Supplemental Material can be found at: http://jn.nutrition.org/content/suppl/2014/10/15/jn.114.20289 5.DCSupplemental.html

The Journal of Nutrition Nutritional Immunology

Vitamin D Receptor Signals Regulate Effector and Memory CD8 T Cell Responses to Infections in Mice1–3 Yevgeniy Yuzefpolskiy,4 Florian M Baumann,4 Laura A Penny,4 George P Studzinski,5 Vandana Kalia,4* and Surojit Sarkar4* Center for Molecular Immunology and Infectious Diseases, Department of Veterinary and Biomedical Sciences, and The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, PA; and 5Department of Pathology and Laboratory Medicine, Rutgers New Jersey Medical School, Newark, NJ

Abstract Background: Vitamin D insufficiency is associated with broad-ranging human disease sequelae such as bone disease, cancer, cardiovascular disease, allergy, autoimmune disorders, diabetes, and infectious diseases. Disease risk and severity of a large proportion of the nonskeletal disorders heavily involve the cytotoxic cluster of differentiation (CD) 8 T lymphocyte (CTL) arm of cellular adaptive immunity. Considering the importance of vitamin D in CTL-dependent diseases, there is a critical need for systematic in-depth explorations into the role of vitamin D deficiency in generation and maintenance of CTL immunity during infections and vaccinations. Objective: With the use of wild-type (WT) vitamin D–sufficient mice and the vitamin D receptor knockout (Vdr2/2) mouse model of in vivo deficiency of vitamin D signaling, we systematically analyzed the impact of vitamin D deficiency on antigen-specific effector and memory CD8 T cell responses to acute viral and bacterial infections. Methods: WT and Vdr2/2 mice were infected with lymphocytic choriomeningitis virus, a natural mouse pathogen, and antigen-specific CTL responses were analyzed during priming, expansion, contraction, and memory phases. Magnitude, breadth, cytokine production, and localization of antiviral effector and memory CTLs to lymphoid and nonlymphoid tissues were specifically assessed. Results: The absence of vitamin D signals led to 1) aberrant CD8 T cell effector differentiation (;2-fold lower granzyme B and reduced B cell lymphoma 2; P # 0.05) and enhanced contraction (;15% increase; P # 0.05) in antigen-specific CTLs; 2) a significantly restricted (P # 0.05) breadth of the antigen-specific CD8 T cell effector and memory repertoire; and 3) preferential localization of effector (;2.5-fold increase; P # 0.01) and memory (;5-fold increase; P # 0.001) CD8 T cells to the lymph nodes compared to nonlymphoid tissues. Conclusion: Our data show a previously unrecognized impact of vitamin D deficiency on the quantity, quality, breadth, and location of CD8 T cell immunity to acute viral and bacterial infections. J Nutr 2014;144:2073–82. Keywords: CD8 T cells, effector, infection, memory, vitamin D

Introduction Vitamin D is a member of the steroid superfamily of fatsoluble hormones. The prohormone is either acquired from dietary sources (animal origin, cholecalciferol/vitamin D3 and ergocalciferol/vitamin D2 from yeast/fungi) or photochemically

synthesized in the skin by solar UV-B radiation (1). The biologically active form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D]6, is synthesized from the dietary form through 2 hydroxylation steps: first in the liver to generate 25-hydroxyvitamin D [25(OH)D] and second in the kidney through the action of 25-hydroxyvitamin

1

Supported by funds from The Pennsylvania State University to SS and VK. Author disclosures: Y Yuzefpolskiy, FM Baumann, LA Penny, GP Studzinski, V Kalia, and S Sarkar, no conflicts of interest. 3 Supplemental Figures 1–4 and Supplemental Materials and Methods are available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition. org. * To whom correspondence should be addressed. E-mail: sarkarkalia@gmail. com (S Sarkar and V Kalia). 2

6 Abbreviations used: APC, antigen-presenting cell; Bcl-2, B cell lymphoma 2; CD, cluster of differentiation; CTL, cytotoxic CD8 T lymphocyte; CYP27B1, 25-hydroxyvitamin D-1a-hydroxylase; DC, dendritic cell; GP, glycoprotein; GzmB, granzyme B; KLRG-1, killer cell lectin-like receptor G1; LCMV, lymphocytic choriomeningitis virus; MHC-I, major histocompatibility complex I; NP, nucleoprotein; rLM, recombinant Listeria monocytogenes; TCR, T cell receptor; Vdr, vitamin D receptor; WT, wild-type; 1,25(OH)2D, 1,25-dihydroxyvitamin D; 25(OH) D, 25-hydroxyvitamin D.

