5 to 6-wk old Female BALB/c mice were obtained from the National Cancer Institute, National Institute of Health, Bethesda, MD, USA. Mice belonging to younger group were 8-wk old and mice belonging to aged group were 72-wk old. All mice were maintained in the FDA/CBER AAALAC-accredited facility under standard environmental conditions for this species. Among parasites, the wild type L. donovani (LdWT) (MHOM/SD/62/1S) maintained in golden Syrian hamsters and centrin1 gene–deleted (LdCen-/-) line of L. donovani (Ld1S2D) were used [39, 40]. The parasites were cultured according to the procedure previously described [39, 44]. Red fluorescent protein (RFP)-expressing LdWT parasites were developed using the pA2RFPhyg plasmid for integration of a RFP/ Hygromycin B resistance gene expression cassette into the parasite 18S rRNA gene locus as described previously [45]. mCherry expressing LdCen-/- parasites were generated using the pLEXSY-cherry-sat2 plasmid and following the company’s protocols (Jena Bioscience). The parasites were cultured according to the procedure previously described [44].

The animal protocol for this study has been approved by the Institutional Animal Care and Use Committee at the Center for Biologics Evaluation and Research, US FDA (ASP 1995#26). Further, the animal protocol is in full accordance with ‘The guide for the care and use of animals’ as descried in the US Public Health Service policy on Humane Care and Use of Laboratory Animals 2015 (http://grants.nih.gov/grants/olaw/references/phspolicylabanimals.pdf).

The young and aged mice were immunized via tail vein with 3X10⁶ stationary phase LdCen-/- promastigotes; 5-wk post-immunized mice were then challenged via tail vein with 10⁵ virulent L. donovani (LdWT) metacyclic parasites. In each study, at least 6 mice were used per group. Age matched naive mice used as controls were also similarly challenged with 10⁵ virulent L. donovani metacyclic parasites. After 10-wk of post-challenge period, parasite load was recorded from spleens of challenged mice by culturing the separated host cell preparations by limiting dilutions as previously described [40]. After 4-wk post challenge period proinflammatory cytokines, nitric oxide, antibody titers and T cell effector responses were measured.

Splenocytes were plated in 24-well culture plates and stimulated with freeze-thaw L. donovani Ag (80μg/ml FTAg) in complete RPMI 1640 medium and cells were incubated at 37°C in 5% CO2, with 95% humidity. After 72h of culture, cell supernatants were collected and stored in −80°C until cytokines were analyzed using multiplex kits, MILLIPLEX Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore). The plate was read in a Luminex-100 (Luminex) system using Bio-Plex Manager software 5.0. The cytokine analysis procedure has been performed according to the manufacturer’s instructions, and the level of cytokine concentration was measured using a standard curve of each specific cytokine.

Splenocytes or macrophages obtained from peritoneal fluid were cultured in complete RPMI 1640 medium in the presence of FTAg (80μg/ml) for 24h at 37°C. NO (nitrite/nitrate) production was determined from the supernatants of the cultures by the Griess Reaction Kit (Sigma-Aldrich) [40].

The IgG specific Ab responses were measured by conventional ELISA method. Briefly, ELISA plates were coated overnight at room temperature with FTAg (80 μg/ml). A serial dilution of the sera was carried out to determine the titer, which is defined as the inverse of the highest serum dilution factor giving an absorbance of >0.2. Titers for the Abs were determined using the following HRP-conjugated secondary Abs: Rabbit anti-mouse IgG (H+L)–HRP, Rabbit anti-mouse IgG1-HRP, Rabbit anti-mouse IgG2a-HRP (Southern Biotech; all with 1:1000 dilutions). SureBlue (KPL) was used as a peroxidase substrate. After 15 min, the reaction was stopped by the addition of 100 μl 1M H₂SO₄, and the absorbance was read at 450 nm.

