Fast high yield of pure Leishmania (Leishmania) infantum axenic amastigotes and their infectivity to mouse macrophages

Leishmania (L.) infantum (syn. Leishmania chagasi) is a dimorphic protozoan parasite that lives in promastigote and amastigote form in its sandfly vector and mammalian hosts, respectively. Here, we describe an in vitro culture system for the generation of a pure population of L. infantum axenic amastigotes after only 4 days incubation in culture medium supplemented with fetal calf serum, human urine, l-glutamine, and HEPES at 37oC (pH 5.5). Ultrastrutural analysis and infection assays in two macrophage populations (Kupffer cells (KUP) and peritoneal macrophages (PM)) infected with axenic amastigotes demonstrated that they maintained morphological and biochemical (A2 expression) features and a similar infection pattern to tissue-derived L. infantum amastigotes. The susceptibility of the macrophage lines to axenic or tissue-derived amastigotes and promastigotes was investigated. We found a completely different susceptibility profile for KUP and PM. Liver macrophages, both KUP and immigrant macrophages, are intimately involved in the response to L. infantum infection; this difference in susceptibility is probably related to their capacity to eliminate these parasites. Our in vitro system was thus able to generate axenic amastigotes that resemble tissue-derived amastigotes both in morphology and infectivity pattern; this will help in further investigation of the biological characteristics of the host–parasite relationship as well as the process of pathogenesis.     

Antibiotic Transport in Bacteria

Abstract

Survival or destruction of intramacrophage pathogen Leishmania depends in part on modulation of their host cell phagosome, capabilities of the infected macrophages to present parasite antigen to the host's immune system. Macrophages house these parasites as amastigotes in the acidic phagolysosomal compartment. Leishmania phagolysosome is the potential site for processing and presentation of its antigen as well as being the target site for chemotherapy in leishmaniasis. It is thought that the parasites are killed from macrophage activation by lymphokines secreted from either helper T1 cells or CD8⁺ T cells. Characterization of both the host and parasite molecules in the compartment in the context of biogenesis of Leishmania-phagolysosome and processing of the parasite antigen by this compartment are discussed. Trafficking of different drugs and new agents through this compartment and their role in chemotherapy and necessity of developing new drug carrier are also stressed.     

Leishmania mexicana promastigotes induce cytotoxic T lymphocytes in vivo that do not recognize infected macrophages

The question is addressed whether antigens of Leishmania, a parasite residing in the endosomal compartment of macrophages, can be presented in the context of major histocompatibility complex class I molecules. We used E. coli β-galactosidase as a model antigen which can be expressed in high levels in L. mexicana promastigotes (L. mexicana-gal). Infection of BALB/c mice with L. mexicanagal induces β-galactosidase-specific cytotoxic T cells (CTL), which can be isolated using a β-galactosidase-expressing mastocytoma line as an antigen-presenting cell. These CTL recognize epitopes of β-galactosidase in the context of H-2K^d; however, they do not recognize L. mexicanagal-infected macrophages even after killing of the intracellular amastigotes by drug treatment or macrophage activation by lymphokines, although class I-peptide interaction and the presentation of endogenously produced antigens is normal. It is concluded that parasite antigens can induce a CTL response in vivo but that these CTL cannot recognize infected macrophages because the relevant epitopes cannot gain access to class I molecules. The effect of priming in vivo may be explained by the well-known but ill-understood phenomenon of cross-priming.     

Leishmania-infected macrophages sequester endogenously synthesized parasite antigens from presentation to CD4+ T cells

CD4⁺ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the 14.07 macrophage cell line (H-2^k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4⁺ T cells. In contrast, promastigote-infected macrophages did present endogenous parasite molecules to CD4⁺ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post-infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4⁺ T cells, a major and critical anti-leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.     

