Wild-type C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA) or Jackson Laboratories (Bar Harbor, ME). Mice were housed and used in a specific pathogen-free environment. All experiments were done using both male and female mice of age 6–8 weeks. The animal housing and experiments were performed according to the strict guidelines of the Institutional Animal Care and Use Committee at the University of Illinois Urbana-Champaign.

This work was executed in accordance with the protocols approved by the Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-Champaign.

The mice were vaccinated with an isogenic, attenuated (mutant lacking an essential virulence factor, Blastomyces adhesion-1; BAD1) (38) strain of Blastomyces dermatitidis ATCC 26199 named B.d. #55 (a kind gift from Dr. Bruce Klein, UW-Madison) subcutaneously (s.c.; ~2 × 10^5 CFU) at two sites, dorsally and at the base of the tail. For antigen presentation studies, recombinant B.d. #55 strain engineered to express OT-I epitope SIINFEKL-mCherry (30) was used for vaccination and the cohorts of mice vaccinated with OVA_257–264 peptide (10 μg/mL) pre-mixed with non-recombinant Blastomyces dermatitidis #55 served as positive control. The yeast strains were cultured on Middlebrook 7H10 agar slants supplemented with OADC (oleic acid-albumin complex; Sigma-Aldrich) at 39°C in a humidified incubator.

The draining lymph nodes (dLN) were harvested and processed as described (44, 45). Briefly, the harvested LN were dissected using 22G needles, and the separated tissues were digested in 1 mg/ml of Collagenase, Type IV (Stem-Cell) in 1X PBS containing 10 μg/ml DNAse I (Roche), kept at 37°C for 30 minutes. Digested tissues/cells were washed with 1X PBS, passed through 40μm strainers (BD Biosciences), and resuspended in complete RPMI media (supplemented with 10% FBS/NEAA/PenStrep). The single-cell suspensions were stained with fluorochrome-conjugated antibodies and analyzed by flow cytometry.

Yeasts were labeled with the Red Fluorescent Cell Linker Dye Kit, PKH26-MIDI26 (Sigma, St. Louis). The yeasts were harvested from the slants and incubated with diluted PKH26 (2 × 10^ (6) yeasts/ml) for 5 minutes at room temperature. To quench excess PKH26 dye, an equal volume of FBS (Corning) was added, mixed, and kept for a minute before washing with complete RPMI medium followed by 1X PBS.

For depletion of CD4⁺ T cells, GK1.5 MAb (BioXCell Inc., Lebanon, NH) was injected intravenously (i.v.) at every 3–4 days with a dose of 200 μg/mouse. The depletion efficiency of CD4⁺ T cells was verified by flow cytometry using MAb Clone RM4-5 (30).

All the antibodies were purchased from BD Biosciences, Biolegend, and Invitrogen. Single-cell suspensions of the dLNs were first incubated with anti-FCγ receptor MAbs (Fc Block; BD Biosciences) for 10 minutes before staining with fluorophore-conjugated antibodies for surface markers and Live/Dead stain (Invitrogen) in FACS buffer (2% BSA in 1X PBS with 0.1% NaN₃) for 30 minutes on ice. For measuring antigen cross-presentation, single-cell suspensions were stained with anti-mouse H-2K^b/SIINFEKL antibody (Clone: 25-D1.16) during surface staining for markers. The stained cells were washed three times with FACS buffer and fixed with 2% Paraformaldehyde in 1X PBS. The cells were analyzed using a full-spectrum flow cytometer, Cytek Aurora. The data were analyzed with FlowJo v10.10 (BD Biosciences).

Statistical analyses of DC dynamics, activation status, and antigen presentation of DC subsets in the unvaccinated vs. vaccinated mice and the presence versus absence of CD4⁺ T cells were conducted using a two-tailed unpaired Student’s t-test. DC kinetics and fungal antigen uptakes were analyzed by one-way ANOVA with post-hoc tests of Dunnett’s correction and Tukey’s correction for multiple comparisons, respectively. We performed all the statistical analyses using Prism 10.2 (GraphPad Software, LLC) software. A two-tailed p-value of ≤ 0.05 was considered statistically significant.

