The fate of heterologous CD4+ T cells during Leishmania donovani infection

Little is currently understood about the consequences of chronic parasitic infection for the fate of memory CD4+ T cells that recognize heterologous antigens, e.g. resulting from prior infections or vaccination. Here, we address how Leishmania donovani infection affected the fate of non-cross-reactive (OVA)-specific memory CD4+ T cells. DO11 cells were adoptively transferred into naive recipient mice, which were then immunized to generate memory DO11 cells. After 6 weeks, mice were infected with L. donovani and the fate of DO11 cells was determined. L. donovani infection stimulated an approximately threefold expansion in the total number of CD4+ T cells and DO11 cells, compared to that observed in uninfected mice. DO11 T cells were more actively dividing in infected mice, as judged by 5-bromo-2' deoxyuridine labeling, whereas their rate of apoptosis in control and infected mice was identical. Both CD45RBhiCD44lo naive T cells and to a greater extent CD45RBloCD44hi memory DO11 cells increased in number in the spleens of infected mice, whereas no changes occurred to DO11 cell number or phenotype in the draining lymph nodes. These data indicate that heterologous CD4+ T cells may actively divide during chronic infectious diseases, with important implications for how chronic infection may impact on heterologous immunity.     

Activated NKT cells increase dendritic cell migration and enhance CD8+ T cell responses in the skin

Activated NKT cells produce cytokines such as IL-4 and IFN-gamma that function locally to influence the strength and functional development of antigen-specific T cells. Here we identify an alternative mechanism by which NKT cells influence the strength of T cell responses: through modulation of peripheral dendritic cell (DC) trafficking. NKT cell activation with alpha-galactosylceramide induced high systemic levels of TNF-alpha that mediated increased DC migration from skin to draining lymph nodes. This increased DC trafficking led to a threefold increase in effector T cell priming and in the immune response elicited to antigen challenge when alpha-galactosylceramide was given at the time of immunization of the skin. These studies provide important implications for the use of NKT cell activation strategies to manipulate T cell-mediated responses including responses to cutaneous tumors and graft vs. host disease.     

NKT cell activation by Leishmania mexicana LPG: Description of a novel pathway

NKT cells have been associated with protection against Leishmania donovani, yet their role in infections with Leishmania mexicana has not been addressed, nor has the activation pathway been defined after stimulation with Leishmania mexicana lipophosphoglycan (LPG). We analyzed the activation of NKT cells and their cytokine production in response to Leishmania mexicana LPG. Additionally we compared NKT-cell numbers and cytokine profile in lymph nodes of skin lesions induced by Leishmania mexicana in BALB/c and C57BL/6 mice. We show that LPG activates NKT cells primarily through the indirect pathway, initiating with TLR2 stimulation of dendritic cells (DC), thereby enhancing TLR2, MHC II, and CD86 expressions and IL-12p70 production. This leads to IFN-γ production by NKT cells. C57BL/6 mice showed enhanced DC activation, which correlated with augmented IFN-γ production by NKT cells. Additionally, infected C57BL/6 mice showed elevated percentages of NKT cells with higher IFN-γ and IL-4 production in lymph nodes.We conclude that the response of NKT cells towards Leishmania mexicana LPG initiates with the indirect activation, after binding of LPG to TLR2 in DC. This indirect activation pathway enables NKT cells to produce IFN-γ during the innate phase of Leishmania infection, the magnitude of which differs between mouse strains.     

Adjuvant effect of Taenia crassiceps glycans against leishmanial antigens in mice infected with Leishmania mexicana

Complex glycans derived from lipid, nucleic acid and protein free extracts of Taenia crassiceps metacestode larvae were found to have adjuvant effect against Leishmania mexicana antigens in BALB/c mice experimentally infected with L. mexicana. A single intraperitoneal or subcutaneous injection of Taenia glycans altered the Th-1/Th-2 balance in experimentally infected mice as determined by Western blot analysis of IgG 1 and IgG 2a antibodies to L. mexicana antigens. Leishmania antigens which were immunogenic in Taenia glycan vaccinated mice were different from those of non-vaccinated mice. Vaccination induced leishmania antigen specific IFN-γ expression in vitro culture by spleen cells from Taenia glycan vaccinated-leishmania infected mice and not from mock vaccinated-leishmania infected BALB/c mice. We conclude that T. crassiceps glycans have immunoadjuvant effects against leishmania and may be developed as adjuvants in anti-leishmania vaccines.     