免疫小鼠及正常小鼠巨噬细胞对利什曼无鞭毛期的吞噬作用

内脏利什曼原虫主要寄生在巨噬细胞系统的单核吞噬细胞内,在一般情况下其无鞭毛期能抵抗巨噬细胞的杀灭作用。 为了观察经杜氏利什曼原虫免疫后的小鼠其巨噬细胞的作用,我们采用了CFW纯系小鼠,经不同免疫方法于免疫后不同时间观察了体外培养中巨噬细胞的吞噬功能。实验采用的巨噬细胞与杜氏利什曼原虫前鞭毛期的比例为1:4。从每24小时吞噬功能的结果表明,经利什曼鞭毛体纯抗原免疫及福氏佐剂加利什曼抗原免疫的两组小鼠,均以免疫后3周的吞噬率最高,分别为72％及96％;两组吞噬指数的均值±SD(4.46±1.72,6.99±4.36)亦较正常组小鼠(1.68±1.25,1.72±1.15)为高,并具有显著差异(P<0.05)。提示了特异性抗原以及与佐剂合并具有对吞噬功能的激活作用。实验并观察了巨噬细胞内利什曼原虫无鞭毛期的活力作用,从吞噬原虫后20小时开始至 144小时,正常小鼠巨噬细胞内的无鞭毛期再经三恩氏培养基培养后均能恢复为前鞭毛期,而经免疫小鼠巨噬细胞内的利什曼原虫无鞭毛期在72小时后即消失活力。 另外,对小鼠腹腔巨噬细胞吞噬利什曼原虫的动态亦作了仔细观察。 实验结果说明了经过免疫的小鼠,由于被淋巴细胞激活后的巨噬细胞能杀死利什曼原虫,巨噬细胞在宿主对感染应答中是一个重要部分,对于探索黑热病的免疫机理具有一     

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface

Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.     

Leishmania mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages

Macrophages of the cell line J774 were used in a comparative study of virulence involving amastigote stages ofLeishmania mexicana pifanoi isolated from macrophages (AMA-M) of the aforementioned cell line, amastigote forms grown in the UM-54 cell-free medium (AMA-C), and promastigote stages. The macrophage cultures were inoculated with AMA-M and AMA-C at the culture cell to parasite ratios of 13, 15, and 110. The macrophages were exposed to either kind of amastigotes for 24, 48, and 72 h. At the end of each of these periods, and for each dilution, the percentages of macrophages harboring the parasites within their cytoplasm and the mean numbers of intracellular parasites/macrophage were estimated on the basis of examination of 200 phagocytes. When either AMA-M or AMA-C were employed, after 24 h, the percentages of infected macrophages were, respectively, 84.5%, 89.0%, and 94.5% for the three aforementioned dilutions, the majority of the phagocytes containing 1–5 parasites. After 48- and 72-h exposures, the macrophages harbored 6–11 and 11–20 amastigotes/cell, respectively. Evidently intracellular multiplication of the amastigotes has taken place. In contrast to the results obtained with the amastigote forms, after inoculations of the macrophage cultures with promastigotes at the dilutions previously used for amastigotes, only 48–78 phagocytes were found to contain intracellular stages within their cytoplasm. Many macrophages were parasite-free, especially when exposed to fewer promastigotes. Experiments in which 5×10⁶ promastigotes, AMA-M, or AMA-C were inoculated into the footpads of hamsters yielded the following results with regard to terminal footpad volumes: 1.57, 3.31, and 3.32 cm³, respectively. Evidently both kinds of amastigotes had equal virulence for hamsters; however, the promastigote stages were much less virulent for these experimental hosts.This report is part of a dissertation submitted to the Graduate School, University of Massachusetts at Amherst, in partial fulfillment of the requirements for the Doctor of Philosophy degree in Zoology     

Intracellular co-localization of Trypanosoma cruzi and inducible nitric oxide synthase (iNOS): evidence for dual pathway of iNOS induction

Evidence is presented from studies in vitro and in vivo for a dual pathway of inducible nitric oxide synthase (iNOS) induction during Trypanosoma cruzi infection, one of which is interferon (IFN)-γ dependent and the other not. In vitro, the IFN-γ-dependent iNOS induction decreases parasite multiplication, and is in vivo associated with protection. iNOS induced by this pathway mediated a high NO output and showed a diffuse, cytoplasmic immunostaining in IFN-γ-activated macrophages in vitro as well as in cell infiltrates or infected tissues. Surprisingly, in such tissues, iNOS co-localized with parasite nests, and by immunoelectromicroscopy, iNOS was demonstrated on the parasite surface. iNOS co-localization with parasites was also seen in tissues from T. cruzi-infected IFN-γ receptor (R) knockout mice suggesting an IFN-γ-independent pathway of induction. However, no cytoplasmic iNOS was seen in inflammatory infiltrates of these tissues. IFN-γR^−/− mice displayed a dramatically enhanced susceptibility to infection with T. cruzi, diminished accumulation of iNOS mRNA in skeletal muscle and spleen cells, and reduced release of NO and per-oxynitrite. Expression of iNOS around intracellular parasites was also observed after infection of peritoneal macrophages or L-929 fibroblasts in vitro in the absence of other exogenous stimuli. A time-dependent NO release and enhanced accumulation of iNOS mRNA also was observed in infected peritoneal cells and fibroblasts. Cultured T. cruzi amastigotes, trypomastigotes, and epimastigotes were not labeled by the anti-iNOS antibodies and contained no iNOS mRNA, indicating that the iNOS detected actually originated from the mammalian cell. A pathogenic effect of low NO levels is suggested by the arresting effect of NOS inhibitors and the enhancing consequences of low concentrations of NO donors on intracellular parasite multiplication.     

