Female BALB/c (National Cancer Institute/Charles River Laboratories) 4–6 weeks of age were used throughout these studies. Mice were housed at the University of Texas at San Antonio Small Animal Laboratory Vivarium and all animal experiments were conducted following NIH guidelines for housing and care of laboratory animals and in accordance with protocols approved by the Institutional Animal Care and Use Committee (protocol number MU021) of the University of Texas at San Antonio.

Cryptococcus neoformans strains H99 (serotype A, mating type α), C. deuterogattii strain R265 (serotype B; a kind gift from Dr. Joseph Heitman of Duke University Medical Center in Durham, NC, USA), C. bacillisporus strain WSA87 (serotype C), and C. deneoformans strain R4247 (serotype D) (each kind gifts from Dr. Brian Wickes of the UT Health San Antonio, San Antonio, TX, USA), and H99γ [an IFN-gamma producing C. neoformans strain derived from H99 (38)] were recovered from 15% glycerol stocks stored at −80°C prior to use in the experiments described herein. The strains were maintained on yeast-extract-peptone-dextrose (YPD) media (1% yeast extract, 2% peptone, 2% dextrose, and 2% Bacto agar). Yeast cells were grown for 16 h at 30°C with shaking in YPD broth, collected by centrifugation, washed three times with sterile PBS, and viable yeasts were quantified using trypan blue dye exclusion on a hemacytometer.

Cryptococcal infections were initiated as previously described (25, 41). Briefly, mice were anesthetized with 2% isoflurane using a rodent anesthesia device (Eagle Eye Anesthesia, Jacksonville, FL, USA) and given an intranasal inoculation with 1 × 10⁴ CFU of Cryptococcus strains H99, R265, WSA87, or R4247 in 50 µl of sterile PBS. Alternatively, mice were given an intranasal immunization with 1 × 10⁴ CFU of C. neoformans strain H99γ or heat-killed H99γ (HKH99γ) yeasts in 50 µl of sterile PBS, allowed 70 days to resolve the infection and subsequently given an intranasal challenge with 1 × 10⁴ CFU of H99, R265, WSA87, or R4247 in 50 µl of sterile PBS. The inocula used for immunizations and challenges were verified by quantitative culture on YPD agar. The mice were monitored by inspection twice daily. Mice were euthanized on pre-determined days post challenge and lung tissues excised using aseptic technique. Tissues were homogenized in 1 ml of sterile PBS followed by culture of 10-fold dilutions of each tissue on YPD agar supplemented with chloramphenicol (Mediatech Inc., Herndon, VA, USA). CFU were enumerated following incubation at 30°C for 48 h. Alternatively, mice intended for survival analysis were monitored by inspection twice daily and euthanized if they appeared to be in pain or moribund (weight loss, ataxia, listlessness, or failure to groom). Mice were euthanized using CO₂ inhalation followed by cervical dislocation.

Mice were immunized with 1 × 10⁴ CFU of C. neoformans strain H99γ as described above. After 70 days of rest, mice were depleted of CD4⁺ T cells, CD8⁺ T cells, or both CD4⁺/CD8⁺ T cell subsets via intraperitoneal administration of anti-CD4 (clone GK1.5) and anti-CD8 (clone 2.43) antibodies (Table 1) (each from Cell Culture Company/NCCC, Minneapolis, MN, USA) or given isotype control antibody (rat IgG_2b) (BioXCell, Lebanon, NH, USA). Each mouse received 200 µg of GK1.5 and/or 2.43 or control rat IgG_2b antibodies in a volume of 200 µl PBS 48 h prior to challenge with H99, R265, WSA87, or R4247 and weekly thereafter during the observation period. The efficiency of T cell depletion in lungs and spleens was assessed by flow cytometric analysis using anti-CD4 and anti-CD8 antibodies that bind epitopes of the CD4 and CD8 protein at locations distinct from GK1.5 and 2.43. Efficiency was determined to be >98% at each anatomic location for each depletion via comparison of T cell subsets in treated mice with those in control animals.

As previously described (25, 41), cytokine levels in lung tissues were analyzed using the Bio-Plex protein array system (Luminex-based technology; Bio-Rad Laboratories, Hercules, CA, USA). Briefly, the left lobe of the lung was excised and homogenized in ice-cold sterile PBS (1 ml). An aliquot (50 µl) was taken to quantify the pulmonary fungal burden and an anti-protease buffer solution containing PBS, protease inhibitors (inhibiting cysteine, serine, and other metalloproteinases), and 0.05% Triton X-100 was added to the homogenate and then clarified by centrifugation (800 × g) for 10 min. Supernatants from pulmonary homogenates were assayed for the presence of IL-1α, IL-1β, IL-1, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17A, CCL5/RANTES, CCL11/Eotaxin, CXCL1/KC, CCL3/MIP-1α, CCL4/MIP-1β, CCL2/MCP-1, G-CSF, GM-CSF, TNF-α, and IFN-γ.