ã 2014 American Society for Nutrition. Manuscript received August 27, 2014. Initial review completed September 5, 2014. Revision accepted September 8, 2014. First published online October 15, 2014; doi:10.3945/jn.114.202895.

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response to multiple viral epitopes. These comprehensive analyses uncovered a link between 1,25(OH)2D signals and CTL responses and form a strong foundation for further mechanistic studies into CD8 T cell intrinsic metabolism and signaling events and for future investigations in the clinical settings of vitamin D insufficiency.

Methods Mice and diets. Vdr2/2 C57BL/6 mice (2) were purchased from Jackson Laboratory (strain 006133) and were bred in-house as heterozygotes. Pups were screened for homozygosity of knockout allele by PCR as recommended by Jackson Laboratory. WT mice were either purchased from Jackson Laboratory (strain 000664) or WT littermate controls were used from the heterozygous Vdr2/2 breeding colony. Purchased WT and Vdr2/2 littermate WT mice had identical outcomes for the immunologic parameters analyzed and were hence used interchangeably. WT P14 transgenic C57BL/6 mice bearing the T cell receptor (TCR) specific for the glycoprotein (GP)33–41 peptide of LCMV (27) were purchased from Taconic (strain 4138) and bred in-house. All mice used for experiments were between 6 and 8 wk of age and were sexmatched within a given experiment. Mice were maintained in a temperature-controlled facility at 23°C and 50% humidity with 12-h light cycle. WT mice were fed standard pelleted mouse PicoLab Rodent Diet 20 (LabDiet 5053) (28). Vdr2/2 mice were maintained on a 20% lactose, 2% calcium, 1.25% phosphorus rescue diet purchased from Harlan Teklad Research Diets (diet 96348) (29) to prevent hypocalcemia. Mice consumed diets and water ad libitum. Mice were bled under isoflurane anesthesia and killed by using carbon dioxide asphyxiation followed by cervical dislocation. All animal procedures were performed in accordance with approved University Institutional Animal Care and Use Committee guidelines. Virus, bacteria, and infections. The Armstrong strain of LCMV was propagated, titered, and used as previously described (30, 31). Recombinant Listeria monocytogenes (rLM), which expresses the GP33–41 epitope of LCMV (rLM-GP33), was propagated, tittered, and used as previously described (24). Mice were infected with 2 3 105 plaqueforming units of LCMV intraperitoneally or 2500 CFUs of rLM-GP33 intravenously. Cell isolation, staining, and flow cytometry. At indicated times postinfection, mice were either bled or killed for isolation of lymphocytes from blood, spleen, lymph node, lung, or liver as described previously (30, 31); cells were enumerated and stained for tetramer, surface, and intracellular antigens. The details of staining, clones, and sources of staining reagents were published previously (31) and are provided in the Supplemental Materials and Methods. Fluorescently labeled samples were acquired by using BD LSR-II Fortessa Flow cytometer (BD Biosciences), and data were analyzed by using FlowJo software (Treestar). Direct ex vivo production of cytokines. Analysis of intracellular cytokine production was performed as previously described (30, 31). Briefly, 106 splenocytes were stimulated with 0.2 mg/mL peptides (GenScript) from LCMV GP or nucleoprotein (NP) (32) in the presence of brefeldin A for 5 h at 37°C and 5% CO2. Cells were stained for gating markers as noted in CD8 and CD44 and then intracellularly stained for cytokines IFN-g (Clone XMG1.2), TNF-a (Clone MP6-XT22), or IL-2 (Clone JES6-5H4) (30, 31). In vitro T cell priming and activation. CD8 T cell intrinsic differences in activation were tested by using either WT or Vdr2/2 CD8 T cells as responders. Briefly, 1 3 105 WT or Vdr2/2 CD8 T responder cells and 1 3 106 CD82/2 splenocyte filler cells were added to a 96-well flatbottom plate coated with 5 mg/mL each of aCD3 (145-2C11) and aCD28 (PV-1) from Biolegend. CD8 T cell extrinsic differences in activation were analyzed by using 1 3 105 WT P14 CD8 T cells as responders and 1 3 106 WT or Vdr2/2 splenocytes as filler cells in a

