Differential Impact of LPG-and PG-Deficient \u3cem\u3eLeishmania major\u3c/em\u3e Mutants on the Immune Response of Human Dendritic Cells

BACKGROUND: Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction. METHODOLOGY/PRINCIPAL FINDINGS: Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription. CONCLUSIONS/SIGNIFICANCE: These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12

Differential impact of LPG-and PG-deficient Leishmania major mutants on the immune response of human dendritic cells

<div><p>Background</p><p><i>Leishmania major</i> infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of <i>Leishmania</i> parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction.</p><p>Methodology/Principal Findings</p><p>Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating <i>L</i>. <i>major</i> Friedlin V1 mutants defective in LPG alone, (FV1 <i>lpg1-</i>), or generally deficient for all PGs, (FV1 <i>lpg2-</i>). Infection with metacyclic, infective stage, <i>L</i>. <i>major</i> or purified LPG induced high levels of <i>IL12B</i> subunit gene transcripts in hDCs, which was abrogated with FV1 <i>lpg1-</i> infections. In contrast, hDC infections with FV1 <i>lpg2-</i> displayed increased <i>IL12B</i> expression, suggesting other PG-related/<i>LPG2</i> dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 <i>lpg1-</i>, FV1 <i>lpg2-</i> infections revealed that FV1 <i>lpg1-</i> mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription.</p><p>Conclusions/Significance</p><p>These data suggest that <i>L</i>. <i>major</i> LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring <i>Leishmania</i> surface glycoconjugates that result in modulation of host cellular IL12.</p></div

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

Background: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. Objective: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. Methods: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. Results: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD81 suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)–150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH2/2 or miRNA-1502/2 mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. Conclusions: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains

TLR1/2 Activation during Heterologous Prime-Boost Vaccination (DNA-MVA) Enhances CD8+ T Cell Responses Providing Protection against Leishmania (Viannia)

Leishmania (Viannia) are the predominant agents of leishmaniasis in Latin America. Given the fact that leishmaniasis is a zoonosis, eradication is unlikely; a vaccine could provide effective prevention of disease. However, these parasites present a challenge and we do not fully understand what elements of the host immune defense prevent disease. We examined the ability of vaccination to protect against L. (Viannia) infection using the highly immunogenic heterologous prime-boost (DNA-modified vaccinia virus) modality and a single Leishmania antigen (TRYP). Although this mode of vaccination can induce protection against other leishmaniases (cutaneous, visceral), no protection was observed against L. (V.) panamensis. However, we found that if the vaccination was modified and the innate immune response was activated through Toll-like receptor1/2(TLR1/2) during the DNA priming, vaccinated mice were protected. Protection was dependent on CD8 T cells. Vaccinated mice had higher CD8 T cell responses and decreased levels of cytokines known to promote infection. Given the long-term persistence of CD8 T cell memory, these findings are encouraging for vaccine development. Further, these results suggest that modulation of TLR1/2 signaling could improve the efficacy of DNA-based vaccines, especially where CD8 T cell activation is critical, thereby contributing to effective and affordable anti parasitic vaccines

RIPK3 inhibition prevents KRAS mutant p53 deficient lung tumor progression by impeding MDSCs

AbstractKRAS mutant p53 deficient (KP) non-small cell lung carcinoma (NSCLC) lacks targeted therapies. Existing treatments for lung cancer cause resistance and result in toxicities requiring novel effective therapies. By targeting mechanisms causing resistance such as myeloid derived suppressor cells (MDSCs), KP tumors could be inhibited. MDSCs are functionally diverse and suppress T cells in many cancers. RIPK3, a cell death inducing enzyme, also functions as a signaling component producing cytokines that mediate the suppressive function of MDSCs. Partial deletion of RIPK3 in myeloid cells reduced KP tumor growth. This also reduced accumulation of MDSCs with a consistent increase in antigen specific IFNγ producing CD8 T cells. Inhibiting RIPK3 with a small molecule inhibitor such as GSK 872 effectively reduces RIPK3 activity in myeloid cells including MDSCs and reduces growth of small and large KP tumors. GSK 872 in combination with checkpoint inhibitors such as anti PD-1 and anti CTLA-4 further decreased KP tumor size. Together, our findings show that inhibiting RIPK3 in MDSCs is effective in inhibiting KP NSCLC and is a viable therapeutic option for improving existing immunotherapeutic treatments.</jats:p

Supplementary figure 6 from RIPK3-Induced Inflammation by I-MDSCs Promotes Intestinal Tumors

&lt;p&gt;TLR expression on MDSCs and viability&lt;/p&gt;</jats:p

Supplementary methods from RIPK3-Induced Inflammation by I-MDSCs Promotes Intestinal Tumors

&lt;p&gt;Supplementary methods&lt;/p&gt;</jats:p