Progesterone amplifies allergic inflammation and airway pathology in association with higher lung ILC2 responses.

Perimenstrual worsening of asthma occurs in up to 40% of women with asthma, leading to increased acute exacerbations requiring clinical care. The role of sex hormones during these times remains unclear. In the current study, we used a translational approach to determine whether progesterone exacerbates allergic inflammation in the traditional chicken egg ovalbumin (OVA) model in BALB/c mice. Simultaneously, we used peripheral blood mononuclear cells (PBMC) from healthy human donors to assess the effects of progesterone on circulating group 2 innate lymphoid cells (ILC2). Briefly, lungs of ovariectomized (OVX) or sham-operated female (F-Sham) controls were implanted with a progesterone (P4, 25 mg) (OVX-P4) or placebo pellet (OVX-Placebo), followed by sensitization and challenge with ovalbumin (OVA). Progesterone increased total inflammatory histologic scores, increased hyper-responsiveness to methacholine (MCh), increased select chemokines in the bronchoalveolar lavage (BAL) and serum, and increased ILC2 and neutrophil numbers, along the airways compared with F-Sham-OVA and OVX-Placebo-OVA animals. Lung ILC2 were sorted from F-Sham-OVA, OVX-Placebo-OVA and OVX-P4-OVA treated animals and stimulated with IL-33. OVX-P4-OVA lung ILC2 were more responsive to interleukin 33 (IL-33) compared with F-Sham-OVA treated, producing more IL-13 and chemokines following IL-33 stimulation. We confirmed the expression of the progesterone receptor (PR) on human ILC2, and showed that P4 + IL-33 stimulation also increased IL-13 and chemokine production from human ILC2. We establish that murine ILC2 are capable of responding to P4 and thereby contribute to allergic inflammation in the lung. We confirmed that human ILC2 are also hyper-responsive to P4 and IL-33 and likely contribute to airway exacerbations following allergen exposures in asthmatic women with increased symptoms around the time of menstruation.NEW & NOTEWORTHY There is a strong association between female biological sex and severe asthma. We investigated the allergic immune response, lung pathology, and airway mechanics in the well-described chicken egg ovalbumin (OVA) model with steady levels of progesterone delivered throughout the treatment period. We found that progesterone enhances the activation of mouse group 2 innate lymphoid cells (ILC2). Human ILC2 are also hyper-responsive to progesterone and interleukin 33 (IL-33), and likely contribute to airway exacerbations following allergen exposures in women with asthma.

Mucosal-associated invariant T cells modulate innate immune cells and inhibit colon cancer growth.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can be activated by microbial antigens and cytokines and are abundant in mucosal tissues including the colon. MAIT cells have cytotoxic and pro-inflammatory functions and have potentials for use as adoptive cell therapy. However, studies into their anti-cancer activity, including their role in colon cancer, are limited. Using an animal model of colon cancer, we showed that peritumoral injection of in vivo-expanded MAIT cells into RAG1-/- mice with MC38-derived tumours inhibits tumour growth compared to control. Multiplex cytokine analyses showed that tumours from the MAIT cell-treated group have higher expression of markers for eosinophil-activating cytokines, suggesting a potential association between eosinophil recruitment and tumour inhibition. In a human peripheral leukocyte co-culture model, we showed that leukocytes stimulated with MAIT ligand showed an increase in eotaxin-1 production and activation of eosinophils, associated with increased cancer cell killing. In conclusion, we showed that MAIT cells have a protective role in a murine colon cancer model, associated with modulation of the immune response to cancer, potentially involving eosinophil-associated mechanisms. Our results highlight the potential of MAIT cells for non-donor restricted colon cancer immunotherapy.

Recombinant fowlpox virus vector-based vaccines: expression kinetics, dissemination and safety profile following intranasal delivery.