Lungs were excised on days 3, 7, and 14 post challenge and digested enzymatically at 37°C for 30 min in 10 ml of digestion buffer (RPMI 1640 and 1 mg/ml of collagenase type IV) (Sigma Chemical Co., St. Louis, MO, USA) with intermittent (every 10 min) stomacher homogenizations as previously described (25, 41). The digested tissues were then filtered through sterile nylon filters of various pore sizes (70 and 40 µm; BD Biosciences) and washed with sterile HBSS to enrich for leukocytes. Erythrocytes were lysed by incubation in NH₄Cl buffer (0.859% NH₄Cl, 0.1% KHCO₃, 0.0372% Na₂EDTA pH 7.4) for 3 min on ice followed by the addition of a twofold excess of PBS. The leukocyte population was then washed twice with sterile PBS, suspended in sterile PBS + 2% heat-inactivated fetal bovine serum (FACS buffer), and enumerated in a hemacytometer using trypan blue dye exclusion. Flow cytometry analysis was used to determine the percentage of each leukocyte population as well as the absolute number of total leukocytes (CD45⁺) within the lung cell suspension for standardization of hemacytometer counts.

Standard methodology was employed for the direct immunofluorescence of pulmonary leukocytes (25, 41). Briefly, in 96-well U-bottom plates, 100 µl containing 1 × 10⁶ cells in PBS plus 2% FBS (FACS buffer) were incubated with Fc block (BD Biosciences) diluted in FACS buffer for 5 min to block nonspecific binding of antibodies to cellular Fc receptors. An optimal concentration of fluorochrome-conjugated antibodies (0.125—1 µg/1 × 10⁶ cells) as listed in Table 1 was added in various combinations to allow for dual or triple staining, and cells were then incubated for 30 min at 4°C. Cells were washed three times with FACS buffer and fixed in 200 µl of 2% ultrapure formaldehyde (Polysciences) diluted in FACS buffer (fixation buffer). Cells analyzed for intracellular cytokine staining were fixed in fixation buffer for 10 min in the dark at room temp. The cells were then permeabilized with 0.1% saponin (diluted in FACS buffer) and incubated for 10 min in the dark at room temperature. Antibodies listed in Table 1 were subsequently added at optimal concentrations, and cells incubated at 4°C for 30 min. Following incubation, cells were washed three times with 0.1% saponin buffer and resuspended in fixation buffer. Cells incubated with either FACS buffer alone or single fluorochrome-conjugated Abs were used to determine positive staining and spillover/compensation calculations, and background fluorescence determined with FlowJo Software (FlowJo, LLC, Ashland, OR, USA). Raw data were collected with the BD FACSArray flow cytometer (BD Biosciences) and then analyzed using FlowJo Software. Dead cells were excluded on the basis of forward angle and 90° light scatter. For data analyses, 30,000 events (cells) were evaluated from a predominantly leukocyte population identified by back gating from CD45⁺ stained cells. The absolute number of total leukocytes was quantified by multiplying the total number of cells observed by hemacytometer counting by the percentage of CD45⁺ cells determined by flow cytometry. The absolute number of leukocytes (CD45⁺ cells), T cells (CD4⁺/CD3⁺ and CD8⁺/CD3⁺), CD19⁺/CD45⁺, Ly6G⁺/CD45⁺, F4/80⁺/CD45⁺, CD11b^int/CD11c⁺/CD45⁺, CD11b^hiCD11c⁺/CD45⁺, and Siglec-F⁺/CD11b⁺ was determined by multiplying the percentage of each gated population by the total number of CD45⁺ cells. Gating strategies are provided as supplemental materials. Intracellular cytokine staining is represented in histograms with unstained cells as a control.

The unpaired Student’s t test (two-tailed) was used to analyze the immunization studies fungal burden, pulmonary cell populations, and cytokine/chemokine data where appropriate using GraphPad Prism version 5.00 for Windows (GraphPad Prism Software, San Diego, CA, USA). Survival data were analyzed using the log-rank test (GraphPad Software). Significant differences were defined as *p < 0.05, **p < 0.005, or ***p < 0.0001.