The proliferative capacity of T cells was assessed by a CFSE dilution assay in LdCen-/- immunized mice before and after challenge with virulent L. donovani parasites. Age-matched naive mice served as negative controls for Ag-specific proliferation. Splenocytes from different groups of mice were isolated, incubated in 5μM CFSE (Molecular Probes/Invitrogen) for 10 min in RPMI 1640 without fetal calf serum (FCS), followed by 5 min of quenching in ice-cold RPMI 1640 plus 10% FCS, and subsequently washed thoroughly before plating in 96-well tissue culture plates at 2×10⁵ cells/well. Cells were cultured for 5 days at 37°C with 5% CO2 under stimulation with FTAg (80 μg/ml). Cells were harvested, washed, and blocked with anti-CD16/32 (5 μg/ml) for 20 min (4°C) and stained with anti-mouse CD3 allophycocyanin–eFluor 780, anti-mouse CD4 eFluor@450, anti-mouse CD8a eFluor@605NC (eBioscience, USA) (each with 1:200 dilution; 4°C) for 30 min. For analysis, single live cells (dead cells were excluded based on staining with the LIVE/DEAD Aqua dye) were gated for CFSE stained CD4⁺T cells and CD8⁺T cell and proliferation was calculated. Cells were acquired on an LSR II (BD Biosciences) equipped with 405-, 488-, 532-, and 638- nm laser lines using FACSDiva 6.1.2 software. Data were analyzed with FlowJo software version 9.1.5 (Tree Star, San Carlos, CA).

Splenocytes isolated from young and aged mice were cultured in 24-well plates in complete RPMI 1640 medium at 37°C and stimulated with FTAg (80 μg/ml). After 48h at 37°C, protein transport inhibitor (BD GolgiStop; BD Pharmingen) was added to the wells and the plate was incubated at 37°C for 6h. 6h after, cells were then blocked at 4°C with rat anti-mouse CD16/32 (5 μg/ml) from BD Pharmingen for 20 min. For surface staining, cells were then stained with anti-mouse CD3 APC-eFluor@780, anti-mouse CD4 eFluor@450, anti-mouse CD8a eFluor@605NC, anti-mouse CD44 FITC, and anti-mouse CCR7 PE-Cy5 Abs for 30 min (each with 1:200 dilution; 4°C). The cells were then stained with Live/Dead fixable aqua (Invitrogen/Molecular Probes) to stain dead cells. Cells were washed with wash buffer and fixed with the Cytofix/Cytoperm Kit (BD Biosciences) for 20 min (room temperature). Intracellular staining was done with anti-mouse IFN-γ PE-Cy7, anti-mouse TNF PerCp-eFluor@710, anti-mouse IL-2 BV711, anti-mouse IL-10 APC for 30 min (each with 1:300 dilution; 4°C). Cells were acquired on an LSRII (BD Biosciences) equipped with 405, 488, 532, and 638 laser lines using DIVA 6.1.2 software. Data were analyzed with FlowJo software version 9.1.5 (TreeStar). For analysis, first doublets were removed using width parameter; dead cells were excluded based on staining with the LIVE/DEAD Aqua dye. Lymphocytes were identified according to their light-scattering properties. CD4 and CD8 T cells were identified as CD3⁺ lymphocytes uniquely expressing either CD4 or CD8. Upon further gating, intracellular cytokines were measured in CD44^HiCCR7^Low cells. Fluorescence minus one controls were used for proper gating of positive events for designated cytokines.

Dendritic cells were cultured in vitro from bone marrow progenitors. Briefly, both young and aged BALB/c mice were sacrificed and their femurs and tibias were excised, cleaned of tissue, and flushed with RPMI medium. Bone marrow was isolated by depletion of erythrocytes with ACK lysis buffer, and cultured with complete RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin (20 U/ml)/ streptomycin (20 μg/ml) and 20 ng/mL GM-CSF (Peprotech, London, United Kingdom) and IL-4 (Peprotech) for 7 days to obtain >75% purity of CD11c⁺ DCs. For Phagocytosis and parasite clearance assay DCs were infected with parasites (5:1, parasite to DCs ratio). After incubation for 6h at 37°C in 5%CO_2, the cultures were washed with culture media to remove extracellular parasites, and the cultures were incubated in RPMI media for maximum of 72h. At 6, 24, 48 and 72h post-infection, cells were washed with PBS, fixed with methanol, stained with Giemsa, and intracellular parasite numbers were evaluated microscopically. For cytokine and NO measurements, DCs were infected with parasites and stimulated with LPS (1μg/ml) for 24h. Culture supernatants were collected at 24h post infection to evaluate cytokine (IL-12, TNF and IL-6) production with the use of sandwich ELISA kit (ebioscience) and nitric oxide production by the Griess assay. The assay was performed according to the manufacturer's instructions.

The young and aged mice were infected via tail vein with 3 X 10⁶ stationary phase red fluorescent LdWT RFP, or LdCen-/-m-cherry promastigotes. In each study, at least 6 mice were used per group. Age-matched naive mice used as control. At day 4 post infection, mice were sacrificed and parasite load was recorded from spleens of the LdWT and LdCen-/- infected mice by culturing the separated host cell preparations by limiting dilutions as previously described [40].