Glycolipid-activated NKT cells support the induction of persistent plasma cell responses and antibody titers

NKT cell activation with CD1d-binding glycolipid alpha-galactosylceramide (alpha-GC) enhances antibody responses to co-administered T-dependent antigen. The efficacy of alpha-GC relative to other CD1d-binding glycolipids and adjuvants is not known. There is little information on how NKT cells affect antibody production beyond initial booster-stimulated recall responses. We therefore tested the hypothesis that alpha-GC stimulates induction of plasma cells and antibody responses as effectively as Th1- and Th2-skewing variants of alpha-GC and several other adjuvants. C57BL/6 and CD1d-/- mice were immunized with nitrophenol-conjugated keyhole limpet hemocyanin (NP-KLH) plus alpha-GC or NP-KLH plus adjuvants before administration of an NP-KLH booster and assessing antibody responses and plasma cell frequency. alpha-GC boosted long-term antibody responses as efficiently as all other agents tested and induced plasma cells that were detected in bone marrow 13 weeks after immunization. We then determined whether NKT cells were required in the presence of other adjuvants. CD1d-/- mice had a reduced induction of plasma cells in response to NP-KLH/Alum as compared to C57BL/6 mice. However, NKT cells were not required for the continued presence of those cells that were induced. Although NKT cells are capable of inducing persistent plasma cell responses, they may not play a major role in supporting longevity post-induction.     

Natural killer cells support the induction of protective immunity during dendritic cell-mediated vaccination against Leishmania major

Dendritic cell (DC)‐mediated vaccination against Leishmania major induces a parasite‐specific T helper 1 (Th1) response and long‐lasting protective immunity in susceptible mice. As the cytokine interleukin‐12 required for induction of this Th1 response is not derived from the transferred DC, but has to be produced by the vaccinated host, we examined cross‐presentation of transferred DC via resident DC of the host and cross‐activation with natural killer (NK) cells as mechanisms supporting the induction of protective immunity after DC‐mediated vaccination. Co‐culture with DC that had been conditioned ex vivo by loading with L. major lysate and stimulation with CpG‐containing oligodeoxynucleotides did not result in the activation of naive DC in vitro. Furthermore, L. major antigen from conditioned DC was not cross‐presented to a significant extent in vivo. In contrast, co‐culture of DC with NK cells led to cross‐activation of both cell populations with induction of interferon‐γ, which was dependent on the activation status of the conditioned DC. Transient depletion of NK cells during vaccination of L. major‐susceptible mice with conditioned DC resulted in reduced protection. Our findings indicate that cross‐presentation of conditioned DC after DC‐based vaccination against L. major plays a minor role in the induction of protective immunity. However, we demonstrated for the first time that the capacity of DC to mediate protection against L. major is supported by cross‐activation with NK cells of the host and NK‐cell‐derived interferon‐γ.     

DNA-Salmonella enterica serovar Typhimurium primer-booster vaccination biases towards T helper 1 responses and enhances protection against Leishmania major infection in mice

Successful resolution of infections by intracellular pathogens requires gamma interferon (IFN-gamma). DNA vaccines promote T helper 1 (Th1) responses by triggering interleukin-12 (IL-12) release by dendritic cells (DC) through Toll-like receptor 9 (TLR9). In humans TLR9 is restricted to plasmacytoid DC. Here we show that DNA-Salmonella enterica serovar Typhimurium primer-booster vaccination, which provides alternative ligands to bind TLR4 on myeloid DC, strongly biases towards Th1 responses compared to vaccination with DNA alone. This results in higher immunoglobulin G2a (IgG2a) responses compared to IgG1 responses, higher IFN-gamma responses compared to IL-10 CD4(+)-T-cell responses, and enhanced protection against Leishmania major infection in susceptible BALB/c mice.     

