IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.     

Perforin mediated apoptosis of cerebral microvascular endothelial cells during experimental cerebral malaria

Cerebral malaria is a serious complication of Plasmodium falciparum infection. We have investigated the role of perforin in the pathogenesis of cerebral malaria in a murine model (Plasmodium berghei ANKA (PbA) infection). C57BL/6 mice demonstrated the typical neuropathological symptoms of experimental cerebral malaria infection from day 5p.i. and became moribund on day 6p.i. This pathology was not seen in PbA-infected, perforin-deficient (pfp-/-) mice. From days 5-6p.i. onwards there was a significant increase in mRNA for granzyme B and CD8, but not CD4, in brain tissue from PbA-infected C57BL/6 and pfp-/- mouse brains. Perforin mRNA was strongly increased in the brains of PbA-infected C57BL/6 mice on day 6p.i. Immunohistochemistry revealed increased perforin staining and elevated numbers of CD8(+) cells within the cerebral microvessels in PbA-infected C57BL/6 at days 5 and 6p.i. compared with uninfected animals. At day 6p.i., there were TUNEL-positive cells and activated caspase-3 positive cells of endothelial morphology in the CNS of PbA-infected C57BL/6 mice. The TUNEL-positive cells were greatly reduced in pfp-/- mice. These results suggest that CD8(+)T lymphocytes induce apoptosis of endothelial cells via a perforin-dependent process, contributing to the fatal pathogenic process in murine cerebral malaria.     

Malaria-specific and nonspecific activation of CD8+ T cells during blood stage of Plasmodium berghei infection

Cerebral malaria is one of the severe complications of Plasmodium falciparum infection. Studies using a rodent model of Plasmodium berghei ANKA infection established that CD8(+) T cells are involved in the pathogenesis of cerebral malaria. However, it is unclear whether and how Plasmodium-specific CD8(+) T cells can be activated during the erythrocyte stage of malaria infection. We generated recombinant Plasmodium berghei ANKA expressing OVA (OVA-PbA) to investigate the parasite-specific T cell responses during malaria infection. Using this model system, we demonstrate two types of CD8(+) T cell activations during the infection with malaria parasite. Ag (OVA)-specific CD8(+) T cells were activated by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL. These highly activated CD8(+) T cells were preferentially sequestered in the brain, although it was unclear whether these cells were involved in the pathogenesis of cerebral malaria. Activation of OVA-specific CD8(+) T cells in RAG2 knockout TCR-transgenic mice during infection with OVA-PbA did not have a protective role but rather was pathogenic to the host as shown by their higher parasitemia and earlier death when compared with RAG2 knockout mice. The OVA-specific CD8(+) T cells, however, were also activated during infection with wild-type parasites in an Ag-nonspecific manner, although the levels of activation were much lower. This nonspecific activation occurred in a TAP-independent manner, appeared to require NK cells, and was not by itself pathogenic to the host.     

Deficient anti-listerial immunity in the absence of perforin can be restored by increasing memory CD8+ T cell numbers

Compared with wild-type (WT) mice, Listeria monocytogenes (LM)-vaccinated perforin-deficient (PKO) mice have elevated levels of CD8(+) T cell memory, but exhibit reduced levels of protection against virulent LM. In this study, Ag-specific CD8(+) T cells from LM-vaccinated WT and PKO mice were used in adoptive transfer assays to determine the contribution of perforin-dependent cytolysis in protective immunity to LM. Perforin deficiency resulted in an approximately 5-fold reduction in the per-cell protective capacity of Ag-specific memory CD8(+) T cells that was not caused by differences in memory cell quality as measured by CD62L/CD27 expression, TCR repertoire use, functional avidity, differences in expansion of Ag-specific cells upon infection, or maintenance of memory levels over time. However, perforin-deficient CD8(+) T cells exhibited reduced in vivo cytotoxic function compared to WT CD8(+) T cells. Consistent with the existence of perforin-independent effector pathways, double-vaccinated PKO mice were as resistant to challenge with LM as single-vaccinated WT mice. Thus, increasing the number of memory CD8(+) T cells can overcome diminished per-cell protective immunity in the absence of perforin.     

