Intestinal cDC1s provide IL-12 dependent and independent functions required for CD4+ T cell-mediated resistance to Cryptosporidium

Abstract

Cryptosporidium is an enteric pathogen that is a prominent cause of diarrheal disease. Control of this infection requires CD4⁺ T cells, though the processes that lead to T cell-mediated resistance have been difficult to assess. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to dissect the early events that influence CD4⁺ T cell priming and effector function. These studies highlight that parasite-specific CD4⁺ T cells are primed in the draining mesenteric lymph node (mesLN) and differentiate into Th1 cells in the gut, where they mediate IFN-γ-dependent control of the infection. Although type 1 conventional dendritic cells (cDC1s) were not required for initial priming of CD4⁺ T cells, cDC1s were required for CD4⁺ T cell expansion and gut homing. cDC1s were also a major source of IL-12 that was not required for priming but promoted full differentiation of CD4⁺ T cells and local production of IFN-γ. Together, these studies reveal distinct roles for cDC1s in shaping CD4⁺ T cell responses to enteric infection: first to drive early expansion in the mesLN and second to drive effector responses in the gut.

Figure 1.. Engineering Cryptosporidium to express MHCII-restricted model antigens allows for identification of parasite-specific CD4⁺ T cells.

A. Genetic construct of transgenic Cp-2W1S (right) or Cp-gp61 (left) engineered to tag the C-terminus of MEDLE2 with 3xHA-2W1S-SIINFEKL or SIINFEKL-gp61–3xHA tags. The construct also included the neomycin (Neo) selection marker and the nanoluciferase (nluc) reporter to monitor parasite burden, as well as cytoplasmic mNeon. B. HCT-8 cells were infected for 24 hours with 300,000 oocysts of mNeon green Cp-2W1S (right) or Cp-gp61 (left) and then stained for nuclear dye (Hoechst, blue) and HA (red). A white arrow points to the parasite within the cell. Scale bar: 10 μm. C. Ifng^−/− mice received 2×10⁴ CD45.1⁺ SMARTA T cells and were left uninfected or infected with either 1×10⁴ Cp-2W1S or Cp-gp61 oocysts and ileal draining mesenteric lymph node (mesLN), small intestinal lamina propria (SILP), or intraepithelial lymphocytes (IEL) were harvested at 10 dpi for flow cytometry. Representative flow plots show 2W1S:I-Ab+ or CD45.1⁺ SMARTA T cells in the SILP. Gated on Singlets, Live, CD19⁻, NK1.1⁻, CD90.2⁺, CD8a⁻ CD4⁺, CD44-hi, 2W1S:I-Ab⁺ or CD45.1⁺. C. Summary bar graphs from mesLN, SILP, and IEL of mice in (B) showing means of n=3 mice/group from 2 experiments. SMARTA T cells were CD45.1⁺CD44-hi and 2W1S-tetramer⁺ were 2W1S:I-Ab⁺CD44-hi. Statistical significance was determined in (C) by two-way ANOVA and multiple comparisons. p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.

Figure 2.. Cryptosporidium-specific CD4⁺ T cells produce IFN-γ locally to protect against infection.

A-D. IFN-γ-CD90.1 reporter mice were left untreated or treated with 1mg/mouse of αIFN-γ 1 day prior to infection and 2, 5, and 8 dpi with 10⁴ Cp-2W1S. mesLN, SILP, and IEL were harvested at 10 dpi for flow cytometry. Data shown are from 1 experiment representative of 2 independent experiments. A. Representative flow plots from the SILP and IEL of CD4⁺ T cells stained with 2W1S:I-Ab tetramer in PE and APC showing detection of tetramer+ cells when mice are infected and treated with IFN-γ. Gating: Singlets, Live⁺, CD19⁻, NK1.1⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺. B. Quantification/summary of (A) and tetramer (PE and APC double positive) staining from mesLN, SILP, and IEL. C. Representative flow plots showing CD90.1 expression in polyclonal CD4⁺ T cells (first two columns) or 2W1S:I-Ab tetramer⁺ cells (last column) in SILP and IEL of infected mice untreated (first column) or treated with αIFN-γ (last two columns). Gating: Singlets, Live⁺, CD19⁻, NK1.1⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, 2W1S:I-Ab⁺. D. Quantification/summary of (C) showing the percent of cells that were CD90.1⁺ from the following groups: polyclonal CD4⁺ T cells from uninfected mice (white), polyclonal CD4⁺ T cells from untreated mice infected with Cp-2W1S (gray), polyclonal CD4⁺ T cells from αIFN-γ-treated mice infected with Cp-2W1S (white with diagonal stripes), or 2W1S:I-Ab tetramer⁺ CD4⁺ T cells from αIFN-γ-treated mice infected with Cp-2W1S (white with horizontal stripes). E-F. WT B6 mice received 2×10⁴ CD90.1/Ifng reporter SMARTA T cells 1 day prior to infection with 5×10⁴ maCp-ova-gp61 and were left untreated or treated with 1mg/mouse of αIFN-γ 1 day prior to infection and 2, 5, and 8 dpi. At 10 dpi mesLN and SILP were harvested for flow cytometry and cells were stimulated with exogenous gp61 peptide for 3 hours followed by intracellular cytokine staining and flow cytometry. Data shown are from 1 experiment representative of 2 independent experiments. E. Left: representative flow plots and from the SILP showing IFN-γ and TNF-α expression in SMARTA T cells after peptide stimulation. Gating: Singlets, Live⁺, CD19⁻, NK1.1⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, CD45.1⁺. Right: summary of showing the percentage of SMARTA cells from the SILP or mesLN staining IFN-γ+ after peptide stimulation. F. Left: representative flow plots from the SILP showing expression of CD90.1 as a reporter of Ifng expression on SMARTA T cells after peptide stimulation. Gating: Singlets, Live⁺, CD19⁻, NK1.1⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, CD45.1⁺. Right: summary showing the percentage of SMARTA T cells from the SILP or mesLN staining for CD90.1⁺ after peptide stimulation. G. PBS or 10⁶ SMARTA T cells were transferred into Ifng^−/− mice 1 day prior to infection with 10⁴ Cp-gp61 and feces was analyzed by nanoluciferase for parasite burden (relative luminescence) over time. For some mice receiving SMARTA T cells, mice also received 1mg/mouse of αIFN-γ blocking antibody 1 day prior to infection and 2, 5, and 8 dpi. Area under the curve analysis was performed for each treatment for 9–20 dpi. Graphs shown are from 1 experiment, representative of 2 independent experiments. Statistical significance was determined by two-way ANOVA and multiple comparisons. p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.