The immunization routes and the nature of the antigen or pathogen dictate the dominant type of T cell responses associated with the changes in the dendritic cell subsets (11). For example, in a viral infection, where most DCs could be infected, the T cell priming can occur at many regions of the lymph node, and cDC1-lineage predominantly shapes CD8⁺ T cell responses (46, 47), possibly due to their preferential niche or migration into the deep T cell zone (48). Further, several studies have shown that cDC1-lineage dendritic cells are required for cross-presentation (25). Previously, we have shown that antifungal effector and memory CD8⁺ T cells can be induced by subcutaneous route immunization using an experimental vaccine in the absence of CD4⁺ T cells and mediate sterilizing immunity to lethal pulmonary infection (30, 39–41). We noted that the vaccine immunity (efferent phase) directly correlated with the nature and magnitude of effector CD8⁺ T cells generated in the skin-draining lymph nodes (afferent phase) ( 39, 41, 49). However, the dendritic cell subset responses that may dictate antifungal effector CD8⁺ T cell-responses during afferent phase are not known. Therefore, we wanted to determine the dynamics of dendritic cell populations that may dictate the cross-presentation following fungal vaccination during CD4⁺ T cell deficiency during afferent phase. At day 5 post-vaccination, we harvested the draining lymph nodes (dLN), and single-cell suspensions were stained and analyzed by flow cytometry. We classified dendritic cell populations broadly into two groups (I and II) based on the expression levels of CD11c and CD11b (16, 50) ( Figures 1A, B ). When we compared with naïve, non-immunized, controls, the fungal vaccination significantly enhanced the numbers of Groups I and II cells but not the frequency of Group I cells ( Figure 1C ). We found Group I cells mainly composed of migratory cDC1 and resident cDC1 cells and Group II cells comprised primarily of Langerin⁺ DC, migratory cDC2, resident cDC2, and monocyte-derived DCs (moDCs) (13, 51, 52) ( Figure 1D ). CD11c^-CD11b⁺ cells (Group III), mainly consisted of monocytes and neutrophils, significantly increased following vaccination ( Figures 1C, D ). We used XCR1 and SIRPα (CD172a) markers to classify type-1 and -2 cDCs, respectively (51). Additionally, we used the expression of CD11c and MHC-II to classify DC into resident cDCs (CD11c^highMHC-II^int) and migratory cDCs (CD11c^intMHC-II^high) (52, 53). The moDCs and pDCs subsets were classified as CD11c⁺Ly6C⁺CD11b⁺MHC-II^int/lo and CD11c^loCD11b^-Ly6C⁺MHC-II^lo, respectively (13, 54, 55). Since XCR1 and SIRPα can reliably be used to classify cDC1 and cDC2, and XCR1⁺ DCs are instrumental in cross-presentation (56–58), we further evaluated the dynamics of different DC subsets following vaccination. We found an increase in the numbers and frequencies of most subsets of resident and migratory cDC1 and cDC2 cells in vaccinated mice compared with an unvaccinated group ( Figure 1E ; Supplementary Figure 1 ). Notably, the numbers of cDC2 cell subsets were significantly increased compared with a modest increase of cDC1 cells (~6-8X vs. ~2X) following vaccination. The order of significant increase of dendritic cell types following the vaccination was migratory cDC2, resident cDC2, migratory cDC1, and moDCs, followed by resident cDC1 and Langerin⁺ DC ( Figure 1F ). Collectively, the fungal vaccination significantly augmented cDC2 over cDC1-type dendritic cells.