We have previously established that mucosal uptake of recombinant fowlpox virus (rFPV) vaccines is far superior to other vector-based vaccines. Specifically, intranasal priming with rFPV vaccines can recruit unique antigen-presenting cells, which induce excellent mucosal and systemic HIV-specific CD8+ T-cell immunity. In this study, we have for the first time investigated the in vivo dissemination, safety and expression kinetics of rFPV post intranasal delivery using recombinant viruses expressing green fluorescent protein or mCherry. Both confocal microscopy of tissue sections using green fluorescent protein and in vivo Imaging System (IVIS) spectrum live animal and whole organ imaging studies using mCherry revealed that (i) the peak antigen expression occurs 12 to 24 h post vaccination and no active viral gene expression is detected 96 h post vaccination. (ii) The virus only infects the initial vaccination site (lung and nasal cavity) and does not disseminate to distal sites such as the spleen or gut. (iii) More importantly, rFPV does not cross the olfactory receptor neuron pathway. Collectively, our findings indicate that rFPV vector-based vaccines have all the hallmarks of a safe and effective mucosal delivery vector, suitable for clinical evaluation.

An Evaluation of Type 1 Interferon Related Genes in Male and Female-Matched, SARS-CoV-2 Infected Individuals Early in the COVID-19 Pandemic.

SARS-CoV-2 infection has claimed just over 1.1 million lives in the US since 2020. Globally, the SARS-CoV-2 respiratory infection spread to 771 million people and caused mortality in 6.9 million individuals to date. Much of the early literature showed that SARS-CoV-2 immunity was defective in the early stages of the pandemic, leading to heightened and, sometimes, chronic inflammatory responses in the lungs. This lung-associated 'cytokine storm' or 'cytokine release syndrome' led to the need for oxygen supplementation, respiratory distress syndrome, and mechanical ventilation in a relatively high number of people. In this study, we evaluated circulating PBMC from non-hospitalized, male and female, COVID-19+ individuals over the course of infection, from the day of diagnosis (day 0) to one-week post diagnosis (day 7), and finally 4 weeks after diagnosis (day 28). In our early studies, we included hospitalized and critically care patient PBMC; however, most of these individuals were lymphopenic, which limited our assessments of their immune integrity. We chose a panel of 30 interferon-stimulated genes (ISG) to evaluate by PCR and completed flow analysis for immune populations present in those PBMC. Lastly, we assessed immune activation by stimulating PBMC with common TLR ligands. We identified changes in innate cells, primarily the innate lymphoid cells (ILC, NK cells) and adaptive immune cells (CD4+ and CD8+ T cells) over this time course of infection. We found that the TLR-7 agonist, Resiquimod, and the TLR-4 ligand, LPS, induced significantly better IFNα and IFNγ responses in the later phase (day 28) of SARS-CoV-2 infection in those non-hospitalized COVID-19+ individuals as compared to early infection (day 0 and day 7). We concluded that TLR-7 and TLR-4 agonists may be effective adjuvants in COVID-19 vaccines for mounting immunity that is long-lasting against SARS-CoV-2 infection.

MAIT Cells Modulate Innate Immune Cells and Inhibit Colon Cancer Growth.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can be activated by microbial antigens and cytokines and are abundant in mucosal tissues including the colon. MAIT cells have cytotoxic and pro-inflammatory functions and have potentials for use as adoptive cell therapy. However, studies into their anti-cancer activity, including their role in colon cancer, are limited. Using an animal model of colon cancer, we show that peritumoral injection of in vivo-expanded MAIT cells into RAG1-/- mice with MC38-derived tumors inhibits tumor growth compared to control. Multiplex cytokine analyses show that tumors from the MAIT cell-treated group have higher expression of markers for eosinophil-activating cytokines, suggesting an association between eosinophil recruitment and tumor inhibition. In a human peripheral leukocyte co-culture model, we show that leukocytes stimulated with MAIT ligand show an increase in eotaxin-1 production and activation of eosinophils, associated with increased cancer cell killing. In conclusion, we show that MAIT cells have a protective role in a murine colon cancer model, associated with modulation of the immune response to cancer, potentially involving eosinophil-associated mechanisms. Our results highlight the potential of MAIT cells for non-donor restricted colon cancer immunotherapy.

Steady-state estradiol triggers a unique innate immune response to allergen resulting in increased airway resistance.