Previous studies in our lab demonstrated that experimental pulmonary infection with C. neoformans strain H99γ in mice results in clearance of the acute infection and induction of protective immunity against a subsequent otherwise lethal challenge with a WT C. neoformans strain H99 that does not produce IFN-γ (25, 36–40). Nevertheless, studies to date have not demonstrated that protective immunity generated against one serotype of Cryptococcus affords protection against multiple disparate Cryptococcus serotypes. Consequently, we sought to determine the level of cross-protection that is generated in mice immunized with C. neoformans strain H99γ, a serotype A strain, against pulmonary infection with disparate serotypes of Cryptococcus. To establish a baseline prior to evaluating if immunization with Cryptococcus strain H99γ, serotype A, enhanced protection against challenge with disparate Cryptococcus serotypes, we first determined the relative virulence of each serotype in non-immunized mice. To do this, BALB/c mice were given an experimental pulmonary infection with either Cryptococcus strain H99 (serotype A), R265 (serotype B), WSA87 (serotype C), or R4247 (serotype D) and evaluated for mortality (Figure 1A). As shown in Figure 1A, experimental pulmonary infection with Cryptococcus strains H99, R265, and R4247 resulted in 100% mortality with median survival times of 28, 32, and 33 days, respectively. In contrast, we observed 87.5% survival in mice given an experimental pulmonary infection with WSA87. Cryptococcus strain WSA87, serotype C, appeared to be significantly less virulent compared to all the other strains tested during our analysis.

Next, we tested the potential of mice immunized with C. neoformans strain H99γ (serotype A) to be protected against challenge with Cryptococcus serotypes B, C, or D. For this, mice were immunized with C. neoformans strain H99γ or heat-killed H99γ (HKH99γ), rested for 70 days, and separate groups subsequently given an experimental pulmonary challenge with each Cryptococcus serotype. We observed a 100% survival rate in mice immunized with H99γ and challenged with H99, while the HKH99γ immunized mice challenged with H99 demonstrated a median survival time of 28 days post challenge (Figure 1B), confirming our previous observations (38, 40). Mice immunized with H99γ and then challenged with either R265 (C. gattii serotype B) or R4247 (C. deneoformans serotype D) exhibited median survival times of 59 and 72 days post challenge, respectively, and significantly increased survival rates compared to HKH99γ immunized mice challenged with the same strain (median survival of 33 and 34.5 days in R265 and R4247 challenged mice, respectively; Figure 1B). Additionally, we observed a 20% survival rate in protectively immunized mice challenged with R4247 at 100 days post challenge. Figure 1B shows that no significant difference in mortality was observed in H99γ immunized mice challenged with WSA87 (median survival time of 90.5 days) compared to HKH99γ immunized mice challenged with the same strain (median survival time of 87.5 days). The mortality of mice immunized with HKH99γ and challenged with each Cryptococcus serotype mirrored the mortality rates of non-immunized mice given an experimental infection with each serotype and provided no additional protection (Figures 1A,B).

Pulmonary fungal burden was also quantified at days 3, 7, and 14 post challenge in H99γ or HKH99γ immunized mice challenged with each individual serotype (Figure 1C). Mice immunized with HKH99γ and challenged with each species showed progressive growth of each serotype in the lungs. In contrast, we observed significantly less pulmonary fungal burden as early as day 3 in H99γ immunized mice challenged with R265 and WSA87 compared to HKH99γ immunized mice (Figure 1C). Additionally, H99γ immunized mice showed significantly less pulmonary fungal burden following challenge with all serotypes on days 7 and 14 post challenge compared to HKH99γ immunized mice challenged with the same strain. Brain fungal burden was also quantified at day 14 post challenge and a trend (although not statistically significant) toward a reduction of brain fungal burden together with the number of mice negative for Cryptococcus in the brain was observed in H99γ immunized mice challenged with each serotype compared to their HKH99γ immunized counterparts (Figure 1C). Overall, our data show that mice immunized with H99γ, serotype A, display significant protection against challenge with serotypes A, B, or D of Cryptococcus.