In a separate experiment the splenic DCs were sort selected. Single-cell suspensions were prepared from spleens, and RBCs were lysed using ACK lysing buffer. Dendritic cells were enriched using pan dendritic cell isolation kit (Miltenyi Biotec) and subsequent sort selecting of Leishmania (RFP/m-Cherry) infected splenic DCs and uninfected splenic DCs from different age groups of infected mice were done by high speed FACS cell sorter system (BD FACS Aria-IITM). Infected splenic DCs were sorted by gating live single enriched DCs for Cd11c+Ly6G-Cd11b-RFP/m-Cherry⁺. Uninfected splenic DCs after enrichment [Cd11c+ Ly6G-Cd11b- RFP/ m-Cherry^-] were also sort selected.

Total RNA was extracted from the parasitized/uninfected splenic DCs using RNAqueous Microkit (AM1931) and RNA was extracted from total mouse splenocytes using PureLink RNA Mini kit (Ambion). Total RNA (400 nanograms) was reverse transcribed into cDNA using random hexamers by a high-capacity cDNA reverse transcription kit (Applied Biosytems). Gene expressions were determined by TaqManGene Expression Master Mix and premade TaqMan Gene Expression assays (Applied Biosystems) using a CFX96 Touch real-time system (Bio-Rad, Hercules, CA). The data were analyzed with CFX Manager soft-ware. The TaqMan Gene Expression Assay ID (Applied Biosystems) of different primers used was as follows: TNF, Mm00443258_m1; IL-12, Mm00434174_m1; IFN-γ, Mm01168134_m1; IL-6, Mm00446190_m1; IL-4, Mm00445259_m1; IL-10, Mm00439614_m1; and GAPDH, Mm99999915_m1. Expression values were determined by the 2^{- DD Ct} method where samples were normalized to GAPDH expression and determined relative to untreated sample.

Statistical analysis of differences between means of experimental groups was determined by unpaired two-tailed Student t test, using Graph Pad Prism 5.0 software. A p value <0.05 was considered significant, and a p value <0.005 was considered highly significant.

Our previous studies have shown that BMDCs from young mice manifest heightened effector function in response to LdCen-/- infection in vitro [46]. Hence, in this study we compared the ability of LdCen-/- parasites to modulate DC responses in BMDCs in vitro from both young and aged mice. LdWT and LdCen-/- parasites showed similar rates of infection along with identical parasite loads at 6h post-infection in both young and aged BMDCs (Fig 1A and 1B). These BMDCs cultures were then subsequently examined at 24, 48 and 72h post-infection, and the percentage of infected DCs and parasite loads were calculated. Interestingly, BMDCs from both young and aged mice infected with LdCen-/- displayed significantly lower percentage of infected cells (Fig 1A) and a lower number of parasites per infected cell (Fig 1B), compared to those infected with LdWT at all the time points post-infection. This indicates that LdCen-/- parasites infect both young and aged BMDCs similarly but are unable to persist for prolonged periods compared to LdWT parasites. DCs play a crucial role in coordinating immune response in leishmaniasis by providing the IL-12 necessary for the induction of protective Th1 immune response [47, 48]. LdCen-/- parasite infected young and aged BMDCs produced significantly more IL-12 than did those infected with LdWT parasites (Fig 1C). Next, we analyzed the level of proinflammatory cytokines (TNF, IL-6) and nitric oxide (NO), the key components in Leishmania control, in BMDCs cell supernatants 24h post infection. Interestingly, LdCen-/- infected BMDCs from both age groups secreted significantly more TNF, IL-6 and NO compared to LdWT infected cells (Fig 1D, 1E and 1F). Altogether, these observations suggest that BMDCs from both young and aged mice undergo a more pronounced effector response following infection with LdCen-/- compared to LdWT infected BMDCs. Importantly, BMDCs from aged mice showed similar innate response like that of BMDCs of young mice in response to LdCen-/- infection.