The differential effect of stress on natural killer T (NKT) and NK cell function

When C57Bl/6 mice were exposed to restraint stress for 12 h or 24 h, lymphocytopenia was induced in the liver, spleen, and thymus. We examined which types of lymphocytes were sensitive or resistant to such stress by a immunofluorescence test. T cells of thymic origin were sensitive while NKT and NK cells were resistant. In contrast to the increase in the proportion of NK cells, NK activity of liver lymphocytes against YAC-1 targets decreased at 24 h after stress. On the other hand, their NKT cytotoxicity against syngeneic thymocytes increased in parallel with an increase in their proportion. In perforin -/- B6 mice and B6-gld/gld (Fas ligand-) mice, NK cells were found to mediate cytotoxicity through perforin while NKT cells mediated self-reactive cytotoxicity through Fas ligand. These results suggest that stress increases the proportion of both NK and NKT cells, but that NK cytotoxicity is suppressed while self-reactive NKT cytotoxicity is not, due to a diversity of their functional mechanisms.     

Human NKT cells direct the differentiation of myeloid APCs that regulate T cell responses via expression of programmed cell death ligands

NKT cells are innate lymphocytes that can recognize self or foreign lipids presented by CD1d molecules. NKT cells have been shown to inhibit the development of autoimmunity in murine model systems, however, the pathways by which they foster immune tolerance remain poorly understood. Here we show that autoreactive human NKT cells stimulate monocytes to differentiate into myeloid APCs that have a regulatory phenotype characterized by poor conjugate formation with T cells. The NKT cell instructed myeloid APCs show elevated expression of the inhibitory ligand PD-L2, and blocking PD-L1 and PD-L2 during interactions of the APCs with T cells results in improved cluster formation and significantly increased T cell proliferative responses. The elevated expression of PD-L molecules on NKT-instructed APCs appears to result from exposure to extracellular ATP that is produced during NKT-monocyte interactions, and blocking purinergic signaling during monocyte differentiation results in APCs that form clusters with T cells and stimulate their proliferation. Finally, we show that human monocytes and NKT cells that are injected into immunodeficient mice co-localize together in spleen and liver, and after 3 days in vivo in the presence of NKT cells a fraction of the myeloid cells have upregulated markers associated with differentiation into professional APCs. These results suggest that autoreactive human NKT cells may promote tolerance by inducing the differentiation of regulatory myeloid APCs that limit T cell proliferation through expression of PD-L molecules.     

Immunology in the Clinic Review Series; focus on host responses: invariant natural killer T cell activation following transplantation

Invariant natural killer T (iNKT) cells have been shown to play a key role in the regulation of immunity in health and disease. However, iNKT cell responses have also been found to influence both rejection and the induction of tolerance following transplantation of allogeneic cells or organs. Although a number of mechanisms have been identified that lead to iNKT cell activation, how iNKT cells are activated following transplantation remains unknown. This review will attempt to identify potential mechanisms of iNKT cell activation in the context of transplantation by applying knowledge garnered from other disease situations. Furthermore, we put forward a novel mechanism of iNKT cell activation which we believe may be the dominant mechanism responsible for iNKT activation in this setting, i.e. bystander activation by interleukin-2 secreted by recently activated conventional T cells.     