Figure 3.. Cryptosporidium-specific CD4⁺ T cells resemble polyclonal T-bet⁺ cells in the gut.

A. WT B6 mice received 2×10⁴ SMARTA T cells 1 day prior to infection with 5×10⁴ maCp-ova-gp61 oocysts and mesLN, SILP, and IEL were harvested at 10 dpi for flow cytometry. A. UMAP of mesLN, SILP, and IEL CD4⁺ T cells (Singlets, Live⁺, CD8a-EpCAM-NK1.1⁻CD19⁻CD4⁺TCRβ⁺) based on surface expression of the following markers: IL-18Ra, SLAM, CXCR3, T-bet, RORγT, Foxp3, CD40L, CD44, CD69, CD103, Ly6A/E, and LPAM-1. B. X-shift cluster analysis of concatenated CD4⁺ T cells from all tissues revealed 11 clusters. Heatmap of the Z-scores of surface marker expression by X-shift cluster. C. Overlays of each cluster onto the UMAP from Figure 3A. D. The percentage of cells in each cluster coming from mesLN, SILP or IEL. E. The percentage of total CD4⁺ T cells represented by each cluster in uninfected or infected mice. F. UMAP from Figure 3A colored by expression levels of each marker. G. Percentage of SMARTA T cells that fell into each cluster, with the total across all clusters equalling 100%. H. UMAP showing where SMARTA T cells fell in the UMAP (black dots).

Figure 4.. Progressive Th1 skewing of Cryptosporidium-specific CD4⁺ T cells

WT B6 mice received 2×10⁴ SMARTA T cells 1 day prior to infection with 5×10⁴ maCp-ova-gp61 oocysts and mesLN, SILP, and IEL were harvested at 10 dpi for flow cytometry. A. Representative flow plots of T-bet vs. RORγγ in polyclonal CD4⁺ T cells from uninfected mice, polyclonal CD4⁺ T cells from infected mice, or SMARTA T cells (CD45.1⁺) from infected mice organized by tissue (rows). Gating: Singlets, Live⁺, CD19⁻, NK1.1⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, CD45.1⁺. B. Percentage of T-bet⁺, or percentage negative for all lineage-defining TFs (T-bet, RORγγ, Foxp3, GATA3; TF⁻) in polyclonal CD4⁺ T cells from uninfected mice (white), polyclonal CD4⁺ T cells from infected mice (gray), or SMARTA T cells (horizontal lines). C. Percentage of cells positive for LPAM-1, CD44, IL-18Ra, Ly6A/E, or CD103 of polyclonal CD4⁺ T cells from uninfected mice, infected mice, or SMARTA T cells colored as in (B). D. Histograms of protein expression by flow cytometry among SMARTA T cells from the mesLN (teal), SILP (orange) or IEL (purple)). Gating: Singlets, Live+, CD8a⁻EpCAM⁻ NK1.1⁻CD19⁻CD4⁺TCRβ⁺CD45.1⁺. For A-B, representative of 2 independent experiments; for C-D, one independent experiment. n=3 mice/group. Statistical significance was determined by two-way ANOVA and multiple comparisons. p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.

Figure 5.. IL-12p40 is produced in the gut by activated cDC1s.