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D-1a-hydroxylase (CYP27B1). The 1,25(OH)2D binds to the vitamin D receptor (Vdr), a member of the steroid hormone nuclear receptor superfamily, and directly regulates transcription of target genes. Because most biological effects of vitamin D are mediated through Vdr, the Vdr gene knockout (Vdr2/2) mouse (2) has emerged as a key laboratory model for human vitamin D deficiency where Vdr-mediated 1,25(OH)2D signals are missing altogether. Vdr2/2 mice replicate classic human clinical vitamin D deficiency symptoms of rickets, hypocalcemia, hypophosphatemia, osteomalacia, and hyperparathyroidism (2). Large-scale, multiyear cohort studies show increasing vitamin D insufficiency worldwide (3, 4), as assessed by serum 25(OH)D concentrations. However, the link between vitamin D and immunity to diseases and vaccines remains a clinically relevant question that is staked in history: Dr. Niels FinsenÕs Nobel Prize (Physiology or Medicine, 1903)–winning studies (5, 6) provided the first evidence that concentrated light irradiation and vitamin D supplementation could cure an epidermal form of tuberculosis. Over the last decade, there have been clear data that autoimmune disorders (7), infections [e.g., tuberculosis (8, 9), leprosy (10, 11), influenza (12), HIV (13, 14), etc.], and cancers (15), which are dominantly dependent on T cell function for protection, are exacerbated by inadequate levels of vitamin D (16). The immunomodulatory role (17) of vitamin D on T cells is further underscored by Vdr expression in activated cluster of differentiation (CD) 8 T cells (18) and in other immune cells (19– 21). Additionally, CYP27B1 is expressed by many immune cells, indicating an intracrine or paracrine role for extrarenally produced 1,25(OH)2D in immune cells (1) in addition to its classic endocrine role. On the basis of this, it has been proposed that inadequate dietary consumption of cholecalciferol or ergocalciferol may cause significant deficiency in localized 1,25(OH)2D in the immune system due to reduced in situ production (22). Reduced 25(OH)D and 1,25(OH)2D concentrations are in turn associated with lower Vdr expression, thus predicting profound immune effects of widely prevalent vitamin D insufficiency in the human population (3, 4). Despite such correlative advances, the direct impact of 1,25(OH)2D signals on CD8 T cell immunity during infections remains largely unexplored. CD8 T lymphocyte (CTL) immunity is the cytotoxic arm of our adaptive immune response, which is critical for protection against viruses, intracellular bacteria, and cancer. After infection or vaccination, naive CD8 T cells rapidly proliferate and differentiate into effector CTLs to mediate pathogen clearance. Thereafter, differentiation of long-lived protective memory ensues (23–25). Memory CD8 T cells can be maintained for the lifetime of an individual through antigen-independent homeostatic proliferation and can rapidly expand, elaborate heightened effector functions, and expeditiously cull the disease upon secondary encounter with the same pathogen. Hence, generation of immune memory is the basis of prophylactic vaccination and long-term immunity to infectious diseases, and underlying mechanisms are much sought after. In this study, we used the Vdr2/2 mouse model of vitamin D deficiency [due to lack of Vdr-mediated 1,25(OH)2D signaling] to ask the novel question: What is the impact of vitamin D deficiency on CD8 T cell effector and memory responses to acute infections? We used a well-established murine model of infection with a natural pathogen, lymphocytic choriomeningitis virus (LCMV) (26). We compared the quantity and quality of epitopespecific CTL responses during distinct stages in wild-type (WT) and Vdr2/2 mice with the use of major histocompatibility complex I (MHC-I) tetramers and cytokine production in

96-well flat-bottom plate. The 5 3 104 WT or Vdr2/2 splenocytes loaded with GP33 peptide (33) were then used as stimulators. Cells were incubated for 2.5 d at 37°C and 5% CO2 and then analyzed by flow cytometry for blast size via forward scatter as described previously (34).

Statistical analysis. All statistical analyses including the computation of means and SEMs for WT and Vdr2/2 groups were conducted by using Prism 5 software (GraphPad). Differences between the static means of WT and Vdr2/2 groups were compared for significance by using onetailed parametric StudentÕs t test. Because of the normal distribution of in vivo experimental samples, homogeneous variance, and independence of the Vdr2/2 data set from WT mice, we used one-tailed unpaired StudentÕs parametric t test to evaluate differences between sample means of WT and Vdr2/2 mice. Power analysis was used to determine that n $ 3 mice were needed in each experimental group to observe significant differences in phenotype. To objectively analyze the representation of each epitope-specific CD8 T cell species to the global diversity of the breadth of CD8 T cell response, SimpsonÕs diversity index was calculated as described previously (35). In vitro differences in forward scatter were compared by using a probability-binning algorithm in FlowJo as described previously (34). All statistical analyses were conducted by using a = 0.05. Further details of the statistical methods used, sample size, assumptions, and justifications are provided in the Supplemental Materials and Methods.