Role of human non-invariant NKT lymphocytes in the maintenance of type 2 T helper environment during pregnancy

The molecular and cellular mechanisms that generate the T(h)2 cytokine environment necessary for the maintenance of pregnancy are still not fully understood. We herein show that the human decidua is highly enriched for TCR alpha beta(+)CD161(+) NKT cells. They express non-invariant antigen receptors encoded by diverse TCRV alpha- and V beta-chain gene segments, thereby referred to as non-invariant NKT (non-iNKT) cells. In spite of their diverse TCR expression, they do not recognize fetal allo-antigens but specifically responded to CD1d-transfected cell lines. In contrast to the peripheral blood non-iNKT cells, the decidua-residing non-iNKT cells had a marked T(h)2 bias. In addition, they suppress the mixed leukocyte reaction directed against the paternal antigens. The T(h)2 cytokines have been known to stimulate trophoblast outgrowth and invasion. Thereby, the non-iNKT cells residing in the decidual tissue may have a functionally important interaction with the villous and extravillous trophoblast cells expressing CD1d and may therefore play a pivotal role in successful pregnancy by inhibiting fetal rejection and enhancing placental growth. These findings may reflect one mechanism that is an essential component for the T(h)2 environment necessary for the maintenance of pregnancy.     

Harnessing natural killer T (NKT) cells in human myeloma: progress and challenges

Multiple myeloma is a hematologic malignancy characterized by growth of malignant plasma cells in the bone marrow. Tumor cells in myeloma express CD1d and are sensitive to lysis by CD1d restricted natural killer T (NKT) cells. Here we discuss recent studies to harness the properties of these cells in the context of human myeloma. In spite of large body of preclinical data, attempts to fully harness the properties of these cells in the clinic are in early stages. Early phase clinical studies document the capacity of human monocyte-derived dendritic cells to expand NKT cells in vivo in myeloma patients. These results have set the stage for ongoing studies combining NKT activation with immune-modulatory drugs. Lessons learnt from these studies may inform the optimal application of human NKT based therapies in other settings as well.     

Distinct roles for IL-6 and IL-12p40 in mediating protection against Leishmania donovani and the expansion of IL-10+ CD4+ T cells

Adoptive dendritic cell (DC) immunotherapy provides a useful experimental tool to evaluate immunoregulation in vivo and has previously been successfully used to enhance host resistance in a variety of experimental models of leishmaniasis. Here, we used this approach to identify IL-6 and IL-12p40 as critical cytokines that cooperate to mediate host protection to Leishmania donovani but which act independently to regulate expansion of IL-10(+) CD4(+) T cells, shown here for the first time to be associated with this infection. Adoptive transfer of LPS-activated bone marrow-derived DC (BMDC) from wild-type mice was therapeutically beneficial and led to enhanced resistance as measured by spleen parasite burden. In contrast, IL-6- or IL-12p40-deficient BMDC had no protective benefit, indicating that production of both cytokines was essential for the therapeutic efficacy of DC. IL-10 production by CD25(-) FoxP3(-) IL-10(+) CD4(+) T cells is a strong correlate of disease progression, and BMDC from wild-type mice inhibited expansion of these cells. Strikingly, IL-12-deficient BMDC could also inhibit the expansion of this T cell population whereas IL-6-deficient BMDC could not, indicating that IL-6 played a key role in this aspect of DC function in vivo. Breadth of cytokine production is thus an important factor when considering strategies for DC-based interventions.     

Ag85A DNA疫苗加强免疫显著提高卡介苗初免小鼠的抗结核T细胞免疫应答

目的 构建表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)免疫优势抗原Ag85A的DNA疫苗,分析其加强免疫后提高卡介苗(BCG)初免小鼠的抗结核T细胞免疫应答.方法 以Mtb毒株H37Rv基因组DNA为模板,PCR扩增Ag85A抗原编码的结构基因并克隆至真核表达载体pVAX1中构建其DNA疫苗;接着,将纯化后的该DNA疫苗加强免疫BCG初免小鼠2针,以BCG和DNA单独免疫小鼠为对照,免疫8周后无菌分离脾淋巴细胞,分别应用IFN-γ ELISPOT和多因子胞内流式细胞术(intracellular staining)分析免疫小鼠的Mtb抗原特异性效应细胞免疫水平与分泌IFN-γ/TNF-α/IL-2的多功能CD4+T细胞频率及其强度以及CD8+T细胞免疫应答.结果 与BCG免疫及DNA单独免疫组相比,Ag85A DNA加强免疫不仅能显著提高小鼠IFN-γ+TNF-α+IL-2+多功能T细胞,IFN-γ+IL-2+、IL-2+TNF-α+双功能T细胞与IL-2+单功能T细胞的频率以及IL-2的分泌能力,还能显著诱导小鼠产生更多分泌IFN-γ和IL-2的CD8+T细胞.结论 本研究成功构建了表达Mtb免疫优势抗原Ag85A的DNA疫苗并分析了其免疫原性,证实了BCG初免-DNA加强的免疫策略可同时显著增强实验小鼠的Mtb抗原特异性CD4+T和CD8+T细胞应答水平,有利于提高BCG的免疫原性,为增强BCG逐渐下降的抗结核保护效果提供新思路.     

