg both in thymus and periphery than their wild-type littermates. This CD62low subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+CD25+ regulatory T cells.

CXCR3 AND CCR5 ARE BOTH REQUIRED FOR T CELL MEDIATED PROTECTION AGAINST C. TRACHOMATIS INFECTION IN THE MURINE GENITAL MUCOSA

Chemokine receptors direct T lymphocytes to the site of an infection by following coordinated chemokine gradients, which allow their recruitment to specific tissues. Although identification of receptors needed for homing to some mucosal sites, such as skin and gut, have been elucidated, the receptors that direct lymphocytes to the genital mucosa remain relatively uncharacterized. In this study we identify that the chemokine receptors CXCR3 (chemokine (C-X-C motif) receptor 3) and CCR5 (chemokine (C-C motif) receptor 5) are pivotal for T-lymphocyte access to the genital tract during Chlamydia trachomatis infection. Chlamydia-specific CD4+ transgenic T cells that lack CXCR3 or CCR5 do not accumulate in the genital mucosa following infection. Loss of either CXCR3 or CCR5 impairs the protective capacity of Chlamydia-specific T cells, whereas T cells lacking both receptors are completely nonprotective. These results show that CXCR3 and CCR5 are the predominant chemokine receptors that act cooperatively to promote homing to the genital mucosa during Chlamydia infection.