A-D. WT B6 mice were left uninfected or infected with 5×10⁵ maCp oocysts. At 4 dpi mesLN and SILP cells were isolated, plated with brefeldin A (BFA) for 6 hours, and analyzed by flow cytometry for surface markers and IL-12p40 expression. Plotted are histograms from mesLN or SILP (A), summary in (B); IL-12p40 flow plots from the mesLN and SILP in (C) summarized in (D) showing selective induction of IL-12p40 in cDC1s in the SILP of infected mice. Data shown are from 1 experiment representative of 2 independent experiments, 3–4 mice per group. Gating: Singlets, Live+, CD3e⁻, NK1.1⁻, EpCAM⁻, B220⁻, CD19⁻, CD90.2⁻, CD64⁻, MHCII-hi, CD11c⁺, CD26⁺, Ly6C⁻, XCR1⁺ (cDC1s) or SIRPα⁺ (cDC2s). Gating is based on FMOs taken from SILP for all colors except IL-12p40, for which positivity is based on samples from each tissue plated without BFA. Statistical significance was determined by two-way ANOVA and multiple comparisons. p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.

Figure 6.. cDC1s are required for expansion and gut homing of Cryptosporidium-specific CD4⁺ T cells.

A-E. 1 day prior to infection with 5×10⁴ maCp-ova-gp61 oocysts, 1×10⁶ CD45.1⁺ Nur77-GFP reporter SMARTA T cells were labeled with CellTrace Violet (CTV) and transferred into WT B6 mice or age/sex-matched Irf8+32^−/− mice. Mice were sacrificed at 1, 4, and 6 dpi and priming of SMARTA T cells was interrogated using flow cytometry. A-B. Representative flow plots from the mesLN of infected mice at 1, 4, and 6 dpi pre-gated on SMARTA T cells showing Nur77-GFP expression compared to cell division, with summary in (B). C-E Representative flow plots from mesLN SMARTAs showing CellTrace Violet versus CXCR3 (C) and LPAM-1 (D) with summary in (E). For A-E, data is from 1 experiment representative of 2 independent experiments. Gating for SMARTAs in A-E: Singlets, Live⁺, NK1.1⁻, CD19⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, TCR Vβ8.3⁺, CD45.1⁺. F-I. 1 day prior to infection with 5×10⁴ maCp-ova-gp61 oocysts, 2×10⁴ CD45.1⁺ SMARTA T cells were transferred into WT B6 mice or age/sex-matched Irf8+32^−/− mice. At 10 dpi, mesLN, SILP, and IEL were isolated and analyzed by flow cytometry. Data are representative of 2 independent experiments, n=3 mice/group. F. Representative flow plots showing presence or absence of SMARTA T cells in the mesLN (top), SILP (middle), or IEL (bottom). Plots are from 1 experiment representative of 3 independent experiments. F. Summary of (G). H-I. Percentage of mesLN SMARTA T cells staining positive for IL-18Ra with representative flow plots in (H) and summary in (I). Gating for SMARTAs in G-I: Singlets, Live⁺, NK1.1⁻, CD19⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, CD44-hi, CD45.1⁺.

Figure 7.. IL-12p40 is not required for gut-homing but is required for gut CD4⁺ T cell accumulation and induction of Th1 functions in Cryptosporidium-specific CD4⁺ T cells.

A-G. 1 day prior to infection with 5×10⁴ maCp-ova-gp61 oocysts, 2×10⁴ CD45.1+ SMARTA T cells were transferred into WT B6 mice that were treated with isotype control (rat IgG2a) or anti-IL-12p40 (1mg/mouse on d-1, or 2, 5, 8 dpi). At 10 dpi, mesLN, SILP, and IEL were isolated and analyzed by flow cytometry. Data shown in A-G is 1 experiment representative of 2 independent experiments, n=3–4 mice/group. A. Infection was monitored by nanluciferase of feces. B-C. Percentage of SMARTA T cells among CD4⁺ T cells and absolute numbers of SMARTA T cells from each treatment with representative flow plots from the SILP in (C). D-E. Percentage of SMARTA T cells staining T-bet+, with representative flow plots from the SILP in (E). F-G. Percentage of SMARTA T cells staining for IL-18Ra, with representative flow plots from the SILP in (G). H-I. 1 day prior to infection with 10⁴ Cp-gp61, 5×10⁴ CD45.1⁺ SMARTA T cells that expressed CD90.1 as a reporter for Ifng expression were transferred into Ifng^−/− mice that were treated with isotype control (rat IgG2a), anti-IL-12p40, anti-IL-18, or anti-IL12p40+anti-IL-18 (1mg/mouse on d-1, or 2, 5, 8 dpi). Mice were also treated with 1mg/mouse anti-IFN-γ on 3 and 7 dpi. At 10 dpi, mesLN, SILP, and IEL were isolated and analyzed by flow cytometry and cells were stained for CD90.1 to assess in vivo IFN-γ production. Data shown for K is pooled from 2 independent experiments, n=3 mice/group/experiment. Gating for SMARTAs in B-K: Singlets, Live+, NK1.1⁻, CD19⁻, EpCAM⁻, CD90.2⁺, CD8a⁻, CD4⁺, CD44-hi, CD45.1⁺. Statistical significance was determined by two-way ANOVA and multiple comparisons. p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.