Given the rise in lymphopenia due to HIV/AIDS, solid organ transplant, chemotherapy (Li et al., 2019; Fierer, 2019; Pathakumari et al., 2020), and immunosuppressive therapies to control chronic diseases (Grebenciucova et al., 2016; Ward et al., 2016; Bryan et al., 2020), opportunistic invasive fungal infections have emerged as a global health concern, killing ~1.5 million people per year. Among these opportunistic fungal infections, cryptococcosis is caused by Cryptococcus neoformans and Cryptococcus gattii (Chang and Chen, 2015; Diaz, 2020; Montoya et al., 2021), resulting in ~220,000 new cases and ~180,000 deaths annually (Rajasingham et al., 2017; Mayer and Kronstad, 2020).

Once the environmentally endemic Cryptococcus spores are inhaled into the lungs, the initial infection is rapidly controlled within lung granulomas in immunocompetent hosts (Shibuya et al., 2005; Brunet et al., 2018). During immunodeficiency, however, the fungus can disseminate to the brain to cause lethal meningoencephalitis. The mortality rate can be attributed to the aggressiveness of the fungus as well as limited antifungal treatment options available to combat cryptococcosis, resulting in high rates of antifungal resistance [reviewed in (McEvoy et al., 2020)] and host toxicity (Bicanic et al., 2015). Despite extensive vaccine development research, no fungal vaccines are currently available for clinical use.

The fungal vaccine development pipeline has explored various antigens, adjuvants, vaccination routes and frequencies, as well as vaccine-inducing mutants to control cryptococcosis, yielding differing levels of host protection [reviewed in (Scorzoni et al., 2017; Caballero Van Dyke and Wormley, 2018; Nami et al., 2019; Ueno et al., 2020; Pathakumari et al., 2020)]. As current fungal vaccine development is not rooted in models of immunodeficiency, there is a major bottleneck in advancing preclinical findings to prevent these opportunistic infections in the clinical setting. To identify the host cell subtypes that may confer protection against opportunistic invasive fungal infections, the efficacy of fungal vaccines should be tested under immunodeficiencies specific to these infections. Cryptococcosis occurs under CD4⁺ T cell immunodeficiency and our previous work showed that immunization of a CD4⁺ deficient host with C. neoformans Δsgl1 mutant accumulating sterylglucosides (SGs) conferred total protection against a lethal challenge with the highly virulent C. neoformans wild-type (WT) H99 strain (Rella et al., 2015).

SGs are known immunomodulatory glycolipids that have been used in a wide variety of therapeutic applications [reviewed in (Grille et al., 2010; Normile et al., 2020b)]. They have been shown to be promising immunoadjuvants for protection against pulmonary tuberculosis in human clinical trials (Donald et al., 1994), enhancement of human T cell differentiation in vivo (Bouic et al., 1996), and protection against mouse models of invasive candidiasis (Lee and Han, 2006; Lee et al., 2007). Collectively, these studies suggest that SGs induce T cell proliferation, direct Th1 cell differentiation, and induce earlier host recognition leading to rapid recruitment of innate immunity. However, the majority of the vaccination studies utilizing SGs have been performed using plants (Thuan et al., 2013; Singh et al., 2016; Castillo et al., 2019) or purified SGs as a standalone compound prior to infection to induce a protective immune response, not as a simultaneous immunoadjuvant present during the active infection stage (Lee and Han, 2006; Lee et al., 2007).

In the present study, we provide a comprehensive analysis of the host immune response against the live, attenuated C. neoformans Δsgl1 mutant during both the immunization phase and the challenge phase under various host immunodeficiencies. We found that our SG-accumulating mutant induced a robust pro-inflammatory lung environment with increased leukocyte recruitment to the lungs. During immunosuppressive conditions, the host was able to eliminate C. neoformans Δsgl1 from the lungs when monocytes, macrophages, and/or neutrophils as well as B or CD8⁺ T cells were depleted. Even when macrophages, B cells, or CD8⁺ T cells depleted, mice were still fully protected against a subsequent WT infection. Although mice depleted of CD4⁺ T cells were unable to eliminate C. neoformans Δsgl1 from the lungs, they showed no signs of morbidity or fungal dissemination and were fully protected against WT infection during which time they clear the mutant strain. Only when both CD4⁺ or CD8⁺ T cells were depleted, mice were unable to clear C. neoformans Δsgl1 and lost complete protection against the WT infection. Our findings emphasize that C. neoformans Δsgl1 induces robust host immunity and highlight a previously unexplored role of SGs as potent immunomodulators for protection against cryptococcosis during severe immunodeficiency.

In our current study, we have shown that C. neoformans Δsgl1 elicits a robust, pro-inflammatory host response in the lungs that stimulates the clearance of the mutant ultimately leading to subsequent protection from a lethal WT challenge. We were able to measure significantly increased levels of SGs during in vivo inoculation with the mutant, providing evidence that SGs are present outside the yeast and may be the driving factor of host immunomodulation. This pro-inflammatory host response was sufficient to clear the mutant from the lungs during combined immunosuppressive conditions as well as protect the host from a lethal WT challenge in the presence of either CD4⁺ or CD8⁺ T cells.

