Enhanced protection by Th17 cells involves more potent qualitative and quantitative helper effects for the development of <i>T</i>. <i>cruzi</i>-specific CD8⁺ T cells.

<p>RAG KO mice were adoptively transferred with polyclonal CD8⁺ T cells and Th1 or Th17 cells and then infected systemically with <i>T</i>. <i>cruzi</i>. (A) Greater absolute numbers of CD8⁺ T cells were recovered from mice co-adoptively transferred with Th17 cells at 7 days post-infection. (B) The mean fluorescence intensity (MFI) of T-bet expression in these same CD8⁺ T cells was increased over mice receiving CD8⁺ T cells alone or CD8⁺ T cells with Th1 cells, as measured by ICS. (C) Co-culture of sub-optimally stimulated CD8⁺ T cells with activated Th17 cell SN <i>in vitro</i> also induced higher T-bet expression than co-culture with Th1 cell SN. (D-E) Antigen-specific total and CD8⁺ T cell responses were studied 7 days post-challenge by IFN-γ ELISPOT (D) and intracellular cytokine staining (E), respectively. Similar results were detected in multiple experiments.</p

IL-17A alone is not responsible for the Th17-mediated enhanced protective effects.

<p>(A-B) <i>T</i>. <i>cruzi</i>-specific Th17 cells were co-transferred with CD8⁺ T cells into RAG KO mice (5/group) prior to <i>T</i>. <i>cruzi</i> challenge. Neutralizing anti-IL-17A or control IgG1 antibodies were injected intraperitoneally every 48 hours. IL-17A neutralization did not reduce protection as measured by both parasitemia (A) and survival (B). *p<0.001 by two-tailed Student t test, **p<0.01 by log-rank test compared with negative controls. (C-D) Polyclonal CD8⁺ T cells were transferred intravenously (i.v.) into RAG KO mice prior to <i>T</i>. <i>cruzi</i> infection. Either control AdV or IL-17 AdV was injected 1 day prior to infection and 7 days post-infection at 5x10⁹ PFU i.v. Protection was measured by parasitemia 18 days post-infection (C) and survival (D).</p

Th17 cell-derived IL-21 can help expand and activate protective CD8⁺ T cells.

<p>(A) WT or IL-21R KO CD8⁺ T cells were sub-optimally stimulated with plate-bound α-CD3 as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005902#ppat.1005902.g004" target="_blank">Fig 4C and 4E</a>. ICS assays indicated the addition of Th17 cell SNs could help activate WT CD8⁺ T cells but not IL-21R KO CD8⁺ T cells. Shown are fold increases compared to WT or IL-21R KO CD8⁺ T cells cultured with sub-optimal α-CD3 alone, without Th17 cell SNs. (B) Total spleen cells (TSCs) recovered from infected RAG KO mice adoptively transferred with Th17 cells and IL-21R KO CD8⁺ T cells had diminished responses to TS stimulation compared to mice receiving Th17 cells and WT CD8⁺ T cells prior to challenge, as measured by IFN-γ ELISPOT. (C) Mice transferred with Th17 cells (n = 4) and IL-21R KO CD8⁺ T cells (n = 8) were unable to control infection, as indicated by increasing parasitemia over time. **p<0.01 by two-tailed Student t test. (D) Transfer of IL-21R KO CD8⁺ T cells failed to protect mice from <i>T</i>. <i>cruzi</i>-mortality. *** p<0.001 by Mantel-Cox log-rank test.</p

Th17 cells can inhibit <i>T</i>. <i>cruzi</i> intracellular growth in macrophages <i>in vitro</i> by inducing NADPH oxidase via IL-17A.