CD4+ natural regulatory T cells prevent experimental cerebral malaria via CTLA-4 when expanded in vivo

Studies in malaria patients indicate that higher frequencies of peripheral blood CD4(+) Foxp3(+) CD25(+) regulatory T (Treg) cells correlate with increased blood parasitemia. This observation implies that Treg cells impair pathogen clearance and thus may be detrimental to the host during infection. In C57BL/6 mice infected with Plasmodium berghei ANKA, depletion of Foxp3(+) cells did not improve parasite control or disease outcome. In contrast, elevating frequencies of natural Treg cells in vivo using IL-2/anti-IL-2 complexes resulted in complete protection against severe disease. This protection was entirely dependent upon Foxp3(+) cells and resulted in lower parasite biomass, impaired antigen-specific CD4(+) T and CD8(+) T cell responses that would normally promote parasite tissue sequestration in this model, and reduced recruitment of conventional T cells to the brain. Furthermore, Foxp3(+) cell-mediated protection was dependent upon CTLA-4 but not IL-10. These data show that T cell-mediated parasite tissue sequestration can be reduced by regulatory T cells in a mouse model of malaria, thereby limiting malaria-induced immune pathology.     

Granzyme B regulates antiviral CD8+ T cell responses

CTLs and NK cells use the perforin/granzyme cytotoxic pathway to kill virally infected cells and tumors. Human regulatory T cells also express functional granzymes and perforin and can induce autologous target cell death in vitro. Perforin-deficient mice die of excessive immune responses after viral challenges, implicating a potential role for this pathway in immune regulation. To further investigate the role of granzyme B in immune regulation in response to viral infections, we characterized the immune response in wild-type, granzyme B-deficient, and perforin-deficient mice infected with Sendai virus. Interestingly, granzyme B-deficient mice, and to a lesser extent perforin-deficient mice, exhibited a significant increase in the number of Ag-specific CD8(+) T cells in the lungs and draining lymph nodes of virally infected animals. This increase was not the result of failure in viral clearance because viral titers in granzyme B-deficient mice were similar to wild-type mice and significantly less than perforin-deficient mice. Regulatory T cells from WT mice expressed high levels of granzyme B in response to infection, and depletion of regulatory T cells from these mice resulted in an increase in the number of Ag-specific CD8(+) T cells, similar to that observed in granzyme B-deficient mice. Furthermore, granzyme B-deficient regulatory T cells displayed defective suppression of CD8(+) T cell proliferation in vitro. Taken together, these results suggest a role for granzyme B in the regulatory T cell compartment in immune regulation to viral infections.     

CD8(+) T lymphocytes mediate Borna disease virus-induced immunopathology independently of perforin

Perforin-mediated lysis of target cells is the major antiviral effector mechanism of CD8(+) T lymphocytes. We have analyzed the role of perforin in a mouse model for CD8(+) T-cell-mediated central nervous system (CNS) immunopathology induced by Borna disease virus. When a defective perforin gene was introduced into the genetic background of the Borna disease-susceptible mouse strain MRL, the resulting perforin-deficient mice developed strong neurological disease in response to infection indistinguishable from that of their perforin-expressing littermates. The onset of disease was slightly delayed. Brains of diseased perforin-deficient mice showed similar amounts and a similar distribution of CD8(+) T cells as wild-type animals. Perforin deficiency had no impact on the kinetics of viral spread through the CNS. Unlike brain lymphocytes from diseased wild-type mice, lymphocytes from perforin-deficient MRL mice showed no in vitro cytolytic activity towards target cells expressing the nucleoprotein of Borna disease virus. Taken together, these results demonstrate that CD8(+) T cells mediate Borna disease independent of perforin. They further suggest that the pathogenic potential of CNS-infiltrating CD8(+) T cells does not primarily reside in their lytic activity but rather in other functions.     

