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No licensed vaccine exists against visceral leishmaniasis (VL), a disease caused by the Leishmania donovani parasite. We have previously reported both macrophages and dendritic cells play important role in the protection induced by a live attenuated centrin gene–deleted L. donovani (LdCen−/−) parasite vaccine. The role of neutrophils in orchestrating the initial innate response to pathogens is widely recognized. To investigate the early interaction of LdCen−/− with neutrophils, we immunized mice intradermally in the ear pinna with LdCen−/−. Compared with LdWT infection, LdCen−/− parasites induced higher recruitment of neutrophils to the ear dermis and ear draining lymph nodes (dLN) as early as 6–18 h after immunization, which were predominantly proinflammatory in nature. Neutrophils from ear dLN of LdCen−/−-immunized mice exhibited heightened expression of costimulatory molecules and attenuated expression of coinhibitory molecules necessary for higher T cell activation. Further phenotypic characterization revealed heterogeneous neutrophil populations containing Nα and Nβ subtypes in the ear dLN. Of the two, the parasitized Nα subset from LdCen−/−-immunized mice exhibited much stronger Ag-specific CD4+ T cell proliferation ex vivo. Adoptive transfer of neutrophils bearing LdCen−/− parasites induced an increased Th1 response in naive mice. Importantly, neutrophil depletion significantly abrogated Ag-specific CD4+ T cell proliferation in LdCen−/−-immunized mice and impaired protection against virulent challenge. Conversely, replenishing of neutrophils significantly restored the LdCen−/− -induced host-protective response. These results suggest that neutrophils are indispensable for protective immunity induced by LdCen−/− parasite vaccine.

BackgroundNeutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuatedLeishmania donovanicentrin deleted parasite vaccine (LdCen-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs duringLdCen-/-infection and compared with wild type parasite (LdWT)bothin vitroandin vivo.Methodology/findingsLdCen-/-parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared toLdWTinfection. To further illustrate neutrophil-DCs interactionsin vivo, we infected LYS-eGFP mice with red fluorescentLdWT/LdCen-/-parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containingLdCen-/-parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4+T cell primingex-vivo. Notably, potent DC activation occurred whenLdCen-/-parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment ofLdCen-/-parasites by DCs. Neutrophil depletion inLdCen-/-infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4+Th1 cell primingex vivothat correlated with attenuated Tbet expression in ear dLN derived CD4+T cellsin vivo.ConclusionsCollectively,LdCen-/-containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.

No vaccine exists against visceral leishmaniasis. To develop effective vaccines, we have previously reported protective role of live attenuated centrin gene–deleted Leishmania donovani (LdCen−/−) parasites through induction of Th1 type immune response in mice, hamsters, and dogs. In this study, we specifically explored the role of Th17 cells in LdCen−/−-induced host protection in mice. Our results showed that compared with wild-type L. donovani infection, LdCen−/− parasites induce significantly higher expression of Th17 differentiation cytokines in splenic dendritic cells. There was also induction of IL-17 and its promoting cytokines in total splenocytes and in both CD4 and CD8 T cells following immunization with LdCen−/−. Upon challenge with wild-type parasites, IL-17 and its differentiating cytokines were significantly higher in LdCen−/−-immunized mice compared with nonimmunized mice that resulted in parasite control. Alongside IL-17 induction, we observed induction of IFN-γ–producing Th1 cells as reported earlier. However, Th17 cells are generated before Th1 cells. Neutralization of either IL-17 or IFN-γ abrogated LdCen−/−-induced host protection further confirming the essential role of Th17 along with Th1 cytokines in host protection. Treatment with recombinant IL-23, which is required for stabilization and maintenance of IL-17, heightened Th17, and Tc17 responses in immunized mice splenocytes. In contrast, Th17 response was absent in immunized IL-23R−/− mice that failed to induce protection upon virulent Leishmania challenge suggesting that IL-23 plays an essential role in IL-17–mediated protection by LdCen−/− parasites. This study unveiled the role of IL-23–dependent IL-17 induction in LdCen−/− parasite-induced immunity and subsequent protection against visceral leishmaniasis.