<p>PEMs were infected with trypomastigotes for 3 hours (MOI = 5). <i>T</i>. <i>cruzi</i>-specific TCR Tg Th1 or Th17 cells were then added to macrophages 1:25. Two days later, slides were Giemsa stained and intracellular parasites counted microscopically. Th1 and Th17 cells (A), as well as IFN-γ or IL-17A alone (B) were able to decrease the number of infected macrophages. *p<0.001 compared to medium alone by two-tailed Student t test. (C) Neutralizing antibodies directed against IFN- γ or IL-17A were able to abolish the direct protection provided by Th1 and Th17 cells, respectively. (D-E) The effects of the iNOS inhibitor L-NIL on Th1- and Th17-induced NO production and <i>T</i>. <i>cruzi</i> protection are shown. **p<0.001 by two-tailed Student t test. (F+G) BMDMs from wild type (WT) and gp91^phox KO mice were infected with <i>T</i>. <i>cruzi</i> for 3 hours (MOI = 10), then cytokines IFN-γ and IL-17A were added. Two days later, NO was measured (F) and intracellular parasites were enumerated (G). **p<0.001 by two-tailed Student t test. These results were reproduced in multiple experiments.</p

<i>T</i>. <i>cruzi</i>-specific Th17 cells provide T helper effects and directly activate CD8⁺ T cells through a soluble mediator.

<p>(A) Th17 cells required co-adoptive transfer with polyclonal CD8⁺ T cells to confer optimal immunity as measured by parasite burden. **p<0.01, ***p<0.001 by two-tailed Student t test. (B) Th17 cells transferred alone were not able to protect mice from <i>T</i>. <i>cruzi</i>-related death, indicating that Th17 cells protect through helper effects on CD8⁺ T cells. **p<0.01 by Mantel-Cox log-rank test. (C) CFSE-labeled, polyclonal CD8⁺ T cells were sub-optimally activated with plate-bound α-CD3 (1 μg/ml) and increasing numbers of dendritic cells. CD8⁺ T cells were cultured with or without Th17 cells for 5 days. Proliferation was measured by CFSE dilution. (D) CD8⁺ T cells were sub-optimally activated with α-CD3 as in C (without added dendritic cells), in the presence of Th17 cells, Th17 SNs or Th17 cells plus IL-17A neutralizing antibody. CD8⁺ T cell proliferation, CD44 expression and MIP-1α/IFN-γ production were measured 5 days later by Flow Cytometry and ICS and shown as fold increases compared with α-CD3 activation alone. (E) Purified Th17 cytokines were individually added to sub-optimally α-CD3 activated CD8⁺ T cells and markers of activation were analyzed 5 days later. (F) High levels of IL-21 could be detected by ELISA in Th17 cell SNs, but not in Th1 cell SNs. Representative results from multiple experiments are shown.</p

Design, Synthesis, and Evaluation of Novel Hybrid Efflux Pump Inhibitors for Use against Mycobacterium tuberculosis

Efflux pumps are considered a major potential contributor to the development of various forms of resistance in Mycobacterium tuberculosis leading to the emergence of multidrug-resistant tuberculosis (TB). Verapamil (VER) and tricyclic chemosensitizers such as the phenothiazines are known to possess efflux pump inhibition properties and have demonstrated significant efficacy in various TB disease models. Novel hybrid molecules based on fusion of the VER substructure with various tricyclic, as well as nontricyclic, chemosensitizer cores or their structural motifs are described. These hybrid compounds were evaluated in vitro and ex vivo individually for their intrinsic activity and in combination for their potentiating potential with the frontline anti-TB drugs, rifampin and isoniazid. In addition, efflux pump inhibition was assessed in an ethidium bromide assay. This study led to the identification of novel compounds, termed hybrid efflux pump inhibitors, with intrinsic antimycobacterial activities (MIC₉₀ ≤ 3.17 μg/mL) and intracellular activity in macrophages at a low concentration (≤6.25 μg/mL)

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

<div><p><i>Trypanosoma cruzi</i> infection is controlled but not eliminated by host immunity. The <i>T</i>. <i>cruzi</i> trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against <i>T</i>. <i>cruzi</i> challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for <i>T</i>. <i>cruzi</i> vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.</p></div

CD4⁺ TSaa57-74 (p7)/IA^d- and CD8⁺ TSaa359-367 (TSKd1)/K^d-specific T cell responses are minimally sufficient for induction of protective <i>T</i>. <i>cruzi</i> immunity.