RATIONALE: Asthma is a chronic airway condition that occurs more often in women than men during reproductive years. Population studies have collectively shown that long-term use of oral contraceptives decreased the onset of asthma in women of reproductive age. In the current study, we hypothesized that steady-state levels of estrogen would reduce airway inflammation and airway hyperresponsiveness to methacholine challenge. METHODS: Ovariectomized BALB/c mice (Ovx) were implanted with subcutaneous hormone pellets (estrogen, OVX-E2) that deliver consistent levels of estrogen [68 ± 2 pg/mL], or placebo pellets (OVX-Placebo), followed by ovalbumin sensitization and challenge. In conjunction with methacholine challenge, immune phenotyping was performed to correlate inflammatory proteins and immune populations with better or worse pulmonary outcomes measured by invasive pulmonary mechanics techniques. RESULTS: Histologic analysis showed an increase in total cell infiltration and mucus staining around the airways leading to an increased inflammatory score in ovarectomized (OVX) animals with steady-state estrogen pellets (OVX-E2-OVA) as compared to other groups including female-sham operated (F-INTACT-OVA) and OVX implanted with a placebo pellet (OVX-Pl-OVA). Airway resistance (Rrs) and lung elastance (Ers) were increased in OVX-E2-OVA in comparison to F-INTACT-OVA following aerosolized intratracheal methacholine challenges. Immune phenotyping revealed that steady-state estrogen reduced CD3+ T cells, CD19+ B cells, ILC2 and eosinophils in the BAL across all experiments. While these commonly described allergic cells were reduced in the BAL, or airways, we found no changes in neutrophils, CD3+ T cells or CD19+ B cells in the remaining lung tissue. Similarly, inflammatory cytokines (IL-5 and IL-13) were also decreased in OVX-E2-OVA-treated animals in comparison to Female-INTACT-OVA mice in the BAL, but in the lung tissue IL-5, IL-13 and IL-33 were comparable in OVX-E2-OVA and F-INTACT OVA mice. ILC2 were sorted from the lungs and stimulated with exogenous IL-33. These ILC2 had reduced cytokine and chemokine expression when they were isolated from OVX-E2-OVA animals, indicating that steady-state estrogen suppresses IL-33-mediated activation of ILC2. CONCLUSIONS: Therapeutically targeting estrogen receptors may have a limiting effect on eosinophils, ILC2 and potentially other immune populations that may improve asthma symptoms in those females that experience perimenstrual worsening of asthma, with the caveat, that long-term use of estrogens or hormone receptor modulators may be detrimental to the lung microenvironment over time.

A subset of follicular helper-like MAIT cells can provide B cell help and support antibody production in the mucosa.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that aid in protection against bacterial pathogens at mucosal surfaces through the release of inflammatory cytokines and cytotoxic molecules. Recent evidence suggests that MAIT cells can also provide B cell help. In this study, we describe a population of CXCR5+ T follicular helper (Tfh)­like MAIT cells (MAITfh) that have the capacity to provide B cell help within mucosal lymphoid organs. MAITfh cells are preferentially located near germinal centers in human tonsils and express the classical Tfh-associated transcription factor, B cell lymphoma 6 (BCL-6), the costimulatory markers inducible T cell costimulatory (ICOS) and programmed death receptor 1 (PD-1), and interleukin-21 (IL-21). We demonstrate the ability of MAIT cells to provide B cell help in vivo after mucosal challenge with Vibrio cholerae. Specifically, we show that adoptive transfer of MAIT cells into αß T cell­deficient mice promoted B cell differentiation and increased serum V. cholerae­specific IgA responses. Our data demonstrate the capacity of MAIT cells to participate in adaptive immune responses and suggest that MAIT cells may be potential targets for mucosal vaccines.

Use of a MAIT-Activating Ligand, 5-OP-RU, as a Mucosal Adjuvant in a Murine Model of Vibrio cholerae O1 Vaccination.