Notably, we determined that SGs were found in vivo during inoculation with C. neoformans Δsgl1 but not the WT strain ( Figure 1C ). These SGs were believed to cause the significantly increased levels of Th1 cytokines, including IL-1α, TNFα, IL-17, and IFNγ, and decreased levels of Th2 cytokines, including IL-4 and IL-5 ( Figure 2 ) since they occurred over the same timeline as the SGs were measurable in vivo ( Figure 1C ). It is widely accepted that host protection against C. neoformans requires a pro-inflammatory type 1 immune response mediated by IFNγ (Mourad and Perfect, 2018; Li et al., 2019; Linyu et al., 2020; Hester et al., 2020; Normile et al., 2020a; Akhtar et al., 2020; Montoya et al., 2021). Interestingly, SGs have been shown to induce Th1 T cell polarization (Bouic et al., 1996; Lee and Han, 2006; Lee et al., 2007), but these studies have used plant-based SGs, not fungal-derived SGs. To our knowledge, this is the first report of fungal-derived SGs being used as an immunoadjuvant inducing a robust type 1 cytokine milieu in the lungs for protective host immunity.

Our SG-accumulating mutant also induced increased effector leukocyte and adaptive lymphocyte recruitment to the lungs compared to WT infected mice ( Figure 3A ). This, too, temporally correlated with the levels of SGs measured in vivo as well as the cytokine profile in the lungs since we observed significantly increased levels of chemotactic chemokines such as G-CSF, KC (CXCL1), MIP-1b (CCL4), and RANTES (CCL5) ( Figure 2 ). Our increased neutrophil, monocyte, and macrophage response post inoculation with C. neoformans Δsgl1 correlated with the same timeline as these leukocyte chemotactic chemokines. Moreover, the significantly increased levels of RANTES (CCL5) from days 4 to 6 post inoculation with C. neoformans Δsgl1 correlated with strong recruitment of lymphocytes to the lungs yielding a robust adaptive immune response, which is a known effect of SGs seen previously in the literature using other forms of this glycolipid (Lee and Han, 2006; Lee et al., 2007).

Interestingly, these chemotactic factors also decrease to baseline levels by day 14 post inoculation with C. neoformans Δsgl1 displaying a resolution of infection correlating with the same time frame for clearance of the mutant strain (and lack of measurable SGs), suggesting a delayed-type hypersensitivity host immune response. This was opposite to what was observed for the WT strain, where the chemotactic factors and pro-inflammatory cytokines were delayed and increased later post infection around day 14 resulting in host pathology. Lung histology confirmed these data showing the lungs of C. neoformans Δsgl1 inoculated mice displayed a decrease in inflammation from day 6 to 14 during the time the mutant was cleared, cytokine levels decreased, and leukocyte recruitment halted ( Supplementary Figure S1 ). Overall, we concluded the increased levels of SGs derived from our mutant sparked these positive host conditions and downstream protective nature observed during the WT challenge.

One interesting caveat that was recently published by our group has shown that C. neoformans Δsgl1 does have a growth defect in low oxygen and glucose conditions (de Sa et al., 2021). We cannot overlook the fact that the lung interstitium is anoxic compared to the alveolar spaces, and the hypoxia induced death of the mutant may be the causative reasoning for the proinflammatory environment with increased leukocyte migration. However, we do not believe this to be the case as the mutant strain would be unable to persist in the lungs to the day 35 timepoint in the CD4-deficient conditions ( Figure 4C ) and past the WT challenge in CD4- and CD8-dual deficiency ( Figure 5E ). We do believe the fungal SGs in combination to the yeast cells induce the augmented host responses observed that result in the mutant clearance and host vaccination to the WT strain.

We utilized an in vivo antibody depletion strategy to assess the necessity of specific cell types for host clearance of C. neoformans Δsgl1. Interestingly, we found that only CD4⁺ T cells were the only cell type examined that delayed host clearance of the mutant during the 30-day immunization period ( Figures 3 , 4 ). Solo or combined innate effector leukocyte depletion (neutrophils, monocytes, and/or macrophages), as well as solo adaptive lymphocyte depletion (B cells and CD8⁺ T cells), resulted in clearance of the mutant within the 30-day immunization period, although at various times compared to the isotype-treated mice. Although the mice were unable to clear the infection in the absence of CD4⁺ T cells, no morbidity or extrapulmonary dissemination was observed and the lung CFU remained relatively stable from day 10 to day 35 ( Figure 4C ) strongly suggesting that mice can control the mutant without displaying sterilizing immunity.