Malaria prevalence metrics in low- and middle-income countries: an assessment of precision in nationally-representative surveys

Maladaptive immune responses during cerebral malaria (CM) result in high mortality despite opportune anti-malarial chemotherapy. Rapamycin, an FDA-approved immunomodulator, protects against experimental cerebral malaria (ECM) in mice through effects on the host. However, the potential for reduced adaptive immunity with chronic use, combined with an incomplete understanding of mechanisms underlying protection, limit translational potential as an adjunctive therapy in CM. The results presented herein demonstrate that a single dose of rapamycin, provided as late as day 4 or 5 post-infection, protected mice from ECM neuropathology and death through modulation of distinct host responses to infection. Rapamycin prevented parasite cytoadherence in peripheral organs, including white adipose tissue, via reduction of CD36 expression. Rapamycin also altered the splenic immune response by reducing the number of activated T cells with migratory phenotype, while increasing local cytotoxic T cell activation. Finally, rapamycin reduced brain endothelial ICAM-1 expression concomitant with reduced brain pathology. Together, these changes potentially contributed to increased parasite elimination while reducing CD8 T cell migration to the brain. Rapamycin exerts pleotropic effects on host immunity, vascular activation and parasite sequestration that rescue mice from ECM, and thus support the potential clinical use of rapamycin as an adjunctive therapy in CM.     

Protection from experimental cerebral malaria with a single dose of radiation-attenuated, blood-stage Plasmodium berghei parasites

Background

Whole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens.

Methodology and Results

We used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice.

Conclusions

This report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.     

In situ activation of antigen-specific CD8+ T cells in the presence of antigen in organotypic brain slices

The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8(+) T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c(+) cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c(+) cells stimulate Ag-specific naive CD8(+) T cells locally in the CNS and may contribute to immune responses in the brain.     

Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis

Experimental cerebral malaria (ECM) resulting from Plasmodium berghei ANKA infection involves T lymphocytes. However, the mechanisms of T cell-mediated pathogenesis remain unknown. We found that, in contrast to ECM-susceptible C57BL6 mice, perforin-deficient (PFP-KO) mice were resistant to ECM in the absence of brain lesions, whereas cytoadherence of parasitized erythrocytes and massive accumulation of activated/effector CD8 lymphocytes were observed in both groups of mice. ECM is induced in PFP-KO mice after adoptive transfer of cytotoxic CD8+ cells from infected C57BL6 mice, which were directed to the brain of PFP-KO mice. This specific recruitment might involve chemokine/chemokine receptors, since their expression was up-regulated on activated CD8 cells, and susceptibility to ECM was delayed in CCR5-KO mice. Thus, lymphocyte cytotoxicity and cell trafficking are key players in ECM pathogenesis.     

Perforin is required for primary immunity to Histoplasma capsulatum

Protective immunity against primary and secondary infection by the fungus Histoplasma capsulatum (HC) is multifactorial, requiring cells of the innate and adaptive immune response. Effector mechanisms that could mediate intracellular killing of HC include cytokines such as IFN-gamma and TNF-alpha and/or direct cytolytic activity by T and NK cells. In this regard, although previous work has clearly demonstrated a critical role for IFN-gamma and TNF-alpha in limiting fungal growth in primary HC infection, less is known regarding the role of cytolytic mechanisms. The studies reported here first address the role of perforin in mediating immunity to HC. Remarkably, perforin-deficient knockout (PfKO) mice were shown to have accelerated mortality and increased fungal burden following a lethal or sublethal primary challenge. These data established an essential role for perforin in primary immunity systemic HC infection. Interestingly, depletion of CD8(+) T cells in PfKO mice caused a further increase in fungal burden and accelerated mortality, suggesting a perforin-independent role for CD8(+) T cells. Moreover, adoptive transfer of CD8(+) T cells from PfKO mice into IFN-gamma(-/-) mice caused a reduction in fungal burden following infectious challenge compared with control IFN-gamma(-/-) mice. Together, these data suggest that CD8(+) T cells can mediate immunity to HC through both perforin-dependent and -independent mechanisms.     