Background: Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in the mucosa with capacity for B-cell help. We hypothesize that targeting MAIT cells, using a MAIT-activating ligand as an adjuvant, could improve mucosal vaccine responses to bacterial pathogens such as Vibrio cholerae. Methods: We utilized murine models of V. cholerae vaccination to test the adjuvant potential of the MAIT-activating ligand, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU). We measured V. cholerae-specific antibody and antibody-secreting cell responses and used flow cytometry to examine MAIT-cell and B-cell phenotype, in blood, bronchoalveolar lavage fluid (BALF), and mucosal tissues, following intranasal vaccination with live V. cholerae O1 or a V. cholerae O1 polysaccharide conjugate vaccine. Results: We report significant expansion of MAIT cells in the lungs (P < 0.001) and BALF (P < 0.001) of 5-OP-RU treated mice, and higher mucosal (BALF, P = 0.045) but not systemic (serum, P = 0.21) V. cholerae O-specific-polysaccharide IgG responses in our conjugate vaccine model when adjuvanted with low-dose 5-OP-RU. In contrast, despite significant MAIT cell expansion, no significant differences in V. cholerae-specific humoral responses were found in our live V. cholerae vaccination model. Conclusions: Using a murine model, we demonstrate the potential, as well as the limitations, of targeting MAIT cells to improve antibody responses to mucosal cholera vaccines. Our study highlights the need for future research optimizing MAIT-cell targeting for improving mucosal vaccines.

Erratum to: Use of a MAIT-Activating Ligand, 5-OP-RU, as a Mucosal Adjuvant in a Murine Model of Vibrio cholerae O1 Vaccination.

[This corrects the article DOI: 10.20411/pai.v7i1.525.].

IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17ß-estradiol.

Asthmatic women tend to develop severe airway disease in their reproductive years, and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells, or ILC2, are readily detected in allergic conditions, such as rhinosinusitis, in individuals that develop nasal polyps do to allergen exposures, and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33, an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in naïve and ovalbumin (OVA) challenged, female BALB/c mice in comparison to male mice following stimulation with IL-33. Here, we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-κB signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated, and had reduced NF-κB activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2, and Nfkb1 was found among top-ranked regulatory interactions. Together, these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases.

Mucosal-Associated Invariant T (MAIT) cells are highly activated in duodenal tissue of humans with Vibrio cholerae O1 infection: A preliminary report.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes with a semi-conserved TCRα, activated by the presentation of vitamin B metabolites by the MHC-I related protein, MR1, and with diverse innate and adaptive effector functions. The role of MAIT cells in acute intestinal infections, especially at the mucosal level, is not well known. Here, we analyzed the presence and phenotype of MAIT cells in duodenal biopsies and paired peripheral blood samples, in patients during and after culture-confirmed Vibrio cholerae O1 infection. Immunohistochemical staining of duodenal biopsies from cholera patients (n = 5, median age 32 years, range 26-44, 1 female) identified MAIT cells in the lamina propria of the crypts, but not the villi. By flow cytometry (n = 10, median age 31 years, range 23-36, 1 female), we showed that duodenal MAIT cells are more activated than peripheral MAIT cells (p < 0.01 across time points), although there were no significant differences between duodenal MAIT cells at day 2 and day 30. We found fecal markers of intestinal permeability and inflammation to be correlated with the loss of duodenal (but not peripheral) MAIT cells, and single-cell sequencing revealed differing T cell receptor usage between the duodenal and peripheral blood MAIT cells. In this preliminary report limited by a small sample size, we show that MAIT cells are present in the lamina propria of the duodenum during V. cholerae infection, and more activated than those in the blood. Future work into the trafficking and tissue-resident function of MAIT cells is warranted.

Diverse Mucosal-Associated Invariant TCR Usage in HIV Infection.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that specifically target bacterial metabolites but are also identified as innate-like sensors of viral infection. Individuals with chronic HIV-1 infection have lower numbers of circulating MAIT cells compared with healthy individuals, yet the features of the MAIT TCR repertoire are not well known. We isolated and stimulated human PBMCs from healthy non-HIV-infected donors (HD), HIV-infected progressors on antiretroviral therapy, and HIV-infected elite controllers (EC). We sorted MAIT cells using flow cytometry and used a high-throughput sequencing method with bar coding to link the expression of TCRα, TCRß, and functional genes of interest at the single-cell level. We show differential patterns of MAIT TCR usage among the groups. We observed expansions of certain dominant MAIT clones in HIV-infected individuals upon Escherichia coli stimulation, which was not observed in clones of HD. We also found different patterns of CDR3 amino acid distributions among the three groups. Furthermore, we found blunted expression of phenotypic genes in HIV individuals; most notably, HD mounted a robust IFNG response to stimulation, whereas both HIV-infected progressors and EC did not. In conclusion, our study describes the diverse MAIT TCR repertoire of persons with chronic HIV-1 infection and suggest that MAIT clones of HIV-infected persons may be primed for expansion more than that of noninfected persons. Further studies are needed to examine the functional significance of unique MAIT cell TCR usage in EC.

Intestinal Infection Is Associated With Impaired Lung Innate Immunity to Secondary Respiratory Infection.

BACKGROUND: Pneumonia and diarrhea are among the leading causes of death worldwide, and epidemiological studies have demonstrated that diarrhea is associated with an increased risk of subsequent pneumonia. Our aim was to determine the impact of intestinal infection on innate immune responses in the lung. METHODS: Using a mouse model of intestinal infection by Salmonella enterica serovar Typhimurium (S. Typhimurium [ST]), we investigated associations between gastrointestinal infections and lung innate immune responses to bacterial (Klebsiella pneumoniae) challenge. RESULTS: We found alterations in frequencies of innate immune cells in the lungs of intestinally infected mice compared with uninfected mice. On subsequent challenge with K. pneumoniae, we found that mice with prior intestinal infection have higher lung bacterial burden and inflammation, increased neutrophil margination, and neutrophil extracellular traps, but lower overall numbers of neutrophils, compared with mice without prior intestinal infection. Total numbers of dendritic cells, innate-like T cells, and natural killer cells were not different between mice with and without prior intestinal infection. CONCLUSIONS: Together, these results suggest that intestinal infection impacts lung innate immune responses, most notably neutrophil characteristics, potentially resulting in increased susceptibility to secondary pneumonia.

Mucosal-associated invariant T (MAIT) cells mediate protective host responses in sepsis.

Sepsis is a systemic inflammatory response to infection and a leading cause of death. Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in mucosal tissues that recognize bacterial ligands. We investigated MAIT cells during clinical and experimental sepsis, and their contribution to host responses. In experimental sepsis, MAIT-deficient mice had significantly increased mortality and bacterial load, and reduced tissue-specific cytokine responses. MAIT cells of WT mice expressed lower levels of IFN-γ and IL-17a during sepsis compared to sham surgery, changes not seen in non-MAIT T cells. MAIT cells of patients at sepsis presentation were significantly reduced in frequency compared to healthy donors, and were more activated, with decreased IFN-γ production, compared to both healthy donors and paired 90-day samples. Our data suggest that MAIT cells are highly activated and become dysfunctional during clinical sepsis, and contribute to tissue-specific cytokine responses that are protective against mortality during experimental sepsis.

Human mucosal-associated invariant T (MAIT) cells possess capacity for B cell help.

Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset, restricted by the nonclassic MHC class I-related protein MR1 and enriched at mucosal sites. Human studies have shown an association between MAIT cells and pathogen-specific antibody responses. In this study, we investigate the effect of human MAIT cells on B cells ex vivo. We found that supernatants from microbe- or cytokine-stimulated MAIT cells, when added to purified autologous B cells, increase frequencies of plasmablasts and promote IgA, IgG, and IgM production. We found effects to be mostly MR1-dependent and that the increases in plasmablasts are likely a result of increased differentiation from memory B cells. Furthermore, microbe-activated MAIT cell supernatant contains multiple cytokines known to stimulate B cells, including IL-6, -10, and -21. This study thus provides the first direct evidence of a newly identified role of MAIT cells in providing help to B cells.

The Influence of Immunization Route, Tissue Microenvironment, and Cytokine Cell Milieu on HIV-Specific CD8+ T Cells Measured Using Fluidigm Dynamic Arrays.

Thirty different genes including cytokines, chemokines, granzymes, perforin and specifically integrins were evaluated in Peyer's patch-KdGag197-205-specific CD8+ T cells (pools of 100 cells) using Fluidigm 48.48 Dynamic arrays following three different prime-boost immunization strategies. Data revealed that the route of prime or the booster immunization differentially influenced the integrin expression profile on gut KdGag197-205-specific CD8+ T cells. Specifically, elevated numbers of integrin αE and αD expressing gut KdGag197-205-specific CD8+ T cells were detected following mucosal but not systemic priming. Also, αE/ß7 and αD/ß2 heterodimerization were more noticeable in an intranasal (i.n.)/i.n. vaccination setting compared to i.n./intramuscular (i.m) or i.m./i.m. vaccinations. Moreover, in all vaccine groups tested α4 appeared to heterodimerize more closely with ß7 then ß1. Also MIP-1ß, RANTES, CCR5, perforin and integrin α4 bio-markers were significantly elevated in i.n./i.m. and i.m./i.m. immunization groups compared to purely mucosal i.n./i.n. delivery. Furthermore, when wild type (WT) BALB/c and IL-13 knockout (KO) mice were immunized using i.n./i.m. strategy, MIP-1α, MIP-1ß, RANTES, integrins α4, ß1 and ß7 mRNA expression levels were found to be significantly different, in mucosal verses systemic KdGag197-205-specific CD8+ T cells. Interestingly, the numbers of gut KdGag197-205-specific CD8+ T cells expressing gut-homing markers α4ß7 and CCR9 protein were also significantly elevated in IL-13 KO compared to WT control. Collectively, our findings further corroborate that the route of vaccine delivery, tissue microenvironment and IL-13 depleted cytokine milieu can significantly alter the antigen-specific CD8+ T cell gene expression profiles and in turn modulate their functional avidities as well as homing capabilities.

IL-17A expression in HIV-specific CD8 T cells is regulated by IL-4/IL-13 following HIV-1 prime-boost immunization.

Although Th1 and Th2 cytokines can inhibit interleukin (IL)-17-secreting T cells, how these cells are regulated under different infectious conditions is still debated. Our previous studies have shown that vaccination of IL-4 and IL-13 gene knockout (KO) mice can induce high-avidity HIV K(d)Gag197-205-specific CD8 T cells with better protective efficacy. In this study, when IL-13, IL-4, STAT6 KO, and wild-type BALB/c mice were prime-boost immunized with an HIV poxviral modality, elevated numbers of IL-17A(+) splenic K(d)Gag197-205-specific CD8 T cells were observed in all the KO mice compared with the wt BALB/c control. Similarly, when wt BALB/c mice were immunized with IL-13Rα2-adjuvanted HIV vaccines (that transiently inhibited IL-13 activity and induced high-avidity CD8 T cells with enhanced protective efficacy), elevated IL-17A(+) K(d)Gag197-205-specific CD8 T cells were detected both in the lung and the spleen. However, at the transcriptional level, elevated TGF-ß, IL-6, ROR-γt, and IL-17A mRNA copy numbers were mainly detected in IL-4 KO, but not the IL-13 KO mice. These data suggested that TGF-ß, IL-6, ROR-γt, but not IL-23a, played a role in IL-17A regulation in K(d)Gag197-205-specific CD8 T cells. Collectively, our findings suggest that IL-4 and IL-13 differentially regulate the expression of IL-17A in K(d)Gag197-205-specific CD8 T cells at the transcriptional and translational level, respectively, implicating IL-17A as an indirect modulator of CD8 T cell avidity and protective immunity.

Identification of biomarkers to measure HIV-specific mucosal and systemic CD8(+) T-cell immunity using single cell Fluidigm 48.48 Dynamic arrays.

Thirty genes composed of cytokines, chemokines, granzymes, perforin and integrins were evaluated in gut and splenic K(d)Gag197-205-specific single CD8(+) T cells using Fluidigm 48.48 Dynamic arrays, with the aim of identifying biomarkers to predict effective mucosal and systemic vaccine efficacy. The mRNA expression profiles were analyzed in three ways: (i) the "number" of K(d)Gag197-205-specific CD8(+) T cells expressing the biomarker, (ii) "level" of mRNA expression using principal component analysis (PCA) and (iii) poly-functionality in relation to RANTES expression. In total, 21 genes were found to be differentially expressed between the vaccine groups and the immune compartments tested. Overall, the PCA indicated that IL-13Rα2 or IL-4R antagonist adjuvanted vaccines that previously induced high-avidity mucosal/systemic CD8(+) T cells with better protective efficacy, the "level" of mRNA expression, specifically RANTES, MIP-1ß, and integrin α4 in gut K(d)Gag197-205-specific single CD8(+) T cells, were significantly elevated compared to unadjuvanted vaccine. Furthermore, significantly elevated granzymes/perforin levels were detected in IL-13(-/-) mice given the unadjuvanted vaccine, indicating that the degree of IL-13 inhibition (total, transient or no inhibition) can considerably alter the level of T-cell activity/poly-functionality. When splenic- and gut-K(d)Gag197-205-specific CD8(+) T cells were compared, PC1 vs. PC2 scores revealed that not only RANTES, MIP-1ß, and integrin α4 mRNA, but also perforin, granzymes A/B, and integrins ß1 and ß2 mRNA were elevated in spleen. Collectively, data suggest that RANTES, MIP-1ß, perforin, and integrins α4, ß1 and ß7 mRNA in single HIV-specific CD8(+) T cells could be used as a measure of effective mucosal and systemic vaccine efficacy.