The keystone factor of our mutant strain is the lasting vaccine protection for the host against lethal WT infection shown previously (Rella et al., 2015). We have added to the robustness of this protection beyond just CD4⁺ T cell depletion. Mice immunized with C. neoformans Δsgl1 depleted of either alveolar macrophages, B cells, or CD8⁺ T cells have all shown complete host protection ( Figure 5 ) without any extrapulmonary dissemination ( Supplementary Figure S4 ). We have also uncovered an interesting facet of our mutant in that complete host protection was not requisite of prior mutant clearance shown by the protection in CD4-depleted mice. Although we had previously reported that C. neoformans Δsgl1 conferred complete protection in CD4-deficient mice, we have now uncovered these mice do not clear the mutant prior to WT infection. The mounted host immune response against the WT challenge was sufficient to clear C. neoformans Δsgl1 from the lungs. This was the opposite for the WT cells that remain in the lungs for at least 75 days post challenge in all mice tested, whether immunocompetent or immunocompromised ( Supplementary Figure S4 ). Nonetheless, these mice show no signs of morbidity or extrapulmonary dissemination, so we attest these mice are controlling replication and dissemination of the yeast. This statement of controlling the WT yeast post challenge was supported with histological data ( Supplementary Figure S5 ). The degree of lung tissue inflammation decreased from day 45 post challenge to 105 post challenge even though no significant changes in lung CFU were observed over this timeline. The WT yeast were shown to be contained within granulomatous regions in the lung, but not within healthy tissue, strongly suggesting a controlled means of host containment. Exploring the immunological niche of these granulomatous regions and clearance of the WT strain from the lungs will be an interesting topic for future experimentation and currently underway in our lab.

Our data strongly suggest that either CD4⁺ or CD8⁺ T cells are required for control of C. neoformans Δsgl1 ( Figure 5E and Supplementary Figure S6 ) and the conferred complete host protection post vaccination ( Figure 5E ). When either of these subsets are depleted alone, full control of the mutant strain ( Figure 4 ) and the WT strain post challenge ( Figure 5 ) are observed with no visible pathology or extrapulmonary dissemination ( Supplementary Figure S4 ). The immunological roles of CD4⁺ and CD8⁺ T cells are not normally redundant but have been shown to be complementary during the absence of the other subset in other work (Lindell et al., 2005; Nanjappa et al., 2012a; Nanjappa et al., 2012b). We conclude here that the roles of CD4⁺ and CD8⁺ T cells are redundant and overlapping post vaccination with C. neoformans Δsgl1 while the other subset is missing. However, we hypothesize that CD4⁺ T cells are more robust in their protective nature since we observed that clearance of C. neoformans Δsgl1 was not accomplished in CD4-deficient hosts ( Figure 4C ), and the endpoint CFU after 75 days post challenge in CD4-deficient hosts was significantly greater than that of the isotype-treated mice ( Supplementary Figure S4C ). The immunological roles of these cells in immunocompetent and immunosuppressed hosts are currently being explored in our lab.

In conclusion, C. neoformans Δsgl1 demonstrates a viable option as a future therapeutic for protection against cryptococcosis, especially due to the fact of being tested under conditions of severe immunosuppression. This is principally due to three main factors: i) the early beneficial properties initiated in the host seen by the lung cytokine profile and early effector cytokine recruitment ( Figures 2 and 3A–B ); ii) the clearance and lack of extrapulmonary dissemination of the mutant during solo or combined immunosuppression ( Figures 3C–G and 4 ); and iii) the host protection against lethal WT challenge during several solo models of immunosuppression without the necessity for initial host clearance of C. neoformans Δsgl1 ( Figure 5 and Supplementary Figure S4 ). Additionally, the notable effect observed by fungal-derived SGs on the host immune response demonstrated a novel immunoadjuvant effect not previously shown before. However, it should be noted that this immunoadjuvant requires T cell priming for control and protection as highlighted through our dual CD4⁺ and CD8⁺ T depletion and TCR β KO mice ( Figure 5E and Supplementary Figure S6 ), so pan T cell immunosuppression would be a limiting factor of our mutant from a clinical viewpoint. Nonetheless, because we show that either CD4⁺ or CD8⁺ T cells are required, C. neoformans Δsgl1 still remains a viable option for the major risk factors during the opportunistic infection with C. neoformans since CD8⁺ T cells remain accessible during CD4-lymphopenia (Collins et al., 2020; Akhtar et al., 2020; Linyu et al., 2020). We are also currently investigating subunit-based vaccination strategies that have the potential to surpass this limitation. Overall, our data strongly suggest that fungal-derived SGs promote beneficial host immunity during the primary infection when SGs are present and leave a lasting immune signature on protection against a normally lethal WT challenge when SGs are absent. Future experimentation on direct host recognition of these fungal SGs and exploring the roles of non-canonical T cells in the protection by our mutant will be exciting points to explore.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The animal study was reviewed and approved by IACUC Stony Brook University.

TGN, AR, and MDP took part in the conceptualization of this study. TGN performed animal infections, depletions, survival studies, CFU quantification, and flow cytometry analysis. AR performed the cytokine determination and SG quantification experiments. All authors contributed to the article and approved the submitted version.

This work was supported by NIH grants AI136934, AI116420, and AI125770, and by the Merit Review Grant I01BX002924 from the Veterans Affairs Program to MDP. MDP is a Burroughs Welcome Investigator in the Pathogenesis of Infectious Diseases and a Research Career Scientist at the Veterans Administration Medical Center in Northport, NY.

MDP is a Co-Founder and Chief Scientific Officer (CSO) of MicroRid Technologies Inc.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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