Definition of Naturally Processed Peptides Reveals Convergent Presentation of Autoantigenic Topoisomerase I Epitopes in Scleroderma.

ObjectiveAutoimmune responses to DNA topoisomerase I (topo I) are found in a subset of scleroderma patients who are at high risk for interstitial lung disease (ILD) and mortality. Anti-topo I antibodies (ATAs) are associated with specific HLA-DRB1 alleles, and the frequency of HLA-DR-restricted topo I-specific CD4+ T cells is associated with the presence, severity, and progression of ILD. Although this strongly implicates the presentation of topo I peptides by HLA-DR in scleroderma pathogenesis, the processing and presentation of topo I has not been studied.MethodsWe developed a natural antigen processing assay (NAPA) to identify putative CD4+ T cell epitopes of topo I presented by monocyte-derived dendritic cells (mo-DCs) from 6 ATA-positive patients with scleroderma. Mo-DCs were pulsed with topo I protein, HLA-DR-peptide complexes were isolated, and eluted peptides were analyzed by mass spectrometry. We then examined the ability of these naturally presented peptides to induce CD4+ T cell activation in 11 ATA-positive and 11 ATA-negative scleroderma patients.ResultsWe found that a common set of 10 topo I epitopes was presented by Mo-DCs from scleroderma patients with diverse HLA-DR variants. Sequence analysis revealed shared peptide-binding motifs within the HLA-DRβ chains of ATA-positive patients and a subset of topo I epitopes with distinct sets of anchor residues capable of binding to multiple different HLA-DR variants. The NAPA-derived epitopes elicited robust CD4+ T cell responses in 73% of ATA-positive patients (8 of 11), and the number of epitopes recognized correlated with ILD severity (P = 0.025).ConclusionThese findings mechanistically implicate the presentation of a convergent set of topo I epitopes in the development of scleroderma

The Grizzly, November 12, 2015

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Dendritic Cells Are Responsible for the Capacity of CpG Oligodeoxynucleotides to Act as an Adjuvant for Protective Vaccine Immunity Against Leishmania major in Mice

Vaccination with leishmanial Ag and CpG oligodeoxynucleotides (ODN) confers sustained cellular immunity and protection to infectious challenge up to 6 mo after immunization. To define the cellular mechanism by which CpG ODN mediate their adjuvant effects in vivo, the functional capacity of distinct dendritic cell (DC) subsets was assessed in the lymph nodes (LNs) of BALB/c mice, 36 h after immunization with the leishmanial antigen (LACK) and CpG ODN. After this immunization, there was a striking decrease in the frequency of the CD11c+B220+ plasmacytoid DCs with a proportionate increase in CD11c+CD8−B220− cells. CD11c+CD8+B220− cells were the most potent producers of interleukin (IL)-12 p70 and interferon (IFN)-γ, while plasmacytoid DCs were the only subset capable of secreting IFN-α. In terms of antigen presenting capacity, plasmacytoid DCs were far less efficient compared with the other DC subsets. To certify that DCs were responsible for effective vaccination, we isolated CD11c+ and CD11c− cells 36 h after immunization and used such cells to elicit protective immunity after adoptive transfer in naive, Leishmania major susceptible BALB/c mice. CD11c+ cells but not 10-fold higher numbers of CD11c− cells from such immunized mice mediated protection. Therefore, the combination of LACK antigen and CpG ODN adjuvant leads to the presence of CD11c+ DCs in the draining LN that are capable of vaccinating naive mice in the absence of further antigen or adjuvant

Cytotoxic polyfunctionality maturation of cytomegalovirus-pp65-specific CD4 + and CD8 + T-cell responses in older adults positively correlates with response size

Cytomegalovirus (CMV) infection is one of the most common persistent viral infections in humans worldwide and is epidemiologically associated with many adverse health consequences during aging. Previous studies yielded conflicting results regarding whether large, CMV-specific T-cell expansions maintain their function during human aging. In the current study, we examined the in vitro CMV-pp65-reactive T-cell response by comprehensively studying five effector functions (i.e., interleukin-2, tumor necrosis factor-α, interferon-γ, perforin, and CD107a expression) in 76 seropositive individuals aged 70 years or older. Two data-driven, polyfunctionality panels (IL-2-associated and cytotoxicity-associated) derived from effector function co-expression patterns were used to analyze the results. We found that, CMV-pp65-reactive CD8 + and CD4 + T cells contained similar polyfunctional subsets, and the level of polyfunctionality was related to the size of antigen-specific response. In both CD8 + and CD4 + cells, polyfunctional cells with high cytotoxic potential accounted for a larger proportion of the total response as the total response size increased. Notably, a higher serum CMV-IgG level was positively associated with a larger T-cell response size and a higher level of cytotoxic polyfunctionality. These findings indicate that CMV-pp65-specific CD4 + and CD8 + T cell undergo simultaneous cytotoxic polyfunctionality maturation during aging

Protective Immunity Induced with the RTS,S/AS Vaccine Is Associated with IL-2 and TNF-α Producing Effector and Central Memory CD4+ T Cells

A phase 2a RTS,S/AS malaria vaccine trial, conducted previously at the Walter Reed Army Institute of Research, conferred sterile immunity against a primary challenge with infectious sporozoites in 40% of the 80 subjects enrolled in the study. The frequency of Plasmodium falciparum circumsporozoite protein (CSP)-specific CD4+ T cells was significantly higher in protected subjects as compared to non-protected subjects. Intrigued by these unique vaccine-related correlates of protection, in the present study we asked whether RTS,S also induced effector/effector memory (TE/EM) and/or central memory (TCM) CD4+ T cells and whether one or both of these sub-populations is the primary source of cytokine production. We showed for the first time that PBMC from malaria-non-exposed RTS,S-immunized subjects contain both TE/EM and TCM cells that generate strong IL-2 responses following re-stimulation in vitro with CSP peptides. Moreover, both the frequencies and the total numbers of IL-2-producing CD4+ TE/EM cells and of CD4+ TCM cells from protected subjects were significantly higher than those from non-protected subjects. We also demonstrated for the first time that there is a strong association between the frequency of CSP peptide-reactive CD4+ T cells producing IL-2 and the titers of CSP-specific antibodies in the same individual, suggesting that IL-2 may be acting as a growth factor for follicular Th cells and/or B cells. The frequencies of CSP peptide-reactive, TNF-α-producing CD4+ TE/EM cells and of CD4+ TE/EM cells secreting both IL-2 and TNF-α were also shown to be higher in protected vs. non-protected individuals. We have, therefore, demonstrated that in addition to TNF-α, IL-2 is also a significant contributing factor to RTS,S/AS vaccine induced immunity and that both TE/EM and TCM cells are major producers of IL-2

Microneedle Array Design Determines the Induction of Protective Memory CD8+ T Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice

BACKGROUND: Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a microneedle patch affects the CD8(+) T cell responses to a malaria antigen induced by a live vaccine. METHODOLOGY AND FINDINGS: Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the design of microneedle patches significantly influences the magnitude and memory of vaccine-induced CD8(+) T cell responses and can be optimised for the induction of desired immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of immunization programmes

Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

BACKGROUND: Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. METHODOLOGY/PRINCIPAL FINDINGS: We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T(H)1 CD4(+) and CD8(+) T cells and a systemic LACK-specific T(H)1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T(H)1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T(H)1 response. CONCLUSIONS/SIGNIFICANCE: This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania