Antigen-specific Th17 cells are primed by distinct and complementary dendritic cell subsets in oropharyngeal candidiasis

Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC) we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs) present in the infected oral mucosa. Candida- derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida- specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity

Cutting edge: IL-17-secreting innate lymphoid cells are essential for host defense against fungal infection

IL-17-mediated immunity has emerged as a crucial host defense mechanism against fungal infections. Although Th cells are generally thought to act as the major source of IL-17 in response to Candida albicans, we show that fungal control is mediated by IL-17-secreting innate lymphoid cells (ILCs) and not by Th17 cells. By using a mouse model of oropharyngeal candidiasis we found that IL-17A and IL-17F, which are both crucial for pathogen clearance, are produced promptly upon infection in an IL-23-dependent manner, and that ILCs in the oral mucosa are the main source for these cytokines. Ab-mediated depletion of ILCs in RAG1-deficient mice or ILC deficiency in retinoic acid-related orphan receptor c(-/-) mice resulted in a complete failure to control the infection. Taken together, our data uncover the cellular basis for the IL-23/IL-17 axis, which acts right at the onset of infection when it is most needed for fungal control and host protection

Arming Th17 cells for antifungal host defense

Interleukin 17 (IL-17)-mediated immunity has emerged as a crucial host defense mechanism against fungal infections. The family of IL-17 cytokines is phylogenetically ancient, but remains the least understood of all cytokine subclasses. The effects mediated by IL-17 are pleiotropic and include the induction of antimicrobial peptides as well as cytokines and chemokines that lead to the recruitment and activation of neutrophils. Neutrophils in turn are key effector cells of the antifungal defense. CD4+ T cells act as a major source of IL-17 and a lot has been learned about these cells since their discovery a decade ago. This review highlights key aspects of the underlying mechanisms regulating the development of Th17 responses during fungal infections. We discuss the impact of different subsets of antigen-presenting cells, innate cytokine signals and tissue-specific factors on Th17 differentiation, and we highlight the prerequisites for the mediation by Th17 cells of vaccine immunity against fungi

Antigen-specific Th17 cells are primed by distinct and complementary dendritic cell subsets in oropharyngeal candidiasis

Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC) we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs) present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity

MHC II^hi CD11c⁺ migratory DCs present <i>C</i>. <i>albicans</i>-derived antigen in the cervical lymph nodes.

<p>(<b>A</b>) Three major populations of CD11c and MHC II positive cells were identified in the cervical lymph nodes of B6 and <i>Ccr7</i>-/- mice on day 2 post-infection: population I (MHC II^high CD11c⁺), population II (MHC II⁺ CD11c^high) and population III (MHC II⁺ CD11c⁺). Representative FACS plots are shown on the left, summary of data from individual mice with mean + SD are shown on the right. (<b>B</b>) CD11b, CD64, CD24 and SIRPα expression in each of the three DC populations in cervical lymph nodes on day 2 post-infection, as defined in (A). Representative FACS plots are shown. <b>(C)</b> The three DC populations defined in (A) were quantified in the cervical lymph nodes of naïve and OPC infected B6 mice. Each symbol represents an individual mouse. Data in (A—C) are representative of at least two independent experiments. (<b>D—E</b>) Mice were infected sublingually with <i>C</i>. <i>albicans</i> and cervical lymph nodes were isolated on day 2 post-infection. The three DC populations defined in (A) were FACS-sorted and co-cultured with CD4⁺ Hector T cells without adding additional exogenous antigen. Thy1.1⁺ CD3⁺ CD4⁺ TCRVα2⁺ cells were analyzed for CD69 expression after 1 day (D) and for dilution of the CFSE signal after 4 days (E). Data show representative FACS plots from at least 3 independent experiments.</p

Transport of <i>C</i>. <i>albicans</i>-derived antigen to the cervical lymph nodes is CCR7-dependent.

<p>(<b>A</b>) Cervical lymph node cells that were isolated from naïve or from sublingually infected mice on day 2 post-infection and either enriched for CD11b⁺ cells or left non-enriched were co-cultured with the <i>C</i>. <i>albicans</i>-specific T cell hybridoma cells. IL-2 production as a read-out for hybridoma activation was quantified by CTLL-2 bioassay. (<b>B</b>) Cervical lymph node cells were isolated from naïve mice or from sublingually infected mice on day 2 p.i., enriched for CD11b⁺ cells, and co-cultured with CFSE-labeled CD4⁺ Hector T cells. CFSE-dilution of the Thy1.1⁺ CD3⁺ CD4⁺ Hector cells was analyzed after 4 days. (<b>C</b>) CD11b⁺ cells were enriched from cervical lymph nodes at different time points after infection as indicated and analyzed for antigen presentation as described in (A). <b>(D—E)</b> cervical lymph node cells were isolated from sublingually infected B6 or <i>Ccr7</i>-/- mice on day 2 post-infection, enriched for CD11b⁺ cells and co-cultured with CD4⁺ Hector T cells. CD69 expression (D) and CFSE dilution (E) of Thy1.1⁺ CD3⁺ CD4⁺ TCRVα2⁺ cells was analyzed on day 1 and day4 respectively. In (B), (D) and (E), representative FACS plots are shown on the left; summary of data from individual mice with mean + SD are shown on the right. Data are from individual experiments that are representative of at least two independent experiments each. <b>(F)</b> CD4⁺ Hector T cells were adoptively transferred into B6 and <i>Ccr7</i>-/- mice one day prior to sublingual infection. Cytokine production by Thy1.1⁺ CD3⁺ CD4⁺ Hector cells in the cervical lymph nodes was analyzed on day 7 post-infection after re-stimulation with DC¹⁹⁴⁰ cells pulsed with pADH1_126-140, heat-killed (h.k.) <i>C</i>. <i>albicans</i> or left unpulsed as indicated. Symbols represent individual mice pooled from 2 independent experiments, the mean + SD is indicated.</p

Antigen-specific Th cell response to <i>C</i>. <i>albicans</i> oropharyngeal infection.

<p>(<b>A, B</b>) CFSE-labelled CD4⁺ Hector T cells were adoptively transferred one day prior to sublingual infection of B6 mice with <i>C</i>. <i>albicans</i>. Proliferation of Thy1.1⁺ CD4⁺ Vα2⁺ Hector cells was analyzed by flow cytometry on day 3 post-infection. A representative FACS plot is shown in (A), quantification of Thy1.1⁺ CD4⁺ Vα2⁺ Hector cells in cervical lymph nodes is shown in (B). Each symbol represents an individual mouse, the mean + SD is indicated, data are pooled form 2 independent experiments. (<b>C—E</b>) CD4⁺ Hector T cells were adoptively transferred one day prior to sublingual infection with <i>C</i>. <i>albicans</i>. In (C), Thy1.1⁺ CD4⁺ Vα2⁺ Hector cells in the cervical lymph nodes were analyzed on day 7 post-infection for expression of CD44 and CD62L. Representative FACS plots from 3 independent experiments are shown. In (D—E), cytokine production by endogenous CD3⁺ CD4⁺ Thy1.2⁺ T cells (left) and Thy1.1⁺ CD4⁺ Vα2⁺ Hector cells (right) on day 7 post-infection was analyzed by flow cytometry after re-stimulation with DC¹⁹⁴⁰ cells pulsed with heat-killed (h.k.) <i>C</i>. <i>albicans</i> or pADH1_126-140 as indicated. Representative FACS plots are shown in C and D, quantification of IL-17-producing cells is shown in (E). Each symbol represents an individual mouse, the mean + SD is indicated, data are pooled form 2 independent experiments. (<b>F, G</b>) B6 mice obtained an adoptive transfer of CD4⁺ Hector T cells one day prior to systemic infection with 5 x 10⁴ cfu <i>C</i>. <i>albicans</i>. Cytokine production by endogenous CD3⁺ CD4⁺ Thy1.2⁺ T cells (left) and Thy1.1⁺ CD4⁺ Vα2⁺ Hector cells (right) in the renal lymph nodes (left) and spleen (right) on day 7 post-infection was analyzed after re-stimulation with DC¹⁹⁴⁰ cells pulsed with heat-killed (h.k.) <i>C</i>. <i>albicans</i> or pADH1_126-140 as indicated. Representative FACS plots are shown in (F), quantification of IFN-γ-producing cells is shown in (G). Each symbol represents an individual mouse, the mean + SD is indicated, data are pooled form 2 independent experiments.</p

Flt3L-dependent migratory DCs and monocyte-derived DCs both present <i>C</i>. <i>albicans</i> derived antigen.

<p><b>(A)</b> Cervical lymph node cells of B6 or <i>Flt3l</i>-/- mice were analyzed on day 2 post-infection. Representative FACS plots from individual mice and quantification of MHC II^high CD11c⁺ (population I), MHC II⁺ CD11c^high (population II) and MHC II⁺ CD11c⁺ cells (population III) from one of two independent experiments are shown. <b>(B, C)</b> Cervical lymph node cells were isolated from naïve B6 mice and from infected B6 and <i>Flt3l</i>-/- mice on day 2 post-infection, enriched for CD11b⁺ cells and co-cultured with CD4⁺ Hector T cells. Thy1.1⁺ CD3⁺ CD4⁺ TCRVα2⁺ cells were analyzed for CD69 expression after 1 day (B) and for proliferation after 4 days, respectively (C). Representative plots are shown on the left, summary of data from individual mice with mean + SD are shown on the right. Each symbol represents one mouse. Data are representative of 2 independent experiments. (<b>D</b>) As in B, but MHC II^high CD11c⁺ migratory DCs were isolated by FACS-sorting from the cervical lymph nodes of infected B6 and <i>Flt3l</i>-/- mice. (<b>E—F</b>) CCR2⁺ CD11b⁺ cells in the cervical lymph nodes of naïve and infected B6 mice on day 2 post-infection were analyzed for the expression of MHC II and CD11c (D). Three distinct subsets of CCR2⁺ CD11b⁺ cells (MHC II^lo/int Ly6C⁺ (Q1), MHC II^int Ly6C^- (Q2) and MHC II^high Ly6C^- (Q3)) from infected mice were further analyzed for the expression of CCR2 and CD11c (E). Representative FACS plots are shown. <b>(G)</b> Cervical lymph nodes were isolated from infected B6 mice on day 2 post-infection. CCR2^hi, CCR2^int and CCR2^lo subsets within the MHC II^high CD11c⁺ population I were FACS-sorted and co-cultured with CD4⁺ Hector T cells for 1 day. Thy1.1⁺ CD3⁺ CD4⁺ TCRVα2⁺ cells were then analyzed for CD69 expression. Representative FACS plots are shown, Data are representative of 2 independent experiments.</p

Flt3L-dependent migratory DCs and monocyte-derived DCs complement each other for Th17 priming during OPC.

<p><b>(A)</b> Cervical lymph nodes were isolated from B6 and <i>Flt3l</i>-/- mice on day 7 post-infection and re-stimulated with heat-killed (h.k.) <i>C</i>. <i>albicans</i> or medium only. IL-17A production by endogenous CD3⁺ CD4⁺ cells was then analyzed by flow cytometry. (<b>B</b>) B6 and <i>Ccr2</i>-/- mice were infected sublingually and IL-17 production by endogenous CD3⁺ CD4⁺ cells was analyzed on day 7 post-infection as described in (A). (<b>C</b>) B6 mice were treated with anti-CSF1R or left untreated prior to sublingual infection with <i>C</i>. <i>albicans</i>. IL-17 production by endogenous CD3⁺ CD4⁺ cervical lymph node cells was analyzed on day 7 post-infection as described in (A). (<b>D</b>) B6 and <i>Flt3l</i>-/- mice were treated with anti-CSF1R or left untreated prior to sublingual infection with <i>C</i>. <i>albicans</i>. IL-17 production by endogenous CD3⁺ CD4⁺ cervical lymph node cells was analyzed on day 7 post-infection as described in (A). Each symbol represents one mouse, the mean + SD for each group is shown.</p