Results Robust antigen-specific CD8 T cell expansion but compromised effector differentiation in the absence of 1,25(OH)2 vitamin D signals. Although an impact of vitamin D signals on CD8 T cell responses may be surmised from clinical associations, a direct assessment of antigen-specific CD8 T cell differentiation in the presence or absence of 1,25(OH)2D signals is lacking. As shown in Figure 1, the overall kinetics of antigenspecific CD8 T cell responses (3 distinct LCMV epitopes: DbGP33, DbNP396, and DbGP276) were largely similar in WT and Vdr2/2 mice infected with LCMV (Figure 1A). All epitope specificities rapidly expanded after infection and reached a peak at ;8 d postinfection. Thereafter, although the majority of effector cells underwent death (contraction), a small proportion survived to form the memory pool at ;30 d postinfection. As observed with individual specificities, CD44hi (higher in MFI for CD44) CD8 T cells, which represent the sum of antigenexperienced cells specific for pathogen-derived epitopes (36), also exhibited a similar kinetics of expansion and contraction in Vdr2/2 and WT mice (Figure 1A). Likewise, longitudinal tracking of the DbGP33-specific CD8 T cell response by tetramer staining in peripheral blood mononuclear cell samples of mice infected with rLM expressing the GP33 epitope of LCMV (rLM-GP33) also exhibited a similar kinetics (Supplemental Figure 1A). Both WT and Vdr2/2 groups exhibited robust antigen-specific CD8 T cell expansion and, consistent with well-known antiproliferative effects of 1,25(OH)2D (18, 37, 38),

Vdr deficiency is associated with a less diverse antigenspecific effector CTL repertoire. The generation of a broad repertoire of CD8 T cells targeting many different pathogen-specific epitopes is critical to thwart pathogen escape mechanisms through mutation of epitope sequences (41, 42). Data presented in Figure 1 focus on 3 major immunodominant epitopes of LCMV for which MHC-I tetramer reagents are available. We next investigated the broader spectrum of LCMV-specific CD8 T cell responses in Vdr2/2 mice by using a panel of 7 different peptide epitopes from the glycoprotein and nucleoprotein of LCMV. Similar to the data obtained by using tetramers, assays measuring intracellular IFN-g production in response to in vitro stimulation with peptide epitopes also showed a modest increase in the proportions of effector CD8 T cells specific to the immunodominant GP33 and NP396 (P # 0.09) epitopes at 8 d postinfection (Figure 2A) in Vdr2/2 mice. However, to our surprise, subdominant GP118 (P = 0.05), GP61 (P # 0.05), and GP92 (P = 0.06) epitope-specific CD8 T cells were diminished in Vdr2/2 mice compared with WT mice (Figure 2A). Calculation of the SimpsonÕs diversity index, an unbiased numeric indicator reflecting the breadth of epitope specificities, exhibited a significant decrease in epitope diversity of the effector CD8 T cell pool in the absence of 1,25(OH)2D signals. In addition to the expression of effector molecules such as GzmB and IFN-g, CTLs also produced effector cytokine TNF-a as well as IL-2, which is known to drive expansion and effector differentiation (30,43). This ability of CD8 T cells to coproduce Vitamin D regulation of CTL immunity to pathogens

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In vivo T cell priming and activation experiments. To analyze the activation of WT CD8 T cells in a WT or Vdr2/2 in vivo environment, 106 GP33-specific WT P14 CD8 T cells (Thy 1.1+) were adoptively transferred into either WT or Vdr2/2 (Thy 1.2+) C57BL/6 mice and infected with LCMV. These 2 experimental groups allowed us to analyze priming of WT antigen-specific CD8 T cells in an infected in vivo setting where all other cell types were WT or Vdr2/2. Altered P14 responses in Vdr2/2 compared with WT mice would indicate a requirement of Vdr on cells other than antigen-specific CD8 T cells. At 2.5 d postinfection, mice were killed and indicated tissues isolated for analysis of lymphocytes as described previously (30, 31).