Histopathological Lesions in 15 Cats with Leishmaniosis

Recent research into the prevalence of Leishmania infantum infection in the Mediterranean basin points to the involvement of cats as a reservoir host, but only sporadic cases of feline leishmaniosis have been reported. Feline leishmaniosis presents primarily as cutaneous disease and diagnosis is based on the demonstration of the parasite by skin biopsy. The present report describes the microscopical changes in tissue biopsies from 15 cats with leishmaniosis. The biopsies were derived from the skin, ocular tissue and mucocutaneous junctions. The most common histopathological feature was diffuse granulomatous inflammation with macrophages containing numerous amastigotes. Other patterns included granulomatous perifolliculitis and lichenoid interface dermatitis, where there were fewer parasitized macrophages. The presence of amastigotes was confirmed by immunohistochemistry in each case. The results of the study confirm the value of histopathological and immunohistochemical techniques for the diagnosis of feline leishmaniosis.     

Antigen presentation by Leishmania mexicana-infected macrophages: Activation of helper T cells specific for amastigote cysteine proteinases requires intracellular killing of the parasites

Leishmania mexicana amastigotes proliferate in the phagolysosomes of mammalian macrophages. The parasites abundantly synthesize lysosomal cysteine proteinases, which are encoded by the lmcpb gene family. One of these genes was overexpressed in Escherichia coli, and the purified recombinant protein was used as an antigen to induce and establish a T helper 1 (T_h1) cell line. The T cells recognize epitopes shared by the native cysteine proteinases and the recombinant protein. Infected bone marrow-derived macrophages induced to express major histocompatibility complex class II molecules by interferon (IFN)-γ do not affect parasite viability. These macrophages fail to stimulate the proliferation of the T cell line. In contrast, strong T cell stimulation is observed after the parasites are killed by treatment with L-leucine methylester, or after activation of macrophages by IFN-γ and tumor necrosis factor-α. It is concluded that infected macrophages efficiently present this lysosomal Leishmania antigen once the parasites are inactivated and degraded. This observation may be of considerable relevance for the outcome of Leishmania infections provided that it can be extended to other parasite antigens.     

Cytophilic and Opsonic Antibodies in Visceral Leishmaniasis in Mice

Although acquired immunity to leishmaniasis is generally considered to be cell mediated, humoral factors may be partially responsible. The present study showed that antisera from C57BL/6J mice superinfected with Leishmania donovani contained cytophilic antibody and opsonins for both the amastigote and promastigote stages of the parasite. Macrophages treated with mouse hyperimmune serum in an in vitro macrophage culture system bound statistically significantly more parasites at 4°C (and subsequently phagocytized them at 37°C) than did macrophage cultures treated with control serum. The percentages of antibody-treated macrophages bearing and containing parasites were also significantly greater than the percentages of control serum-treated macrophages bearing and containing parasites, respectively. These differences persisted in cultures during a 9-day observation period when sera from mice killed 10 or 11 days after superinfection were used. However, when sera from mice killed 24 days after superinfection were tested with amastigotes, by day 9 the number of parasites and the percentage of cells parasitized in the culture decreased to control values or significantly below them. Thioglycolate-stimulated macrophages treated with hyperimmune serum bound more amastigotes at 4°C than did stimulated macrophages treated with control serum. Activated macrophages also demonstrated increased nonspecific binding of amastigotes. Treatment of macrophages with trypsin reduced both cytophilic antibody-specific and nonspecific binding of amastigotes. The demonstration of in vitro effects of anti-leishmanial antibody from superinfected mice might indicate a possible role for humoral antibody in immunity to leishmaniasis in mice.     