Protective anti-mycobacterial T cell responses through exquisite in vivo activation of vaccine-targeted dendritic cells

Vaccine efficacy largely depends upon DC targeting and activation. The most potent TLR soluble ligands induce diffuse DC activation, which may be associated with marked pro-inflammatory responses and possibly adverse effects. This raises the concern that effective vaccine adjuvants may similarly rely on widespread DC activation. Using a promising candidate vaccine against tuberculosis (fusion protein of Ag85B and 6-kDa early secretory antigenic target (ESAT-6)) formulated in the potent IC31 adjuvant, DC targeting and activation was studied in vivo, following the fate of antigen and adjuvant in the draining lymph nodes, to define the magnitude of DC targeting/activation required in vivo to induce protective vaccine responses. Unexpectedly, protective IFN-gamma-mediated Ag85B-ESAT-6/IC31 responses were associated to the activation of a minute population (less than 0.3%) of CD11c(+) lymph node DC, without detectable systemic pro-inflammatory responses. This activated peripheral tissue-derived DC population, characterized by enhanced CD80, CD86, CD40 and IL-12p40 expression, was only identified when focusing on adjuvant- or antigen-labeled CD11c(+) DC, which were found to support T cell proliferation. Immunization with aluminum hydroxide adjuvant (Alum) resulted in a similar proportion of antigen-associated DC but without detectable enhancement of CD80, CD86, CD40 or IL-12p40 expression. Thus, potent protective IFN-gamma-producing responses may be elicited by the exquisite activation of a minute number of in vivo targeted DC.     

CD1d-restricted NKT cell activation enhanced homeostatic proliferation of CD8+ T cells in a manner dependent on IL-4

CD1d-restricted NKT cells are activated by TCR-mediated stimulation via CD1d plus lipid antigens such as alpha-galactosylceramide (alpha-GalCer). These cells suppressed autoimmunity and graft rejection, but sometimes enhanced resistance to infection and tumor immunity. This double-action phenomenon of NKT cells is partly explained by cytokines produced by NKT cells. Therefore, roles of cytokines from activated NKT cells have been extensively examined; however, their roles on T cell homeostatic proliferation in lymphopenic condition have not been investigated. Here, we showed that alpha-GalCer enhanced homeostatic proliferation of CD8+ but not CD4+ T cells and this effect of alpha-GalCer was required for NKT cells. IL-4 was essential and sufficient for this NKT cell action on CD8+ T cell homeostatic proliferation. Importantly, the expression of IL-4Ralpha and STAT6 in CD8+ T cells was essential for the NKT activity, indicating a direct action of IL-4 on CD8+ T cells. Consistent with this, the level of IL-4Ralpha expression on memory phenotype CD8(+) T cells was higher than that on naive phenotype one and CD4+ T cells. Thus, these results showed the 'involvement' of IL-4 that is produced from activated NKT cells for CD8+ T cell homeostatic proliferation in vivo.     