The subcutaneous route of immunization with large microbial antigens will induce migratory cells to engulf antigens and shuttle them to draining lymph nodes, an orchestrated process distinct from the quick passive transfer of soluble small antigens (59). Skin or local DCs play a larger role in shuttling the antigen cargo into the draining lymph nodes for antigen deposition and presentation. Since several dendritic cells carry the antigen cargo from the subcutaneous space and influence the resident and hematopoietic cell-derived dendritic cell populations for antigen presentation and T cell activation in a time-dependent manner, we determined the kinetics of dendritic cell subsets following subcutaneous vaccination in the draining lymph nodes. As expected, we did not find major changes in the resident cDC subsets at day 2 post-vaccination ( Figures 2A, B ). However, there was a significant increase in their numbers by day 5 post-vaccination (PV) that stabilized till day 8 before tapering off. The number of resident cDC subsets remained elevated even at day 15 PV. In contrast, migratory DCs and Langerin⁺ DC numbers were steadily increased starting as early as day 2 PV, peaking around days 5 to 8 PV before reducing to the basal levels by day 15 PV ( Figures 2A, B ). The kinetics of moDCs were similarly reflected. Not surprisingly, the kinetics of the monocyte population were distinct and steadily increasing through day 15 PV. Although the overall kinetics of various subsets of the dendritic cell population mirrored each other in the dLN ( Figure 2B ; Supplementary Figure 2 ), the numbers of each subset were noticeably different. Therefore, we evaluated the relative abundance of dendritic cell subsets, including monocytes, over time, which may reflect their ability to engage with the T cells. The relative proportions of potent cross-presenting DC subsets and resident/migratory cDC1 subsets were stable or decreased following the vaccination ( Figure 2C ). The significant and most abundant DC cell subset was migratory cDC2 that started to inflate as early as day 2 PV and remained as such till day 8 PV and was replaced by accumulating CD11b⁺Ly6C⁺ monocytes. By day 15 PV, CD11b⁺Ly6C⁺ monocytes were the most abundant cells. Interestingly, resident cDC2, rather than conventional cross-presenting cDC1, population was the third most abundant cell type throughout the vaccine response period.

Collectively, cDC2, but not cDC1, subsets were abundant in the early phase of vaccine response and gradually replaced by the monocytic population later.

DC status largely dictates its phagocytosis, antigen presentation, and the activation of T cells’ abilities. While immature DCs are highly phagocytic, their activation and maturation are accompanied by higher expression of costimulatory and MHC molecules optimized for activation of T cells (60). Here, we evaluated the expression of different costimulatory molecules and MHC molecules on different dendritic cell subsets following fungal vaccination. We found that vaccination induced the upregulation, albeit in a disparate manner, of CD80 and CD86 molecules on resident DC subsets ( Figure 3A ). Similarly, the upregulation of costimulatory molecules, CD80 and CD86, was significant on migratory DC and moDC subsets following vaccination ( Figures 3B, C ) However, the expression of the costimulatory molecule CD40 remained unaltered following the vaccination in all the subsets. Despite DCs constitutively expressing high levels of MHC-I and MHC-II molecules (61), their expression may increase following their activation (62). However, we did not find significant upregulation of either of the MHC molecules on any DC subsets ( Figures 3A, B ). Thus, our data suggest that fungal vaccination efficiently fosters the activation of DC subsets by upregulating CD80 and CD86 but not CD40 molecules in the absence of CD4⁺ T cells.