Next, we compared the antigen presentation capability of LdCen-/- infected BMDCs by measuring their ability to present LdCen-/- antigens and activate CFSE-labeled naïve CD4⁺ T cells as measured by IFN-γ and IL-10 production. BMDCs from young and aged mice were pulsed with OVA peptide followed by infection with LdWT or LdCen-/- and then co-cultured with OVA specific (DO11.10) CFSE labeled CD4⁺T cells. CD4⁺T cell proliferation was evaluated after 5 days by flow cytometry. LdCen-/- infection induced the proliferation of T cells in both young and aged BMDCs and was significantly higher compared to those co-cultured with LdWT infected BMDCs as demonstrated by the histogram overlay depicting the individual % proliferated CD4⁺T cells from different sets (Fig 1G) and the subsequent quantitative bar diagram (Fig 1H). Interestingly, LdCen-/- mediated induction of CD4⁺T cell proliferation was similar in young and aged BMDCs thereby highlighting the similar dendritic cell activation. Cytokine production resulting from CD4⁺T cell response was measured in culture supernatants after 5 days of co-culture. IFNγ levels from LdWT and LdCen-/- infected BMDCs–T cell co-cultures did not differ significantly between the two age groups and were comparatively higher in LdCen-/- infected BMDCs (Fig 1I). On the other hand, LdWT infected BMDCs–T cell co-cultures significantly induced IL-10 production compared to LdCen-/- infected BMDCs-T cell co-cultures in both age groups (Fig 1J). The IFN-γ: IL-10 ratio was significantly higher in LdCen-/- infected BMDCs-T cell cultures compared to LdWT infected BMDCs-T cells co-cultures (Fig 1K). Thus in vitro results suggest that LdCen-/- infected BMDCs from both age groups are capable of inducing the proliferation of Th1 type of CD4⁺T cells in vitro that may lead to enhanced adaptive immune response and also there is no diminution of LdCen-/- induced innate response between two age groups.

Further, we analyzed LdCen-/- mediated modulation of the dendritic cell (DCs) response in spleen using an in vivo mouse model. We infected young and aged mice intravenously with red fluorescent LdWT RFP, or LdCen-/- m-Cherry parasites. After 4 days we enriched DCs from spleen and sort selected uninfected DCs (UI DC) by gating live single cells for Cd11c+Ly6G-Cd11b-RFP/m-Cherry^- whereas parasitized DCs (Inf DC) were sorted by gating live single cells for Cd11c+Ly6G-Cd11b-RFP/m-Cherry⁺ (Fig 1L). We observed, LdCen-/- infected young and aged mice had comparatively lower although not statistically significant level of splenic parasite burden compared to LdWT infected mice (Fig 1M) which is in agreement with our in vitro observation. We also assessed the expression of proinflammatory and anti-inflammatory cytokine gene expression in LdCen-/- infected DC population and compared them with LdWT parasitized DCs. RT-PCR analysis showed that 4 major proinflammatory cytokines such as IL-12 (Fig 1N), IL-6 (Fig 1O), IFN-γ (Fig 1P) and TNF (Fig 1Q) were significantly elevated in parasitized splenic DCs isolated from LdCen-/- infected young and aged mice compared to LdWT infected mice. In contrast, anti-inflammatory cytokine IL-10 (Fig 1R) expression was significantly reduced in parasitized DCs isolated from LdCen-/- infected young and aged mice compared to LdWT infected mice. We did not see any significant difference in the behavior between infected DCs (Inf DC) and bystander DCs (UI DC) from LdWT infected young and aged mice. However, we observed that bystander DCs from LdCen-/- infected mice resulted in comparatively lower production of proinflammatory cytokines and higher production of anti-inflammatory cytokines compared to infected DCs from both age groups (Fig 1N–1R). Moreover, un-infected splenic DCs (UI DC) from LdCen-/- infected mice showed slightly higher but not statistically significant level of proinflammatory cytokines (IL-12, IL-6, IFN-γ and TNF) along with down regulation of IL-10, compared to uninfected DCs from LdWT infected young and aged mice (Fig 1N–1R).

Additionally, we have investigated the antigen presentation capability of dendritic cells sort selected from young and aged mice which were infected with LdWT or LdCen-/- parasites for 4 days. We pulsed these DCs with OVA peptide followed by co-culture with OVA-specific CD4⁺T cells from both age groups for 5 days to analyze OVA-specific proliferation of T cells. Consistent with our in vitro study, the percentage of proliferating CD4⁺T cells after 5 days were comparatively higher upon co-culture with OVA-pulsed DCs from LdCen-/- infected young and aged mice compared to LdWT infected mice (S1B Fig). Cytokine production resulting from such proliferated CD4⁺T cell response was measured by flow cytometry. The results showed that DCs from LdCen-/- parasite infected young and aged mice stimulated CD4⁺ T cells to produce significantly higher levels of IFN-γ/IL-10 ratio compared to CD4⁺T cells cocultured with DCs from LdWT infected mice (S1C Fig), thereby indicating a similar Th1 response predominance in both age groups of mice. Thus, from the in vivo gene expression profile and antigen presentation assay, it is also evident that LdCen-/- infection results in similar and significant enhancement of innate effector responses in young and aged mice.