Differentiation of CD8+ T cells from tumor-invaded and tumor-free lymph nodes of melanoma patients: role of common gamma-chain cytokines

Differentiation of CD8(+) T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy of antitumor response developing naturally or induced through immunotherapy. To address this issue, CD8(+) T lymphocytes from tumor-invaded (n = 142) and tumor-free (n = 42) lymph nodes removed from the same nodal basin of melanoma patients were analyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8(+) T cells from all tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainly by CCR7(+) CD45RA(+/-) cytotoxic factor(-) cells. The remaining three clusters contained only samples from tumor-invaded lymph nodes and showed a progressive shift of the CD8(+) T cell population toward CCR7(-) CD45RA(-/+) perforin(+) granzyme B(+) differentiation stages. Distinct CD8(+) T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, were identified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 days of CCR7(+) perforin(-) CD8(+) T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly in CCR7(+)CD45RA(-) cells, proliferation coupled to differentiation to the CCR7(-) perforin(+) stage and acquisition of melanoma Ag-specific effector functions. Taken together, these results indicate that CD8(+) T cells differentiated toward CCR7(-) cytotoxic factor(+) stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients and suggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8(+) T cells.     

Dynamics of T cell responses in HIV infection

Cytotoxic CD8(+) T cells play a major role in the immune response against viruses. However, the dynamics of CD8(+) T cell responses during the course of a human infection are not well understood. Using tetrameric complexes in combination with a range of intracellular and extracellular markers, we present a detailed analysis of the changes in activation and differentiation undergone by Ag-specific CD8(+) T cells, in relation to Ag-specific CD4(+) T cell responses, in the context of a human infection: HIV-1. During primary HIV-1 infection, the initial population of HIV-specific CD8(+) T cells is highly activated and prone to apoptosis. The Ag-specific cells differentiate rapidly from naive to cells at a perforin low intermediate stage of differentiation, later forming a stable pool of resting cells as viral load decreases during chronic infection. These observations have significant implications for our understanding of T cell responses in human viral infections in general and indicate that the definition of effector and memory subsets in humans may need revision.     

A role for IFN-gamma from antigen-specific CD8+ T cells in protective immunity to Listeria monocytogenes

Whether IFN-gamma contributes to the per-cell protective capacity of memory CD8(+) T cells against Listeria monocytogenes (LM) has not been formally tested. In this study, we generated LM Ag-specific memory CD8(+) T cells via immunization of wild-type (WT) and IFN-gamma-deficient (gamma knockout (GKO)) mice with LM peptide-coated dendritic cells and compared them phenotypically and functionally. Immunization of WT and GKO mice resulted in memory CD8(+) T cells that were similar in number, functional avidity, TCR repertoire use, and memory phenotype. The protective capacity of memory CD8(+) T cells from immunized WT and GKO mice was evaluated after adoptive transfer of equal numbers of WT or GKO cells into naive BALB/c mice followed by LM challenge. The adoptively transferred CD8(+) T cells from GKO donors exhibited a decreased ability to reduce bacterial numbers in the organs of recipient mice when compared with an equivalent number of Ag-matched WT CD8(+) T cells. This deficiency was most evident early (day 3) after infection if a relatively low infectious dose was used; however, transferring fewer memory CD8(+) T cells or increasing the LM challenge dose revealed a more pronounced defect in protective immunity mediated by the CD8(+) T cells from GKO mice. Our studies identified a decrease in Ag-specific target cell lysis in vivo by CD8(+) T cells from GKO mice as the mechanism for the decreased protective immunity after LM challenge. Further studies suggest that the lack of IFN-gamma production by the Ag-specific CD8 T cells themselves diminishes target cell sensitivity to cytolysis, thereby reducing the lytic potency of IFN-gamma-deficient LM-specific memory CD8(+) T cells.     