Vaccination with plasmacytoid dendritic cells induces protection against infection with Leishmania major in mice

DC-based vaccination against Leishmania major induces a parasite-specific Th1 response and long-lasting protective immunity in susceptible mice. Since distinct DC subsets have been proposed to direct the predominant development of either Th1 or Th2 cells, we analyzed the capability of plasmacytoid DC (pDC) to induce protection and elicit a Th1 response against L. major. Pulsing with L. major lysate induced the activation and maturation of semi-mature murine pDC that had been isolated from the spleen, as indicated by up-regulation of the co-stimulatory molecules CD86 and CD80, but did not enhance the level of IFN-alpha secretion by pDC. Vaccination of susceptible mice with L. major lysate-pulsed pDC induced highly effective T cell-mediated immunity against subsequent infection with L. major parasites. Surprisingly, the protection was not accompanied by a polarized Th1 cytokine profile. Co-activation of pDC with CpG-containing oligodeoxynucleotides, which has been shown to be critical for activating the protective potential of myeloid DC, was not required for the protective effect of L. major antigen-pulsed pDC. These findings demonstrate that antigen-loaded pDC are able to induce T cell-mediated protection against a parasite disease and that experimental leishmaniasis is a suitable model to elucidate the mechanisms underlying DC-based vaccination against infections.     

Innate and adaptive stimulation of murine diverse NKT cells result in distinct cellular responses

Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα‐chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll‐like receptor (TLR) ligand activation of TCR‐transgenic murine dNKT cells. IFN‐γ production by dNKT cells required dendritic cells (DC), cell‐to‐cell contact and presence of TLR ligands. TLR‐stimulated DC activated dNKT cells to secrete IFN‐γ in a CD1d‐, CD80/86‐ and type I IFN‐independent manner. In contrast, a requirement for IL‐12p40, and a TLR ligand‐selective dependence on IL‐18 or IL‐15 was observed. TLR ligand/DC stimulation provoked early secretion of pro‐inflammatory cytokines by both CD62L⁺ and CD62L^? dNKT cells. However, proliferation was limited. In contrast, TCR/co‐receptor‐mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L^? dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co‐receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory.     

gammadelta T cells condition dendritic cells in vivo for priming pulmonary CD8 T cell responses against Mycobacterium tuberculosis

gammadelta T cells and dendritic cells are quickly recruited to the lungs shortly after intranasal vaccination with BCG, but the functional in vivo interplay between these two cell populations and its role in the induction of adaptive immune responses is unclear. Using TCR-deficient mice and bone marrow chimeras, we show here that gammadelta T cells provide a non-redundant early source of IFN-gamma in vivo, which enhances IL-12 production by lung dendritic cells. The in vivo-conditioned dendritic cells, in turn, prime a more efficient lung CD8 T cell response against Mycobacterium tuberculosis. Thus, strategies exploiting gammadelta T cell function and IFN-gamma production could be valuable for the design and testing of mucosal vaccines.     

Decreased IL-10 and IL-13 production and increased CD44hi T cell recruitment contribute to Leishmania major immunity induced by non-persistent parasites

Leishmaniasis is currently classified as category 1 disease, i.e. emerging and uncontrolled. Since the importance of persistent infection for maintaining an effective long-lasting protective response is controversial, the present study asks whether immunisation with non-persistent parasites leads to protection against Leishmania infection and to the recruitment of T cells of a specific phenotype. Our study shows that vaccination of susceptible BALB/c mice with live Leishmania major phosphomannomutase-deficient parasites, which are avirulent and non-persistent in vivo, leads to protection against infection. Immunisation with phosphomannomutase-deficient parasites neither leads to differences in IFN-gamma, IL-12, IL-4 production nor alters the expression of effector and memory markers, including CD62L, IL-7Ralpha and IL-2Ralpha, when compared with unvaccinated controls. Observed protection is due to the ability of vaccinated animals to suppress early IL-10 and IL-13 production and to recruit a higher number of antigen-experienced CD44hiCD4+ and CD44hiCD8+ T cells into draining LN following infection. Thus, expansion of T-cell numbers and their rapid recruitment to LN upon infection as well as the restriction of IL-13 and IL-10 production leading to high IFN-gamma/IL-10 ratio play an important role in protection against Leishmania affecting the outcome of the disease in favour of the host.