Subverted transferrin trafficking in Leishmania- infected macrophages

The intracellular fate of human transferrin (HTf) in macrophages infected by Leishmania was investigated. Binding of HTf-gold complexes at 4 °C was competitively inhibited by native holoHTf but not by apoHTf. Infected and uninfected macrophages displayed rather distinct HTf trafficking. Pulse-chase experiments using uninfected macrophages loaded with 15-nm gold-conjugated bovine serum albumin (BSA) and then incubated with 5-nm gold-conjugated HTf revealed a remarkable segregation of these tracers in distinct compartments. Nevertheless, Leishmania-infected macrophages presented extensive particle colocalization at both 60 min and 18 h. Light and electron microscopy immunolabeling indicated that HTf was delivered to the parasitophorous vacuole, formed patches on the amastigote surface, and was endocytosed via the flagellar pocket. Double-staining assays showed the colocalization of biotinylated HTf and its receptor in association with the parasitophorous vacuole. To approach the Tf-binding sites of amastigotes we performed HTf-fluorescein isothiocyanate (FITC) assays. Staining was diffuse at 4 °C and punctate at 35 °C, and only the former was sensitive to ethidium bromide, indicating an eventual temperature-dependent endocytic process. Within parasites, HTf was found in cysteine-proteinase-rich structures, suggesting that the protein can be endocytosed by intracellular amastigotes and sorted to the parasite endosomal-lysosomal compartments rather than being recycled. The treatment of infected macrophages with holoHTf, but not apoHTf, promoted the parasite's intracellular survival. These results suggest that Leishmania amastigotes can exploit and subvert the host-cell endocytic system and indicate the role of Tf-carried iron in the outcome of leishmanial infection. Received: 20 May 1998 / Accepted: 18 June 1998     

Comparative analysis of the nitric oxide production by<Emphasis Type="Italic"> Leishmania</Emphasis> sp.

The present report explores a comparative analysis of nitric oxide (NO^·) production by three different species of Leishmania (L. amazonensis, L. braziliensis and L. chagasi). Among these species, L. braziliensis produced the highest amount of NO^·, measured in the supernatants of promastigotes cultures as nitrite, a stable by-product derived from NO^·. We have previously described the expression of a constitutive nitric oxide synthase (cNOS) in L. amazonensis promastigotes and axenic amastigotes. Comparing those results with the present work, using immunofluorescence assay, it was shown that both L. braziliensis and L. chagasi also express a cNOS. Immunostaining experiments showed that promastigotes from early passages of these species in culture had a strong immunoreactivity against anti-cNOS and anti-endothelial cell NOS, in comparison with the same parasite cultured for long time, suggesting a correlation between the NO^· production and the presence of metacyclic forms prominent in those newly isolated parasites. These data corroborate findings of a higher NO^· production by those parasites, following the growth curve. The relationship between the two NO^·-generating systems in the parasite and in their host cell warrants further investigation. The presence of cNOS raises the possibility of a similar type of cross-talk or down-regulation between the NO^· signaling systems in host cells and the lower eukaryotic-like Leishmania sp.     

Leishmania expressed lipophosphoglycan interacts with Toll‐like receptor (TLR)‐2 to decrease TLR‐9 expression and reduce anti‐leishmanial responses

Two different Toll‐like receptors (TLRs) have been shown to play a role in host responses to Leishmania infection. TLR‐2 is involved in parasite survival in macrophages upon activation by lipophosphoglycan (LPG), a virulence factor expressed by Leishmania. In contrast, activation of TLR‐9 has been shown to promote a host‐protective response. However, whether there is a relationship between the interaction of LPG and TLR‐2, on one hand, with the effect of TLR‐9, on the other hand, remains unknown. In this study, we report that in‐vitro infection of macrophages with a L. major parasite with high expression levels of LPG results in decreased TLR‐9 expression compared to infection with a L. major parasite with lower expression levels of LPG. Addition of anti‐LPG as well as anti‐TLR‐2 antibodies prevents this reduction of TLR‐9 expression. Also, the addition of purified LPG to macrophages results in a decrease of TLR‐9 expression, which is shown to be mediated by transforming growth factor (TGF)‐β and interleukin (IL)‐10. Finally, in‐vitro treatment of macrophages with anti‐LPG and/or anti‐TLR‐2 antibodies before infection reduces the number of amastigotes in macrophages and co‐treatment of mice with anti‐TLR‐2 antibodies and cytosine–phosphate–guanosine (CpG) reduces footpad swelling and parasite load in the draining lymph nodes, accompanied by an interferon (IFN)‐γ‐predominant T cell response. Thus, for the first time, we show how interactions between LPG and TLR‐2 reduce anti‐leishmanial responses via cytokine‐mediated decrease of TLR‐9 expression.     

