Posttranscriptional regulation of II10 gene expression allows natural killer cells to express immunoregulatory function.

Natural killer (NK) cells play a well-recognized role in early pathogen containment and in shaping acquired cell-mediated immunity. However, indirect evidence in humans and experimental models has suggested that NK cells also play negative regulatory roles during chronic disease. To formally test this hypothesis, we employed a well-defined experimental model of visceral leishmaniasis. Our data demonstrated that NKp46(+)CD49b(+)CD3(-) NK cells were recruited to the spleen and into hepatic granulomas, where they inhibited host protective immunity in an interleukin-10 (IL-10)-dependent manner. Although IL-10 mRNA could be detected in activated NK cells 24 hr after infection, the inhibitory function of NK cells was only acquired later during infection, coincident with increased IL-10 mRNA stability and an enhanced capacity to secrete IL-10 protein. Our data support a growing body of literature that implicates NK cells as negative regulators of cell-mediated immunity and suggest that NK cells, like CD4(+) T helper 1 cells, may acquire immunoregulatory functions as a consequence of extensive activation.

Leishmania donovani-induced expression of signal regulatory protein alpha on Kupffer cells enhances hepatic invariant NKT-cell activation.

Signal regulatory protein alpha (SIRPalpha) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRPalpha-CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN-gamma from splenic iNKT cells following exposure to the alphaGalCer analogue PBS-57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRPalpha was undetectable in the liver of uninfected mice, the hepatic iNKT-cell response to infection was also impaired in CD47-/- mice. However, we found that SIRPalpha was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G-protein-coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection.

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.

Loss of dendritic cell migration and impaired resistance to Leishmania donovani infection in mice deficient in CCL19 and CCL21.

The encounter between APC and T cells is crucial for initiating immune responses to infectious microorganisms. In the spleen, interaction between dendritic cells (DC) and T cells occurs in the periarteriolar lymphoid sheath (PALS) into which DC and T cells migrate from the marginal zone (MZ) along chemokine gradients. However, the importance of DC migration from the MZ into the PALS for immune responses and host resistance to microbial infection has not yet been elucidated. In this study, we report that following Leishmania donovani infection of mice, the migration of splenic DC is regulated by the CCR7 ligands CCL19/CCL21. DC in plt/plt mutant mice that lack these chemokines are less activated and produce less IL-12, compared with those in wild-type mice. Similar findings are seen when mice are treated with pertussis toxin, which blocks chemokine signaling in vivo. plt/plt mice had increased susceptibility to L. donovani infection compared with wild-type mice, as determined by spleen and liver parasite burden. Analysis of splenic cytokine profiles at day 14 postinfection demonstrated that IFN-gamma and IL-4 mRNA accumulation was comparable in wild-type and plt/plt mice. In contrast, accumulation of mRNA for IL-10 was elevated in plt/plt mice. In addition, plt/plt mice mounted a delayed hepatic granulomatous response and fewer effector T cells migrated into the liver. Taken together, we conclude that DC migration from the MZ to the PALS is necessary for full activation of DC and the optimal induction of protective immunity against L. donovani.

Adoptive immunotherapy against experimental visceral leishmaniasis with CD8+ T cells requires the presence of cognate antigen.

CD8+ T cells have a protective role in experimental visceral leishmaniasis. However, the observation that inflammatory cytokines induce bystander activation of CD8+ T cells questions the need for antigen-dependent effector function. Here, we demonstrate that successful adoptive immunotherapy with CD8+ T cells is strictly dependent upon the presence of cognate antigen.

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.

Invariant NKT cells are essential for the regulation of hepatic CXCL10 gene expression during Leishmania donovani infection.

Gamma interferon (IFN-gamma)-regulated chemokines of the CXC family have been implicated as key regulators of a variety of T-cell-dependent inflammatory processes. However, the cellular source(s) of IFN-gamma that regulates their early expression has rarely been defined. Here, we have directly addressed this question in mice after Leishmania donovani infection. Comparison of CXCL10 mRNA accumulation in normal and IFN-gamma-deficient mice confirmed an absolute requirement for IFN-gamma for sustained (24 h) expression of CXCL10 mRNA accumulation in this model. In normal mice, IFN-gamma was produced by both CD3int NK1.1+ NKT cells and CD3- NK1.1+ NK cells, as detected by intracellular flow cytometry. Strikingly, B6.Jalpha281-/- mice lacking NKT cells that express the invariant Valpha14Jalpha18 T-cell-receptor alpha chain, although retaining a significant population of IFN-gamma-producing NK cells and NKT cells, were unable to sustain CXCL10 mRNA accumulation. These data indicate that invariant NKT cells are indispensable for the regulation of hepatic CXCL10 gene expression during L. donovani infection.

Immunotherapy with OX40L-Fc or anti-CTLA-4 enhances local tissue responses and killing of Leishmania donovani.

Enhancing granuloma development and effector function, but without inducing the pathology associated with excess granulomatous inflammation, poses a major challenge for immunotherapeutic intervention against diseases such as visceral leishmaniasis (VL). Here, we demonstrate that a chimeric fusion protein (OX40L-Fc) which stimulates T cells through OX40 and a monoclonal antibody which blocks CTLA-4, an inhibitory receptor on T cells, both enhanced the rate of granuloma maturation, CD4(+) T cell proliferation, and killing of Leishmania. Costimulation-based therapy induced no adverse fibrotic or necrotic reactions, and had no significant effect on the levels of endogenous anti-inflammatory cytokines (IL-10 and TGF-beta). Furthermore, both OX40L-Fc and anti-CTLA4 could be co-administered with conventional anti-leishmanial drugs. Until now, enhancing T cell immunity by the manipulation of costimulatory pathways has only received serious attention for cancer immunotherapy, but our data provide a compelling argument for the evaluation of this approach in human VL and other infectious diseases.

The immunopathology of experimental visceral leishmaniasis.

Experimental murine infection with the parasites that cause human visceral leishmaniasis (VL) results in the establishment of infection in the liver, spleen, and bone marrow. In most strains of mice, parasites are eventually cleared from the liver, and hepatic resistance to infection results from a coordinated host response involving a broad range of effector and regulatory pathways targeted within defined tissue structures called granulomas. In contrast, parasites persist in the spleen and bone marrow by mechanisms that are less well understood. Parasite persistence is accompanied by the failure of granuloma formation and by a variety of pathologic changes, including splenomegaly, disruption of lymphoid tissue microarchitecture, and enhanced hematopoietic activity. Here, we review the salient features of these distinct tissue responses and highlight the varied roles that cytokines of the tumor necrosis factor family play in immunity to this infection. In addition, we also discuss recent studies aimed at understanding how splenomegaly affects the survival and function of memory cells specific for heterologous antigens, an issue of considerable importance for our understanding of the disease-associated increase in secondary infections characteristic of human VL.