The ability of LdCen-/- to protect against virulent L.donovani infection was determined in 5-wk immunized mice followed by 10-wk of challenge with virulent L. donovani parasites. The results showed that immunization with LdCen-/- significantly reduced spleen and liver parasite burden in both young and aged mice compared to naive-challenged mice (Fig 2A and 2B). The parasite burden in spleen and liver of naive challenged aged mice was similar like that of naïve challenged young mice (Fig 2A and 2B). However, the reduction in parasite burden was significantly less in the LdCen-/- immunized–challenged aged group (13 fold in spleen and 8 fold in liver) than the immunized–challenged young mice (49 fold in spleen and 19 fold in liver; data in Fig 2A and 2B). Overall, these data suggest that LdCen-/- immunization confers substantial protection in young and aged mice against virulent L. donovani challenge; however, the protection is reduced in aged mice.

Effective clearance of Leishmania parasites requires a proinflammatory response which is mainly mediated by IFN-γ [49–53]. In order to evaluate the protective immunity induced after LdCen-/- immunization in both young and aged mice, we analyzed Ag-specific cytokine secretion by splenocytes from naive, immunized (5-wk), non-immunized challenged (naïve challenged) and immunized challenged mice (5-wk immunization + 4-wk of challenge) (Fig 3). Comparative cytokine profiles demonstrated a significant induction of Leishmania Ag–specific IFN-γ, IL-12, and TNF secretion in the culture supernatants of 5-wk post-immunized young and aged mice compared to naïve mice at protein level (Fig 3A–3C). Additionally, the steady state cytokine gene expression pattern at mRNA level (S2A–S2C Fig) was similar with Ag stimulated cytokine secretion at protein level. Interestingly, following challenge with virulent L. donovani parasites, the immunized young mice showed significantly enhanced IFN-γ, IL-12 and TNF secretion at both protein (Fig 3A–3C) and mRNA level (S2A–S2C Fig), while in aged mice significantly enhanced secretion of IL-12, TNF but not IFN-γ was observed (Figs 3A–3C and S2A–S2C). Importantly, overall induction of all the proinflammatory cytokines (IFN-γ, IL-12 and TNF) was significantly lower in aged mice compared to young mice from all the groups at both protein (Fig 3A–3C) and mRNA level (S2A–S2C Fig). Additionally, in immunized young and aged mice, compared with naive mice, significant induction of anti-inflammatory cytokines like IL-10 and IL-4 was observed at protein level (Fig 3D and 3E) whereas at mRNA level comparatively higher although statistically non-significant levels of IL-10 and IL-4 were observed (S2D and S2E Fig). However, young and aged immunized-challenged mice showed slightly higher although not statistically significant levels of IL-10 and IL-4, compared to naïve challenged groups at both the protein (Fig 3D and 3E) and mRNA levels (S2D and S2E Fig). Importantly, the IFN-γ: IL-10 or IFN-γ: IL-4 ratios were higher in immunized as well as immunized challenged young and aged mice compared to naïve and non-immunized challenged groups respectively (Fig 3F and 3G). Interestingly IFN-γ: IL-10 or IFN-γ: IL-4 ratios in immunized challenged aged mice were significantly less compared to young mice. Taken together, these results suggest that LdCen-/- immunization initiates mixed pro- and anti-inflammatory immune response against virulent L. donovani challenge in both young and aged mice albeit lower proinflammatory response in aged mice.

Since the preferential production of proinflammatory cytokines specifically IFN-γ results in increased synthesis of host protective nitric oxide (NO) by activated macrophages [54, 55], we determined the level of NO produced by Leishmania Ag restimulated splenocytes derived from the LdCen-/- immunized young and aged mice before and after challenge. It is important to note that NO product observed in the splenocytes is mainly from the macrophages present in such cultures. A significant amount of Leishmania Ag specific nitrite production was observed in both young and aged post immunized mice compared with non-immunized mice splenocytes (Fig 4A). Moreover, a much greater amount of NO was observed in LdCen-/- immunized young and aged mice upon virulent L. donovani challenge than in naive challenged mice (Fig 4A). Of note, the induction of nitrite concentration was significantly lower in aged mice compared to young mice from all the groups. Additionally, since, higher level of anti-leishmania antibody (IgG2a) dictates predominant Th1response [56, 57]; we analyzed sera from immunized-challenged young and aged mice for determination of Leishmania–specific IgG, IgG1, and IgG2a titers. The Leishmania Ag-specific IgG_Total, IgG1, and IgG2a production was significantly higher in immune challenged young and aged mice compared to naïve challenged groups (Fig 4B). Of note, specific increase in Th1 mediated IgG2a titers in immunized and challenged mice sera from both groups were observed. Importantly, the levels of all three Ab populations were reduced in aged mice compared to young mice from all the groups.