Our aforementioned data suggested that fungal vaccination predominantly enhances the cDC2 subsets and expression of CD80 and CD86 costimulatory molecules on most DC subsets. Antigen uptake is an essential event before presentation in the context of MHC molecules, and immature DCs readily engulf large antigens by phagocytosis. Subcutaneously inoculated antigens are picked up by local dendritic cells, which facilitates their migration to the draining lymph nodes as part of migratory DC subsets. Although several overlapping markers are noted between migratory and lymph node resident DC subsets, making it difficult to distinguish, migratory DCs can transfer the antigen cargo to tissue-resident DC subsets. Our data suggested that PKH-labelled fungal antigen is readily detected by flow cytometry, which enabled us to assess fungal antigen-engulfed DC subsets ( Figure 4A & Supplementary Figure 4A ). The fungal antigens were found in all the subsets, but predominantly in migratory cDC2 subset, followed by migratory cDC1 and moDCs (Supplementary Figure 4B ), in line with their augmented numbers at day 5 post-vaccination ( Figure 2C ). Next, we evaluated their ability to cross-present the fungal antigens. The MAb staining for SIINFEKL-bearing MHC-I (H-2K^b) following vaccination with non-Tg and Tg yeasts showed the utility of the MAb to assess the presentation ( Figure 4B ). Using this technique, we assessed the cross-presenting ability of different DC subsets ( Figure 4C ) and found that all DC subsets were able to cross-present the fungal antigens, with a modestly higher level by migratory cDCs and lower by moDCs.

Collectively, despite all DC subsets being able to cross-present, the migratory cDC2 population predominates in gorging and presenting the fungal antigens.

Our data so far suggested that cDC2 and moDC subsets were predominantly enriched with higher activation in the model of fungal vaccination. We asked if cDC2 and moDC subsets are enriched due to CD4 depletion. We evaluated the kinetics of cDC2 and moDC subsets. We found that despite there being an increase in these subsets in the CD4⁺ T cell-sufficient group, the CD4⁺ T cell-deficient group had augmented numbers that were sustained through days 5 to 8 post-vaccination ( Figure 5A ). Similarly, the frequency and numbers of monocytes were significantly increased in the CD4⁺ T cell-deficient group compared with the sufficient group ( Supplementary Figures 5A, B ). Similarly, the frequency of cDC2 subsets and moDCs was significantly lower in the CD4⁺ T cell-sufficient group than in the deficient group ( Figure 5B ). Since CD4⁺ T cells are known to help activation of dendritic cells, we evaluated the upregulation of costimulatory molecules on dendritic cells. We found that the presence of CD4 T cells did not help in upregulating costimulatory molecules, including CD40 and MHC-II ( Figure 5C ; Supplementary Figure 5C ). Since the expression of MHC-I is essential for augmenting CD8⁺ T cell responses, we measured its expression levels. We found similarities in dendritic cell subsets of both CD4⁺ T cell-sufficient and -deficient groups ( Supplementary Figure 5D ). To exclude the possibility of depletion-independent antibody effects of GK1.5 MAb, we used isotype control MAb, and we found non-significant effects of depletion-independent functions of MAb on dendritic cell responses to the immunization.

Thus, the depletion of CD4⁺ T cells did not alter the kinetics but the dynamics and activation of cDC2 and moDC subsets.

Since CD4⁺ T cells “license” cDC1 for cross-presentation of exogenous antigen, we next evaluated if CD4⁺ T cells are required to bolster the cDC1 that are relevant to the model of fungal vaccination targeted to elicit CD8⁺ T cell responses. The kinetics of the resident and migratory cDC1 subsets remained similar in CD4⁺ T cell-sufficient and -deficient groups through day 8 post-vaccination ( Figure 6A ). Further, the frequency of cDC1 subsets were relatively similar in CD4⁺ T cell-sufficient and -deficient groups at days 0, 5, and 8 post-vaccination ( Figure 6B ). Notably, the expression of classical licensing costimulatory molecule, CD40, on cDC1 subsets, were similar in CD4⁺ vs. CD4^- groups ( Figure 6C ). However, the activation status of cDC1 subsets were higher in CD4⁺ T cell-deficient group than in CD4⁺ T cell-sufficient group. We further comprehensively analyzed the landscape of different DC subsets in the CD4⁺ T cell-deficient group, contrasting with the CD4⁺ T cell-sufficient group ( Figure 6D ). We found that proportions of the DC subsets remained largely similar, with a significant increase in cDC2 subsets in the CD4⁺ T cell-deficient group.

Collectively, CD4⁺ T cell help is dispensable following fungal vaccination to augment dendritic cell responses.