Here we demonstrate the feasibility of direct, quantitative measurement of cytokine proteins in single human CD8 lymphocytes from fresh peripheral blood of healthy donors. Cytokine-secreting cells were identified using cell surface “catch” reagents. Single cell data were obtained by sorting of cytokine-secreting cells into 96 well plates containing lysis buffer followed by analysis using ultrasensitive immunoassays for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). CD8 T cells displayed an average of 5.97 femtogram (fg) IFN-γ per cell, with a range of 1.27-15.47 fg (n=28). Per cell averages of IFN-γ in CD8 T cells showed similar results when sorting 2, 5, or 10 cells per well (6.00 fg/cell, 5.73 fg/cell, and 5.25 fg/cell, respectively) (n=28), with an average of all measurements of 5.74 fg IFN-γ per cell (SD=1.96), equal to 178,660 molecules. Surface IFN-γ negative cells revealed a mean of 1.40 fg/cell, suggesting the baseline background. Experiments from TNF-α-producing CD8 T cells showed a mean of 2.49 fg/cell TNF-α (n=28) (0.675 – 5.13 fg/cell) equals to 59,654 molecules, while sorting 2, 5, and 10 cells per well-lysate have means of 1.59 fg/cell, 1.30 fg/cell, and 1.16 fg/cell (n=28). TNF-α-negative cells showed a mean on 0.61 fg/cell (n=28). This study demonstrates that it is feasible to quantify cytokines from individual primary cells. A shift from qualitative to quantitative determinations of cytokine protein levels in single cells will permit more precise and reproducible studies of heterogeneity in the immune system and can be accomplished with readily available instrumentation.

Visceral leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that genetically modified Leishmania donovani parasites, here described as live attenuated parasites, induce a host protective adaptive immune response in various animal models. In this study, we demonstrate an innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites (compared to that with wild-type [WT] L. donovani parasites) induced significantly higher production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], gamma interferon [IFN-γ], and IL-6), chemokines (monocyte chemoattractant protein 1/CCL-2, macrophage inflammatory protein 1α/CCL-3, and IP-10), reactive oxygen species (ROS), and nitric oxide, while concomitantly reducing anti-inflammatory cytokine IL-10 and arginase-1 activities, suggesting a dominant classically activated/M1 macrophage response. The classically activated response in turn helps in presenting antigen to T cells, as observed with robust CD4 + T cell activation in vitro . Similarly, parasitized splenic macrophages from live attenuated parasite-infected mice also demonstrated induction of an M1 macrophage phenotype, indicated by upregulation of IL-1β, TNF-α, IL-12, and inducible nitric oxide synthase 2 and downregulation of genes associated with the M2 phenotype, i.e., the IL-10, YM1, Arg-1, and MRC-1 genes, compared to WT L. donovani -infected mice. Furthermore, an ex vivo antigen presentation assay showed macrophages from live attenuated parasite-infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by ovalbumin-specific CD4 + T cells, resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasites promotes a state of classical activation (M1 dominant) in macrophages that leads to the generation of protective Th1 responses in BALB/c mice.