Aging is known to be associated with a decline in T cell proliferation [11, 58]. Hence to investigate if LdCen-/- vaccination induces Leishmania Ag specific T cell proliferation in young and aged mice, we performed proliferation assays using splenocytes from naïve, 8-wk post immunized, naive challenged (4-wk of challenge) and immunized challenged (8-wk immunization + 4-wk of challenge) mice that were stimulated with Leishmania FTAg. Results showed that both CD4 and CD8 T cells isolated from both young and aged mice immunized with LdCen-/-, have significantly higher proliferative capacity in response to Leishmania FTAg compared to T cells isolated from naive mice (Fig 5B). Further, T cells isolated from mice immunized and challenged with virulent L. donovani parasites have significantly higher proliferative capacity than naive challenged group. The significant proliferative capacity of T cells even in 8-wk post-immunized young and aged mice suggests that some of them may have derived from a memory T cell response since by that time the majority of mice have cleared the LdCen-/- parasite [40]. Interestingly significantly lower proliferative capacity of only CD8 T cells was observed in the spleen of immunized aged mice compared to young mice before and after challenge.

Since efficacy of several experimental Leishmania vaccine candidates has been shown to correlate with the T cells secreting multiple pro-inflammatory cytokines [59], we investigated whether LdCen-/- immunization could induce splenic CD4⁺ and CD8⁺ T cells to secrete multiple cytokines such as IFN-γ, IL-2 and TNF in infected aged mice as was previously observed in young mice [40]. Specifically, we demonstrated cytokine production from the antigen experienced effector memory T cells (CD44^Hi/CCR7^Low) in 8-wk post-immunized young and aged animals. Spleen cells cultured in vitro in presence of Leishmania FTAg followed by multicolor staining were gated according to their surface expression of CD44 and CCR7. Seven distinct populations of cytokine-producing cells were defined from the CD44^Hi/CCR7^Low cells based on different combinations of IFN-γ, IL-2 or TNF (Fig 6A). The results showed that at 8-wk post-immunization in both young and old mice, significantly higher number of CD4 and CD8 T cells were single, (IFN-γ, IL-2 and TNF) double (IFN-γ⁺ TNF⁺, IFN-γ⁺IL-2⁺, TNF⁺IL-2⁺) and triple cytokine producing cells (IFN-γ⁺ TNF⁺ IL-2⁺) (Fig 6B and 6C) compared to naïve mice. However, the number of single and multiple cytokine producing cells were significantly lower in aged mice compared to young mice. Similar analysis conducted with spleen cells of immunized and challenged young and aged mice showed significantly higher percent of the single and multiple cytokine-producing CD4 and CD8 T cells compared to naive challenged mice (Fig 7A and 7B). Again we observed a reduced percent of single and multiple cytokine expressing CD4 and CD8 T cell in aged immunized challenged mice.

We also quantified Ag-experienced CD4 T cells that produce IL-10, a crucial anti-inflammatory cytokine in the pathogenesis of VL from the spleen of young and aged mice (Fig 7C). Ag-experienced CD4 T cells from LdCen-/- immunized and challenged mice produced significantly lower IL-10 in both age groups compared to naïve challenged mice (Fig 7C). Since the balance between pro and anti-inflammatory cytokines predicts the outcome of protective immune response, we measured IFN-γ/IL-10 ratio. In Ag-stimulated CD4 T cells, the IFN-γ/IL-10 ratio was significantly higher in the immunized mice both before challenge and after challenge compared with either naive or naive-challenged controls (Fig 7D). However in aged mice the IFN-γ/IL-10 ratio was significantly lower compared to young mice. In summary, LdCen-/- immunized young and aged mice induced a strong antigen experienced effector T cell memory response after 8-wk post-immunization, at a time point when the majority of mice have cleared the LdCen-/- parasite. However, the effector T cell mediated immune response is lower in aged mice.