Leishmania donovani Ornithine Decarboxylase Is Indispensable for Parasite Survival in the Mammalian Host

Mutations within the polyamine biosynthetic pathway of Leishmania donovani, the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal Δodc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani. ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the Δodc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent Δodc knockout lines were decreased ~80% compared to their wild-type counterpart. Furthermore, α-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both Δodc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the Δodc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.     

Secreted proteophosphoglycan of Leishmania mexicana amastigotes activates complement by triggering the mannan binding lectin pathway

Cutaneous lesions induced by infection of mice with the protozoan parasite, Leishmania mexicana, contain abundant amounts of a high molecular mass proteophosphoglycan (PPG), which is secreted by the amastigote stage residing in phagolysosomes of macrophages and can then be released into the tissue upon rupture of the infected cells. Amastigote PPG forms sausage-shaped but soluble particles and belongs to a novel class of serine-rich proteins that are extensively O-glycosylated by phosphooligosaccharides capped by mannooligosaccharides. The purified molecule is shown here to efficiently activate complement (C) and deplete hemolytic activity of normal serum and may prevent the opsonization of L. mexicana amastigotes. Complement activation is Ca²⁺ dependent but does not depend on antibodies or the complement component C1. PPG binds to serum mannan binding protein (MBP), thus activating the MBP-associated serine protease, P100. Subsequently, the C cascade is triggered through C4 leading to covalent modification probably of carbohydrate hydroxyls of PPG by C3 fragments. Thus, PPG is able to activate C via the mannan binding lectin pathway which is unusual for secreted, soluble products of microbial origin. The proteophosphoglycan-induced complement activation is postulated to contribute to the lesion development and pathology caused by the parasite.     

Leishmania infantum-specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex-restricted manner

Protective immunity to leishmaniasis has been demonstrated in murine models to be mediated by T cells and the cytokines they produce. We have previously shown that resistance to experimental Leishmania infantum infection in the dog, a natural host and reservoir of the parasite, is associated with the proliferation of peripheral blood mononuclear cells (PBMC) to parasite antigen and to the production of interleukin-2 and tumour necrosis factor. In this study we show that PBMC from asymptomatic experimentally infected dogs produce interferon-γ upon parasite antigen-specific stimulation, whereas lymphocytes from symptomatic dogs do not. In addition, we report for the first time the lysis of L. infantum-infected macrophages by PBMC from asymptomatic dogs and by parasite-specific T cell lines derived from these animals. These T cell lines were generated by restimulation in vitro with parasite soluble antigen and irradiated autologous PBMC as antigen-presenting cells. We show that lysis of infected macrophages by T cell lines is major histocompatibility complex restricted. Characterization of parasite-specific cytotoxic T cell lines revealed that the responding cells are CD8⁺. However, for some animals, CD4⁺ T cells that lyse infected macrophages were also found. In contrast to asymptomatic dogs, lymphocytes from symptomatic dogs failed to proliferate and produce interferon-γ after Leishmania antigen stimulation in vitro and were not capable of lysing infected macrophages. These results suggest that both the production of interferon-γ and the destruction of the parasitized host cells by Leishmania-specific T cells play an important role in resistance to visceral leishmaniasis.     

Interleukin-10 from T Cells,but Not Macrophages and Granulocytes,Is Required for Chronic Disease in Leishmania mexicana Infection

Chronic cutaneous disease of mice caused by the protozoan parasite Leishmania mexicana requires interleukin-10 (IL-10) and FcγRIII (an activating IgG receptor). Macrophages readily secrete IL-10 in response to IgG-coated amastigotes, making macrophages a prime candidate as the critical source of IL-10. However, indirect evidence suggested that macrophage IL-10 is not essential for chronic disease. I now show directly that mice lacking IL-10 from macrophages and granulocytes still have chronic disease, like wild-type C57BL/6 mice. However, T cell-derived IL-10 is required for chronic disease. CD4-cre IL-10^flox/flox mice lack IL-10 from T cells (both CD4⁺ and CD8⁺) and heal their L. mexicana lesions, with parasite control. I had previously shown that depletion of CD25⁺ T cells had no effect on chronic disease, and thus, T cells other than CD25⁺ regulatory T (T_reg) cells should be the important source of IL-10. Given that conventional T cells do not express FcγRs, there is likely to be an indirect pathway by which FcγRIII on some other cell engaged by IgG1-amastigote immune complexes induces IL-10 from T cells. Further work is needed to delineate these pathways.