David L. Sacks

CD4 + CD25 + regulatory T cells control Leishmania major persistence and immunity

The long-term persistence of pathogens in a host that is also able to maintain strong resistance to reinfection, referred to as concomitant immunity, is a hallmark of certain infectious diseases, including tuberculosis and leishmaniasis. The ability of pathogens to establish latency in immune individuals often has severe consequences for disease reactivation. Here we show that the persistence of Leishmania major in the skin after healing in resistant C57BL/6 mice is controlled by an endogenous population of CD4+CD25+ regulatory T cells. These cells constitute 5–10% of peripheral CD4+ T cells in naive mice and humans, and suppress several potentially pathogenic responses in vivo, particularly T-cell responses directed against self-antigens. During infection by L. major, CD4+CD25+ T cells accumulate in the dermis, where they suppress—by both interleukin-10-dependent and interleukin-10-independent mechanisms—the ability of CD4+CD25- effector T cells to eliminate the parasite from the site. The sterilizing immunity achieved in mice with impaired IL-10 activity is followed by the loss of immunity to reinfection, indicating that the equilibrium established between effector and regulatory T cells in sites of chronic infection might reflect both parasite and host survival strategies.

The immunology of susceptibility and resistance to Leishmania major in mice

Established models of T-helper-2-cell dominance in BALB/c mice infected with Leishmania major — involving the early production of interleukin-4 by a small subset of Leishmania-specific CD4+ T cells — have been refined by accumulating evidence that this response is not sufficient and, under some circumstances, not required to promote susceptibility. In addition, more recent studies in L. major-resistant mice have revealed complexities in the mechanisms responsible for acquired immunity, which necessitate the redesign of vaccines against Leishmania and other pathogens that require sustained cell-mediated immune responses.

Phagosomes are competent organelles for antigen cross-presentation

The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections. It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic reticulum and presented at the cell surface on major histocompatibility complex (MHC) class I molecules, whereas peptides present in endo/phagocytic compartments are presented on MHC class II molecules. Despite the apparent segregation of the class I and class II pathways, antigens from intracellular pathogens including mycobacteria, Escherichia coli, Salmonella typhimurium, Brucella abortus and Leishmania, have been shown to elicit an MHC class-I-dependent CD8+ T-cell response, a process referred to as cross-presentation. The cellular mechanisms allowing the cross-presentation pathway are poorly understood. Here we show that phagosomes display the elements and properties needed to be self-sufficient for the cross-presentation of exogenous antigens, a newly ascribed function linked to phagocytosis mediated by the endoplasmic reticulum.

In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.

Infection with the obligate intracellular protozoan Leishmania is thought to be initiated by direct parasitization of macrophages, but the early events following transmission to the skin by vector sand flies have been difficult to examine directly. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and infected neutrophils efficiently initiated infection. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.m. appears to have evolved to both evade and exploit the innate host response to sand fly bite in order to establish and promote disease.

CD4+CD25-Foxp3- Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis

Nonhealing forms of leishmaniasis in humans are commonly associated with elevated levels of the deactivating cytokine IL-10, and in the mouse, normally chronic infections can be cleared in the absence of IL-10. Using a Leishmania major strain that produces nonhealing dermal lesions in a T helper type 1 (Th1) cell–polarized setting, we have analyzed the cellular sources of IL-10 and their relative contribution to immune suppression. IL-10 was produced by innate cells, as well as CD4+CD25+Foxp3+ and CD4+CD25−Foxp3− T cells in the chronic lesion. Nonetheless, only IL-10 production by antigen-specific CD4+CD25−Foxp3− T cells, the majority of which also produced IFN-γ, was necessary for suppression of acquired immunity in Rag−/− reconstituted mice. Surprisingly, Rag−/− mice reconstituted with naive CD4+ T cells depleted of natural T regulatory cells developed more severe infections, associated with elevated levels of IL-10 and, especially, Th2 cytokines in the site. The data demonstrate that IL-10–producing Th1 cells, activated early in a strong inflammatory setting as a mechanism of feedback control, are the principal mediators of T cell–derived IL-10–dependent immune suppression in a chronic intracellular infection.

A Natural Model of Leishmania major Infection Reveals a Prolonged “Silent” Phase of Parasite Amplification in the Skin Before the Onset of Lesion Formation and Immunity

A model of Leishmania major infection in C57BL/6 mice has been established that combines two main features of natural transmission: low dose (100 metacyclic promastigotes) and inoculation into a dermal site (the ear dermis). The evolution of the dermal lesion could be dissociated into two distinct phases. The initial “silent” phase, lasting 4–5 wk, favored establishment of the peak load of parasites in the dermis in the absence of lesion formation or any overt histopathologic changes in the site. The second phase corresponds to the development of a lesion associated with an acute infiltration of neutrophils, macrophages, and eosinophils into the dermis and was coincident with the killing of parasites in the site. The onset of immunity/pathology was correlated with the appearance of cells staining for IL-12p40 and IFN-γ in the epidermal compartment, and an expansion of T cells capable of producing IFN-γ in the draining lymph node. Parasite growth was not enhanced over the first 4.5 wk in anti-CD4-treated mice, SCID mice, or C57BL/6 mice deficient in IL-12p40, IFN-γ, CD40 ligand, or inducible NO synthase. These mice all failed to ultimately control infection in the site, but in some cases (anti-CD4 treated, IL-12p40−/−, CD40 ligand−/−, and SCID) high dermal parasite loads were associated with little or no pathology. These results extend to a natural infection model a role for Th1 cells in both acquired resistance and lesion formation, and document the remarkable avoidance of this response during a prolonged phase of parasite amplification in the skin.