Involvement of CD4(+) Foxp3(+) Regulatory T Cells in Persistence of Leishmania donovani in the Liver of Alymphoplastic aly/aly Mice

Visceral leishmaniasis (VL) is a chronic and fatal disease in humans and dogs caused by the intracellular protozoan parasites, Leishmania donovani and L. infantum (L. chagasi). Relapse of disease is frequent in immunocompromised patients, in which the number of VL cases has been increasing recently. The present study is aimed to improve the understanding of mechanisms of L. donovani persistence in immunocompromised conditions using alymphoplastic aly/aly mice. Hepatic parasite burden, granuloma formation and induction of regulatory T cells were determined for up to 7 months after the intravenous inoculation with L. donovani promastigotes. While control aly/+ mice showed a peak of hepatic parasite growth at 4 weeks post infection (WPI) and resolved the infection by 8 WPI, aly/aly mice showed a similar peak in hepatic parasite burden but maintained persistent in the chronic phase of infection, which was associated with delayed and impaired granuloma maturation. Although hepatic CD4(+)Foxp3(+) but not CD8(+)Foxp3(+) T cells were first detected at 4 WPI in both strains of mice, the number of CD4(+)Foxp3(+) T cells was significantly increased in aly/aly mice from 8 WPI. Immunohistochemical analysis demonstrated the presence of Foxp3(+) T cells in L. donovani-induced hepatic granulomas and perivascular neo-lymphoid aggregates. Quantitative real-time PCR analysis of mature granulomas collected by laser microdissection revealed the correlation of Foxp3 and IL-10 mRNA level. Furthermore, treatment of infected aly/aly mice with anti-CD25 or anti-FR4 mAb resulted in significant reductions in both hepatic Foxp3(+) cells and parasite burden. Thus, we provide the first evidence that CD4(+)Foxp3(+) Tregs mediate L. donovani persistence in the liver during VL in immunodeficient murine model, a result that will help to establish new strategies of immunotherapy against this intracellular protozoan pathogen.     

Delayed contraction of the CD8+ T cell response toward lymphocytic choriomeningitis virus infection in mice lacking serglycin

We previously reported that the lack of serglycin proteoglycan affects secretory granule morphology and granzyme B (GrB) storage in in vitro generated CTLs. In this study, the role of serglycin during viral infection was studied by infecting wild-type (wt) mice and serglycin-deficient (SG(-/-)) mice with lymphocytic choriomeningitis virus (LCMV). Wt and SG(-/-) mice cleared 10(3) PFU of highly invasive LCMV with the same kinetics, and the CD8(+) T lymphocytes from wt and SG(-/-) animals did not differ in GrB, perforin, IFN-gamma, or TNF-alpha content. However, when a less invasive LCMV strain was used, SG(-/-) GrB(+) CD8(+) T cells contained approximately 30% less GrB than wt GrB(+) CD8(+) T cells. Interestingly, the contraction of the antiviral CD8(+) T cell response to highly invasive LCMV was markedly delayed in SG(-/-) mice, and a delayed contraction of the virus-specific CD8(+) T cell response was also seen after infection with vesicular stomatitis virus. BrdU labeling of cells in vivo revealed that the delayed contraction was associated with sustained proliferation of Ag-specific CD8(+) T cells in SG(-/-) mice. Moreover, wt LCMV-specific CD8(+) T cells from TCR318 transgenic mice expanded much more extensively in virus-infected SG(-/-) mice than in matched wt mice, indicating that the delayed contraction represents a T cell extrinsic phenomenon. In summary, the present report points to a novel, previously unrecognized role for serglycin proteoglycan in regulating the kinetics of antiviral CD8(+) T cell responses.