Figure 1D demonstrates the fungal burden of moribund mice or mice sacrificed at the conclusion of the study. The HKH99γ immunized mice challenged with R265 appeared to have similar pulmonary fungal burden and less brain fungal burden at time of death compared to H99γ immunized mice challenged with R265 (Figure 1D). No significant differences in pulmonary or brain fungal burden were observed between H99γ and HKH99γ immunized mice challenged with WSA87 (Figure 1D). Both groups of mice immunized with HKH99γ or H99γ and challenged with R4247 appear to have similar pulmonary fungal burden and lower brain fungal burden at sacrifice with the H99γ immunized mice surviving longer. However, we cannot definitively state if the mice succumbed to pulmonary and/or brain fungal burden. Overall, immunization with H99γ appeared to prolong the survival of mice challenged with each serotype.

Our next goal was to examine the pulmonary inflammatory response in immunized mice during infection with the various representative Cryptococcus serotypes. We observed a significant increase in the total number of leukocytes recruited to the lungs of H99γ immunized mice challenged with H99 at days 3 and 7 post challenge compared to HKH99γ immunized mice (Figure 2A). Additionally, we observed significant increases in leukocyte recruitment to the lungs of H99γ immunized mice challenged with R265 on days 7 and 14 post challenge, H99γ immunized mice challenged with WSA87 on days 3 and 7 post challenge, and H99γ immunized mice challenged with R4247 at each time point post challenge compared to their HKH99γ immunized counterparts (Figure 2A). We observed an increased total cell number of F4/80⁺ cells in the lungs of H99γ immunized mice challenged with H99 at day 3, R4247 at days 3 and 7, and WSA87 at day 14 post challenge compared to HKH99γ immunized mice (Figure 2B). We observed a significant increase in the total number of CD11b^hi/CD11c⁺ cells recruited to the lungs of H99γ immunized mice challenged with H99, WSA87, or R4247 at day 3, each serotype at day 7 post challenge, and in H99γ immunized mice challenged with WSA87 at day 14 post challenge compared to HKH99γ immunized mice challenged with the same serotype (Figure 2C). Figure 2C also shows a significant increase in the percentage of CD11b^hi/CD11c⁺ cells in H99γ immunized mice challenged with R265 or WSA87 at day 7, and WSA87 at day 14 post challenge. There was also a significant increase in the total number of CD11b^intCD11c⁺ cells in H99γ immunized mice challenged with H99 at days 3 and 7, R265 at day 7 post challenge, and WSA87 at days 7 and 14 post challenge (Figure 2D). Figure 2D also shows a significant increase in CD11b^intCD11c⁺ cells in H99γ immunized mice challenged with H99 at day 3, while we observe a significant decrease in these cells at day 7 in H99 challenged mice, and across each serotype at day 14 post challenge. The recruitment of Ly6G⁺ cells to the lungs was significantly higher in H99γ immunized mice challenged with H99 at days 3 and 7 post challenge, R265 or WSA87 at day 7 post challenge, and in H99γ immunized mice challenged with R4247 on day 14 post challenge compared to their HKH99γ immunized counterparts (Figure 2E). While mice immunized with H99γ and challenged with H99 are clearing the infection, these mice show a significant decrease in the total number and percentage of Ly6G⁺ cells at day 14 post infection compared to HKH99γ immunized mice (Figure 2E). We observed a significant increase in the total number of Siglec-F⁺ cells in mice immunized with H99γ and challenged with H99 at day 3 post challenge and WSA87 challenged mice at day 14 post challenge (Figure 2F). We observed a significant increase in the total number of B cells in mice immunized with H99γ and challenged with H99, WSA87, or R4247 at day 3 post challenge, across each serotype at day 7 post challenge, and R265 or WSA87 at day 14 post challenge (Figure 2G). For the percentage of B cells, we observed a significant increase in H99γ immunized mice challenged with WSA87 or R4247 across each time point and in mice challenged with R265 at days 7 and 14 post challenge (Figure 2G). Overall, our data show significantly increased pulmonary recruitment of F4/80⁺ cells, CD11b⁺/CD11c⁺ cells, B cells, and Ly6G⁺ cells in mice immunized with the serotype A Cryptococcus strain H99γ following challenge with various other Cryptococcus serotypes which, altogether, may contribute to the increased protective responses observed in these mice.

