Fig 2: Hip1 impedes DLL4 expression on lung DCs via a CD40-dependent mechanismC57BL/6 (CD40+/+) or CD40-/- mice were infected via the aerosol route with a low-dose of Mtb or hip1mutant. Following 2 weeks post-infection, mice were euthanized and lung ex vivo responses were measured using flow cytometry.(A) Fold of infected mice over uninfected mice (from the same mouse strain) for DLL4+ and DLL4+Jagged1+ in the CD103+ DC population.(B) Fold over uninfected for cytokine-positive CD4 T cells.(C) Fold over uninfected for NOTCH receptor-positive CD4 T cells. All CD11b+ DC populations are singlets/live cells/CD45+/CD3-/CD64-F4/80-/MHCII+CD11c+/CD11b+CD103-. All CD103+ DC populations are singlets/live cells/CD45+/CD3-/CD64-F4/80-/MHCII+CD11c+/CD11b-CD103+. All T cell populations are singlets/live cells/CD3+/CD4+. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Data are presented as fold over uninfected mean ± SD. Data are representative of 2 independent experiments. Statistical significance p value key is the following: * = = 0.05, ** = = 0.01, *** = = 0.001.

Fig 3: DLL4 is required for Th17 polarization but is dispensable for Th1 polarizationBMDCs were stimulated with either media alone (UI), or HK Mtb (MOI 30) with or without 1 µg/mL of CD40LT. Following 24H of stimulation, cells were pulsed with 10 µg/mL of cognate peptide (OVA323-339) for one hour and then co-cultured with purified naïve CD4 OT-II Tg Thy1.1 T cells at a ratio of 4:1 T cells to DCs. For blockade conditions, blocking antibodies to either DLL4, Jagged1, or both DLL4 and Jagged1 combined, were added at the time of co-culture at the following concentrations: 15, 30, or 60 µg/mL. After 72H of co-culture, cell-free supernatants were harvested and assayed for cytokines by ELISA.(A) IFN-? (Th1).(B) IL-17 (Th17).(C) IL-2. Data are presented as mean ± SD. Data are representative of 3 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons (all data points were compared to Mtb + CD40LT). Statistical significance p value key is the following: *** = = 0.001, **** = = 0.0001. See also Figures S5–S7.

Fig 4: Th17 responses correlate with NOTCH2 expression and lower lung CFUBMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 µg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry and lung homogenates were plated to enumerate Mtb burdens.(A) Correlogram using R package “corrplot” showing correlation between the frequency of marker-positive CD4 T cells and frequency of cytokine-positive CD4 T cells.(B) Correlations in R using “ggscatter” between the frequency of marker-positive CD4 T cells and frequency of cytokine-positive CD4 T cells.(C) Mtb lung colony-forming unit (CFU) and correlations between CFU and frequencies of marker-positive or cytokine-positive CD4 T cells using “ggscatter” in R. All correlations presented are Pearson’s correlations. Data in C) are presented as mean ± SD and were analyzed using an unpaired Student’s t test. Data are representative of 2 independent experiments. Statistical significance p value key is the following: * = = 0.05, ** = = 0.01, *** = = 0.001, **** = = 0.0001. See also Figure S10.

Fig 5: Blocking DLL4 singly or in combination with Jagged1 on DCs reduces antigen-specific IL-17+ CD4 T cell frequencies in vivo(A) Experimental schema. BMDCs we stimulated with either media alone (UI), or HK Mtb (MOI 30) with or without 1 µg/mL of CD40LT for 24H. At this time in the CD40LT condition, either DLL4 or DLL4 and Jagged1 blocking antibodies (30 µg/mL) were added. One day before transfer, 1E6 ESAT-6 transgenic T cells were transferred into mice via the intravenous (IV) route. On the day of intratracheal (IT) transfer, 1E6 DCs were transferred. At 6 days after transfer, mice were euthanized and lung single cell suspensions were stimulated with 10 µg/mL ESAT-61-20 peptide to assess antigen-specific responses. Cells were then stained for flow cytometry.(B) Representative flow cytometry plots of IFN-?+ and IL-17+ frequencies.(C) Cytokine-positive frequency of CD4 T cells. Populations shown are singlets/live cells/CD3+/CD4+. Experimental schema was made with BioRender.com. Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ns = no significance, * = = 0.05, ** = = 0.01, *** = = 0.001, **** = = 0.0001.