Different HIV pox viral vector-based vaccines and adjuvants can induce unique antigen presenting cells that modulate CD8 T cell avidity.

The lung-derived dendritic cell (LDC) recruitment following intranasal (i.n.) vaccination of different poxviral vector-based vaccines/adjuvants were evaluated to decipher how these factors influenced CD8(+) T cell avidity. Compared to the standard i.n. recombinant fowlpox virus (FPV)-HIV vaccination, the FPV-HIV IL-13Rα2 or IL-4Rα antagonist adjuvanted vaccines that induced higher avidity CD8(+) T cells, also recruited significantly elevated MHCII(+) CD11c(+) CD11b(+) CD103(-) CD64(-) MAR-1(-) conventional DC (cDCs) to the lung mucosae (hierarchy: IL-4R antagonist>IL-13Rα2>unadjuvanted). In contrast, elevated CD11b(-) CD103(+) LDCs were detected in animals that received recombinant HIV vaccinia virus (rVV) or Modified Vaccinia Ankara virus (MVA) vector-based vaccines. Adoptive transfer studies indicated that CD11b(-) CD103(+) LDCs significantly dampened HIV-specific CD8(+) T cell avidity compared to CD11b(+) CD103(-) LDCs. Collectively; our observations revealed that rFPV vector prime and transient inhibition of IL-4/IL-13 at the vaccination site favoured the recruitment of unique LDCs, associated with the induction of high quality immunity.

Novel HIV IL-4R antagonist vaccine strategy can induce both high avidity CD8 T and B cell immunity with greater protective efficacy.

We have established that the efficacy of a heterologous poxvirus vectored HIV vaccine, fowlpox virus (FPV)-HIV gag/pol prime followed by attenuated vaccinia virus (VV)-HIV gag/pol booster immunisation, is strongly influenced by the cytokine milieu at the priming vaccination site, with endogenous IL-13 detrimental to the quality of the HIV specific CD8+ T cell response induced. We have now developed a novel HIV vaccine that co-expresses a C-terminal deletion mutant of the mouse IL-4, deleted for the essential tyrosine (Y119) required for signalling. In our vaccine system, the mutant IL-4C118 can bind to IL-4 type I and II receptors with high affinity, and transiently prevent the signalling of both IL-4 and IL-13 at the vaccination site. When this IL-4C118 adjuvanted vaccine was used in an intranasal rFPV/intramuscular rVV prime-boost immunisation strategy, greatly enhanced mucosal/systemic HIV specific CD8+ T cells with higher functional avidity, expressing IFN-γ, TNF-α and IL-2 and greater protective efficacy were detected. Surprisingly, the IL-4C118 adjuvanted vaccines also induced robust long-lived HIV gag-specific serum antibody responses, specifically IgG1 and IgG2a. The p55-gag IgG2a responses induced were of a higher magnitude relative to the IL-13Rα2 adjuvant vaccine. More interestingly, our recently tested IL-13Rα2 adjuvanted vaccine which only inhibited IL-13 activity, even though induced excellent high avidity HIV-specific CD8+ T cells, had a detrimental impact on the induction of gag-specific IgG2a antibody immunity. Our observations suggest that (i) IL-4 cell-signalling in the absence of IL-13 retarded gag-specific antibody isotype class switching, or (ii) IL-13Rα2 signalling was involved in inducing good gag-specific B cell immunity. Thus, we believe our novel IL-4R antagonist adjuvant strategy offers great promise not only for HIV-1 vaccines, but also against a range of chronic infections where sustained high quality mucosal and systemic T and B cell immunity are required for protection.