<p>Panel A shows a schematic of the TS consensus protein (all 12–15 active TS subfamily members have at least 90% homology within their catalytic domains), and immunodominant TS CD4 and CD8 epitopes. In panels B and C, BALB/c mice were vaccinated with dendritic cells (DC) pulsed (or not) with this pair of CD4 and CD8 epitopes and later challenged with <i>T</i>. <i>cruzi</i>. BALB/c CD11c⁺ splenic DC were purified from BALB/c mice 2 weeks after injection of 5x10⁶ B16-Flt3L-producing cells and 1 day after i.v. injection of 1μg LPS. 1x10⁶ DC pulsed (or left unpulsed) with 50 μg/ml of the indicated peptides were injected i.v. into groups of naïve BALB/c mice 3 times, 2 weeks apart. Mice were challenged 1 month later with <i>T</i>. <i>cruzi</i> (N = 5 in each of the two control groups and N = 10 in DC+TS peptide group). Both parasitemia (B; 2 weeks post-infection) and mortality (C) were significantly reduced in mice given DC pulsed with both TSaa57-74 (p7) and TSaa359-367 (TSKd1) [*p<0.001 by Mann-Whitney U test (B) and *p<0.01 by 2-tailed Fisher exact and Log-Rank [Mantel-Cox] tests(C)]. Survival results are representative of 3 independent experiments.</p

TSKd1 tolerization during WT TS DNA vaccination reduces CD8⁺ T cell responses directed against both immunodominant and subdominant TS epitopes.

<p>BALB/c mice were injected with tERK-1 control or TSKd1 peptide i.v. on indicated days before and after i.m. vaccination with WT TS DNA followed by <i>T</i>. <i>cruzi</i> challenge as shown in panel A. One month following the final immunization, CD8⁺ splenic T cells from representative mice were stimulated with APC (A20) pulsed with TS peptides in overnight IFN-γ ELISPOT assays (B). Results are representative of 2 independent experiments. Other groups of tolerized TS DNA vaccinated mice were challenged with 5,000 <i>T</i>. <i>cruzi</i> BFT and survival monitored (C; 10–12 mice/group; p<0.05 comparing TS DNA vaccinated tolerized with TSKd1 or the tERK-1 peptide by Fisher exact tests).</p

The WT DNA vaccine induces CD8⁺ T cell responses directed against both immunodominant and subdominant T cell epitopes.

<p>BALB/c mice were vaccinated i.m. twice, two weeks apart with 100μg of WT TS DNA or TSKd1 null TS DNA. Four weeks later, CD8⁺ splenic T cells from these vaccinated mice were stimulated overnight in IFN-γ ELISPOT assays with APC (A20 cells) pulsed with TS peptides predicted to bind BALB/c MHC [H2-K^d (A), H2- D^d (B) and H2-L^d(C). Results are representative of 3 experiments. As expected, vaccination of mice with WT but not TSKd1 null TS DNA constructs elicited T cell responses to TSKd1. CD8⁺ T cells from WT TS DNA vaccinated mice also responded to TS peptides Kd2, Kd5, Kd6, Kd7, Kd8, Kd9, Dd2, Dd6, Ld1 and Ld2, while cells from TSKd1 null vaccinate mice responded to only TS Kd8. Mutation of the 2 binding residues of TSKd1 to H2-Kd1 resulted in a marked immunofocusing of CD8⁺ T cell responses. Panel D further shows that WT TS DNA vaccination induced higher levels of TS-specific antibody, compared with TSKd1 null DNA vaccination.</p