Evasion of innate immunity by parasitic protozoa.

Parasitic protozoa are a major cause of global infectious disease. These eukaryotic pathogens have evolved with the vertebrate immune system and typically produce long-lasting chronic infections. A critical step in their host interaction is the evasion of innate immune defenses. The ability to avoid attack by humoral effector mechanisms, such as complement lysis, is of particular importance to extracellular parasites, whereas intracellular protozoa must resist killing by lysosomal enzymes and toxic metabolites. They do so by remodeling the phagosomal compartments in which they reside and by interfering with signaling pathways that lead to cellular activation. In addition, there is growing evidence that protozoan pathogens modify the antigen-presenting and immunoregulatory functions of dendritic cells, a process that facilitates their evasion of both innate and adaptive immunity.

Evidence that the High Incidence of Treatment Failures in Indian Kala-Azar Is Due to the Emergence of Antimony-Resistant Strains of Leishmania donovani

The possibility that the high frequency of treatment failures in Indian kala-azar might be due to infection with antimony-resistant strains of Leishmania donovani has not been experimentally addressed. L. donovani isolates were obtained from splenic aspiration smears of 24 patients in Bihar, India, who either did not respond (15) or did respond (9) to 1 or more full courses of treatment with sodium antimony gluconate (SAG). A strong correlation (P<.001) between clinical response and SAG sensitivity in vitro was observed only when strains were assayed as intracellular amastigotes: responsive isolates ED50=2.4+/-2.6, ED90=6.4+/-7.8 microgram SAG/mL; unresponsive isolates ED50=7.4+/-3.7 microgram SAG/mL, ED90=29.1+/-11.1 SAG/mL. No correlation with clinical response was found by use of extracellular promastigotes (ED50=48+/-22 vs. 52+/-29 microgram/mL). The emergence of antimony-resistant L. donovani strains appears to be a cause of treatment failures in India.

Role for CD4+ CD25+ Regulatory T Cells in Reactivation of Persistent Leishmaniasis and Control of Concomitant Immunity

Reactivation of dormant infections causes an immense burden of morbidity and mortality in the world at large. Reactivation can occur as a result of immunosuppression, environmental insult, or aging; however, the cause of reactivation of such infections is often not clear. We have previously shown that persistence of the parasite Leishmania major is controlled by endogenous CD4+ CD25+ regulatory T (T reg) cells. In this report, we show that despite efficient parasite clearance at secondary sites of infection, Leishmania superinfection can cause disease reactivation at the primary site. Our results strongly suggest that T reg cells, whose numbers increase in sites of reactivation, are directly responsible for such reactivation. Depletion of CD25+ cells at the time of secondary challenge prevented disease reactivation at the site of persistent infection while strengthening the expression of immunity at the site of secondary challenge. Finally, transfer of T reg cells purified from infected mice into chronically infected mice was sufficient to trigger disease reactivation and prevent the expression of an effector memory response. Our results demonstrate that after persistence is achieved, an equilibrium between T reg cells and effector lymphocytes, which can be disturbed by superinfection, controls the efficiency of recall immune responses and disease reactivation.

CD8+ T Cells Are Required for Primary Immunity in C57BL/6 Mice Following Low-Dose, Intradermal Challenge with Leishmania major

Standard murine models of cutaneous leishmaniasis, involving s.c. inoculation of large numbers of Leishmania major promastigotes, have not supported an essential role for CD8+ T cells in the control of primary infection. Recently, a L. major model combining two main features of natural transmission, low parasite dose and inoculation into a dermal site, has been established in resistant C57BL/6 mice. In the present studies, C57BL/6 mice with CD8+ T cell deficiencies, including CD8−/− and CD8-depleted mice, failed to control the growth of L. major following inoculation of 100 metacyclic promastigotes into the ear dermis. The resulting dermal pathology was minor and delayed. Lesion formation in wild-type mice was coincident with the killing of parasites in the inoculation site. Both events were associated with the accumulation of CD8+ T lymphocytes in the skin and with the capacity of CD8+ T cells recovered from draining lymph nodes or infected dermis to release IFN-γ following coculture with infected dendritic cells. Reconstitution of resistance to L. major in RAG−/− mice using T cells from naive donors was optimal when both CD4+ and CD8+ T cells were transferred. Primed CD8+ T lymphocytes obtained from C57BL/6 mice during the acute stage of infection were able to mediate both pathology and immunity when transferred alone. The low dose, intradermal challenge model reveals that CD8+ T cells play an essential role in both pathogenesis of and immunity to primary infection with L. major in the skin.