Background Visceral leishmaniasis (VL) caused by the protozoan parasite Leishmania donovani causes severe disease. Age appears to be critical in determining the clinical outcome of VL and at present there is no effective vaccine available against VL for any age group. Previously, we showed that genetically modified live attenuated L. donovani parasites (LdCen-/-) induced a strong protective innate and adaptive immune response in young mice. In this study we analyzed LdCen-/- parasite mediated modulation of innate and adaptive immune response in aged mice (18 months) and compared to young (2 months) mice. Methodology Analysis of innate immune response in bone marrow derived dendritic cells (BMDCs) from both young and aged mice upon infection with LdCen-/- parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected BMDCs in vitro. Similarly, parasitized splenic dendritic cells from LdCen-/- infected young and aged mice also revealed induction of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) and subsequent down regulation of anti-inflammatory cytokine (IL-10) genes compared to LdWT infected mice. We also evaluated in vivo protection of the LdCen-/- immunized young and aged mice against virulent L. donovani challenge. Immunization with LdCen-/- induced higher IgG2a antibodies, lymphoproliferative response, pro- and anti-inflammatory cytokine responses and stimulated splenocytes for heightened leishmanicidal activity associated with nitric oxide production in young and aged mice. Furthermore, upon virulent L. donovani challenge, LdCen-/- immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells correlating to a significantly reduced parasite burden in the spleen and liver compared to naïve mice. It is interesting to note that even though there was no difference in the LdCen-/- induced innate response in dendritic cells between aged and young mice; the adaptive response specifically in terms of T cell and B cell activation in aged animals was reduced compared to young mice which correlated with less protection in old mice compared to young mice. Conclusions Taken together, LdCen-/- immunization induced a significant but diminished host protective response in aged mice after challenge with virulent L. donovani parasites compared to young mice., Author Summary Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani. There is no effective vaccine available against VL for any age group and importantly, there are no previous studies regarding immune responses against experimental Leishmania vaccines tested in aged animals. We have reported earlier that immunization with a live attenuated L. donovani parasites (LdCen-/-) induced protective immune response in young animals viz, mice, hamsters and dogs. In this study we analyzed LdCen-/- mediated modulation of innate and adaptive responses in aged mice and compared to young mice. We observed that LdCen-/- infected dendritic cells from young and aged mice resulted in enhanced innate effector functions compared to LdWT parasites both in vitro and in vivo. Further, upon virulent challenge, LdCen-/- immunized young and aged mice displayed protective Th1 immune response which correlated with a significantly reduced parasite burden in the visceral organs compared with naïve challenged mice. Although there was no difference in the LdCen-/- induced dendritic cell response between aged and young mice; adaptive response in aged was reduced, compared to young which correlated with less protection in aged compared to young mice. This study supports the potential use of LdCen-/- as vaccine candidate across all age groups against VL.

Cytomegalovirus (CMV) reactivation after stem cell transplantation can be treated with CMV-specific T cells, but current in vitro techniques using dendritic cells as antigen-presenting cells are time-consuming and expensive. To simplify the production of clinical grade CMV-specific T cells, we evaluated gene-modified activated T cells [antigen presenting T cells (T-APCs)] as a reliable and easily produced source of APCs to boost CD4+ and CD8+ T-cell responses against the immunodominant CMV antigen pp65. T-APCs expressing the full-length immunodominant CMV pp65 gene were used to stimulate the expansion of autologous T cells. After 10 to 14 days, the T cell lines were tested for antigen specificity by using the flow cytometric intracellular detection of interferon-gamma after stimulation for 6 hours with a pp65 peptide library of 15-mers, overlapping by 11 amino acids. Under optimal conditions, this technique induced a median 766-fold and a 652-fold expansion of pp65-specific CD4+ and CD8+ responder cells, respectively, in 15 T cell lines. In 13 of 15 T cell lines, over 10 antigen-specific CD4+ plus CD8+ T cells were generated starting with only 5x10 peripheral blood mononuclear cells, representing an over 3-log increase. These data indicate that T-APCs efficiently boost pp65-specific CD4+ and CD8+ T cell numbers to clinically useful levels. The approach has the advantage of using a single leukocyte collection from the donor to generate large numbers of CMV-specific T cells within a total 3-week culture period using only one stimulation of antigen.

CANDLE (Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated Temperature) syndrome is an autoinflammatory disease caused by autosomal recessive mutations in PSMB8 and other subunits of the proteasome. Many IFN regulated genes in CANDLE patients’ whole blood were strongly upregulated and patient monocytes showed STAT1 hyperactivation after IFN-γ stimulation. We hypothesized that cellular stress in CANDLE patients leads to IFN production and asked whether it is restricted to hematopoietic cells. We studied IFN production in PBMCs and fibroblasts from CANDLE patients or in healthy cells treated with proteasome inhibitor epoxomicin. We found increased production of IFN-α and IFN-γ in NK, T and dendritic cells from the two CANDLE patients tested compared with healthy controls. Increased IFN production was reproduced in PBMCs of healthy individuals treated with epoxomicin. We also observed increased IFN-α message in CANDLE patients’ fibroblasts compared to healthy controls. Similarly, epoxomicin treated healthy fibroblasts increased IFN-α message. Interestingly message for other inflammatory cytokines, including TNF-α, IL-1 and IL-6, were either not change or even suppressed. Microarray study showed that IFN regulated genes were upregulated in sorted T, B, NK cells, monocytes and neutrophils. We conclude that proteasome defect causes IFN production in both hemopoietic and non-hemopoietic cells in CANDLE patients that can lead to increased IFN signaling.