The pulmonary fungal burden and mortality analysis suggested that mice immunized with H99γ were able to mount protective responses against challenge with disparate Cryptococcus serotypes. Therefore, we evaluated cytokine levels in the lungs of H99γ or HKH99γ immunized mice following challenge with each Cryptococcus serotype. Lung homogenates were prepared from pulmonary tissues of mice on days 3, 7, and 14 post challenge with Cryptococcus serotypes A, B, C, or D, and the levels of Th1 associated (IFN-γ, IL-2, IL-12p70), Th2 associated (IL-4 and IL-5), immunoregulatory (IL-10 and G-CSF), and proinflammatory (IL-1α, IL-1β, IL-17A, and TNF-α) cytokines and chemokines (CCL3, CCL4, CCL2, CXCL1, and CCL5) were determined. We observed that H99γ immunized mice challenged with C. neoformans strain H99 exhibited a significant increase in Th1 associated cytokines (IFN-γ, IL-2, and IL-12p70) as early as day 3 post challenge that remained significantly elevated, except IL-2, through day 7 post challenge compared to HKH99γ immunized mice challenged with H99 (Tables 2 and 3). However, we observed significantly less Th1 associated cytokine (IL-12p70 and IFN-γ) production at day 14 post challenge in H99γ immunized mice compared to HKH99γ immunized mice (Table 4). Also, proinflammatory (IL-1α and IL-1β) and immunoregulatory (G-CSF) cytokines and chemokine levels (CCL3, CCL4, CCL2, and CXCL1) in H99γ immunized mice challenged with H99 were significantly less compared to levels observed in HKH99γ immunized mice challenged with H99 at day 14 post challenge (Table 4). The significant decrease in Th1 associated and proinflammatory cytokine and chemokine levels in H99γ immunized mice challenged with H99 at day 14 post challenge compared to levels observed in HKH99γ immunized mice likely coincides with the significant reduction in pulmonary fungal burden observed in the H99γ immunized mice; whereas, the HKH99γ immunized mice are experiencing progressive disease (Figure 1C). Th1 associated cytokine levels in the lungs of H99γ immunized mice challenged with each other serotype were generally elevated at day 3 post challenge and remained significantly elevated at days 7 and 14 post challenge, particularly cytokines IFN-γ and IL-12p70, compared to their HKH99γ immunized counterparts (Tables 2–4). For Th2-associated cytokines, we observed a significant increase at day 3 post challenge in mice that were immunized with H99γ and challenged with each serotype [H99 (IL-4 and IL-5), R265, WSA87, or R4247 (IL-5)] (Table 2). At day 7 post challenge in mice immunized with H99γ, we observed an increase in IL-4 in mice challenged with R265 or R4247 (Table 3). By day 14 post challenge, there was a statistically significant decrease in Th2 associated cytokines (IL-4 and IL-5) across each serotype except for WSA87 challenged mice that showed significantly increased IL-5 levels across days 7 and 14 post challenge (Tables 3 and 4). We also observed an overall trend toward significantly increased proinflammatory cytokine (IL-1α, IL-1β, and IL-17A), immunoregulatory cytokine (G-CSF), and chemokine (CCL3, CCL4, CCL2, CXCL1, and CCL5) production as early as day 3 post challenge that remained elevated through day 7 post challenge in H99γ immunized mice challenged with each serotype compared to similarly challenged mice immunized using HKH99γ (Tables 2 and 3). We observed an overall trend toward significantly higher levels of proinflammatory cytokine (IL-1α, IL-1β, IL-17A, and TNF-α), immunoregulatory cytokine (G-CSF), and chemokine levels (CCL3, CCL4, CCL2, CXCL1, and RANTES) was observed in H99γ immunized mice challenged with R265, WSA87, or R4247 compared to similarly challenged HKH99γ immunized at day 14 post challenge except for CCL3 and CXCL1 for R265 challenged mice, and CCL3 and CCL2 for R4247 challenged mice (Table 4). Altogether, the cytokine results suggest that immunization with C. neoformans strain H99γ elicits a heightened cytokine anamnestic response against multiple disparate serotypes of Cryptococcus compared to HKH99γ immunized mice.

We next evaluated T cell responses in the lungs of immunized mice following challenge with the various Cryptococcus serotypes. Flow cytometry analysis was utilized to determine levels of CD4⁺ and CD8⁺ T cell infiltration to the lungs on days 3, 7, and 14 post challenge. Also, intracellular cytokine staining of CD4⁺ T cells for representative T helper (Th) 1, Th2, and Th17-type cytokines IFN-γ, IL-4, and IL-17A, respectively, and subsequent flow cytometry analysis was used to access the CD4⁺ Th cell phenotype during the anamnestic response to pulmonary cryptococcosis. We observed an overall trend toward increased total numbers of CD4⁺ and CD8⁺ T cells in H99γ immunized mice compared to HKH99γ immunized mice following challenge with each serotype as the infection progressed (Figures 3A,B). The total number of CD4⁺ T cells was significantly increased in H99γ immunized mice challenged with H99, R265, or R4247 at day 3 post challenge, and in mice challenged with H99 at day 7 post challenge compared to their HKH99γ immunized counterparts (Figure 3A). However, there was no significant difference in percentage of CD4⁺T cells between groups. We also observed an increase in the total number of CD8⁺ T cells in H99γ immunized mice challenged with H99, WSA87, or R4247 at day 3 post challenge, in mice challenged with H99, R265, or WSA87 at day 7 post challenge, and in R265 or WSA87 challenged mice at day 14 post challenge compared to HKH99γ immunized mice challenged with their equivalent serotype (Figure 3B). A significant increase in the percentage of CD8⁺ T cells was observed in mice immunized with H99γ and challenged with R265 or R4247 at day 14 post challenge compared to their HKH99γ immunized counterparts.

Overall, we observed significant increases in the total number of CD4⁺/IFN-γ⁺ T cells followed by CD4⁺/IL-17A⁺ T cells and then CD4⁺/IL-4⁺ T cells at day 7 post challenge in H99γ immunized mice challenged with each serotype compared to HKH99γ immunized mice challenged with the corresponding serotype (Figures 3C–E). We observed greater total CD4⁺/IFN-γ⁺ T cell numbers in H99γ immunized mice challenged with H99 compared to H99γ immunized mice challenged with R265, WSA87, or R4247 at day 7 post challenge (Figure 3C). These results suggest that mice immunized with H99γ are capable of eliciting a predominantly Th1-type anamnestic response upon challenge with each disparate Cryptococcus serotype. These data show that mice immunized with the serotype A strain exhibit an increased putatively protective Th1-type immune response following challenge with other disparate serotypes.

Previous studies in our lab demonstrated that H99γ immunized mice depleted of CD4⁺ or CD8⁺ T cells were completely protected against an otherwise lethal challenge with the non-IFN-γ producing C. neoformans strain H99 (40). Also, H99γ immunized mice that were subsequently depleted of both CD4⁺ and CD8⁺ T cells were shown to have an 80% survival rate following challenge with C. neoformans strain H99. Therefore, we sought to determine the necessity of T cells in protection against cryptococcosis caused by the different serotypes of Cryptococcus in H99γ immunized mice. For this, BALB/c mice were immunized with C. neoformans strain H99γ and allowed 70 days to resolve the infection. Mice were then depleted of CD4⁺ and/or CD8⁺ T cells or received isotype control antibody two days prior to challenge and weekly thereafter during the observation period. H99γ immunized mice depleted of both CD4⁺ and CD8⁺ T cells prior to and during challenge with H99 demonstrated an 87.5% survival rate upon the conclusion of the study (Figure 4A) as previously demonstrated (40). Figure 4 shows that H99γ immunized mice treated with isotype control antibodies and challenged with R265 (Figure 4B) or R4247 (Figure 4D) had similar prolonged survival (median survival times of 65 and 64.5 days for R265 and R4247 challenged mice, respectively); similar to observations demonstrated in Figure 1B. In contrast, we observed 100% mortality in H99γ immunized mice depleted of CD4⁺ T cells alone during challenge with R265 (median survival of 35.5 days) or depleted of CD4⁺ and CD8⁺ T cells (median survival of 34 days; Figure 4B). Interestingly, H99γ immunized mice depleted of CD8⁺ T cells alone and challenged with R265 showed increased, although not statistically significant, survival (median survival of 80 days) compared to that observed in isotype control-treated mice (median survival of 65 days). We also observed that H99γ immunized mice challenged with R4247 while depleted of CD4⁺ T cells alone experienced 87.5% mortality (median survival time of 30.5 days) and 100% mortality upon depletion of both CD4⁺ and CD8⁺ T cells (median survival time of 31.5 days; Figure 4D). Additionally, H99γ immunized mice depleted of CD8⁺ T cells alone and challenged with R4247 had increased survival although not statistically significant (median survival of 75.5 days) compared to the isotype control-treated mice (median survival of 64.5 days). Mice immunized with H99γ and subsequently depleted of CD4⁺ T and/or CD8⁺ T cells prior to and during challenge with WSA87 did not show any difference in survival compared to the isotype control-treated mice (Figure 4C). Overall, we observed that depletion of CD4⁺ T cells resulted in a significant loss of protection against challenge with R265 (serotype B) or R4247 (serotype D) in H99γ (serotype A) immunized mice, indicating that CD4⁺ T cells are required for broad-spectrum protection against these serotypes.