modestly increased T cell expansion was observed by day 8 in both LCMV (Db-NP396, P = 0.06; Db-GP276, P = 0.026; CD44hi, P = 0.09) and rLM-GP33 (P = 0.07) infections in the absence of Vdr (Figure 1A, Supplemental Figure 1A). Increased expansion of antigen-specific CTLs in the absence of 1,25(OH)2D signals was also reflected in modestly higher numbers (P # 0.09) of DbGP33-, DbNP396-, and DbGP276-specific as well as total CD44hi effector cells in most lymphoid and nonlymphoid tissues analyzed (Supplemental Figure 2), particularly in the lymph nodes. The expansion of naive CD8 T cells after antigenic stimulation is also coupled to rapid differentiation into CTLs capable of producing effector molecules (39) and migrating to peripheral sites of infection (24). Thus, we next assessed the effector differentiation status of antigen-specific CD8 T cells in the absence of 1,25(OH)2D signals. The differentiation of effector CTLs is marked by upregulation of effector molecule granzyme B (GzmB) and terminal differentiation marker killer cell lectinlike receptor G1 (KLRG-1) as well as downregulation of memory marker CD127 and lymph node homing marker CD62L (40). To our surprise, despite robust expansion, Vdr2/2 mice expressed significantly lower levels of GzmB effector molecule at the peak of CTL expansion for all 3 epitope specificities analyzed than did WT mice (Figure 1B). Although lower than in WT cells, GzmB expression in Vdr2/2 antigenspecific CD8 T cells was higher than in naive cells, thus suggesting suboptimal effector differentiation in the absence of 1,25(OH)2D signals. However, CD127 and CD62L were downregulated similarly in WT and Vdr2/2 mice (Figure 1C), and antigen-specific effector CTLs also upregulated the terminal differentiation marker KLRG-1 to a similar extent (Figure 1C). Likewise, migration to peripheral sites was also largely unaffected in the absence of 1,25(OH)2D signals (Supplemental Figure 2). Together, these data demonstrate that 1,25(OH)2D signals exert modest inhibitory effects on the expansion of antigen-specific CD8 T cells during acute viral as well as bacterial infections in vivo and are required for optimal expression of the effector molecule GzmB.

multiple cytokines is commonly referred to as polyfunctionality and is associated with robust CTL responses. Thus, we assessed the ability of IFN-g–producing CTLs to also produce the effector cytokine TNF-a and IL-2. Despite compromised GzmB expression (Figure 1B), no significant defects were observed in the coproduction of effector cytokines IFN-g, TNF-a, or IL-2 (Figure 2B) by Vdr2/2 effector CD8 T cells, and the level of IFN-g production on a per cell basis was also similar in WT and Vdr2/2 antigen-specific CD8 T cells (Figure 2B) as assessed by the mean fluorescence intensity of IFN-g staining. These observations demonstrate that GzmB but not cytokine production is uniquely regulated by 1,25(OH)2D signals. Collectively, these observations of decreased GzmB expression and a polyfunctional, yet less diverse, CTL repertoire in the absence of 1,25(OH)2D signals indicate a critical requirement of 1,25(OH)2D signals in promoting the development of a potent and broad antigen-specific effector CD8 T cell repertoire, possibly through regulation of T cell stimulation, expansion, or survival. Decreased survival of antigen-specific CD8 T cells in the absence of 1,25(OH)2 vitamin D signals. After clearing the antigen, the majority of peak effector CD8 T cells (short-lived effector cells, SLECs) undergo death (contraction), but a small 2076

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subset of antigen-specific CD8 T cells (memory precursor effector cells, MPECs) preferentially survive and differentiate into long-lived functional memory. B cell lymphoma 2 (Bcl-2) is a critical prosurvival/antiapoptotic protein previously shown to promote survival of effector CTLs into memory (44). Thus, we next assessed the extent of effector CTL death in the presence or absence of 1,25(OH)2D signals. As shown in Figure 3A, LCMVspecific effector CD8 T cells underwent enhanced contraction in the absence of 1,25(OH)2D signaling. A similar trend was also observed in the case of rLM-GP33 infection (Supplemental Figure 1B). At a molecular level, increased CTL contraction in Vdr2/2 mice was associated with a modest yet significant reduction in the expression of Bcl-2 compared with WT mice (Figure 3B). A similar trend was also observed in the case of rLM-GP33 infection (P = 0.07) (Supplemental Figure 1C). As shown in Figure 1C, Vdr2/2 and WT mice did not exhibit any differences in the expression of IL-7Ra or KLRG-1 on the surface of antigen-specific CD8 T cells, suggesting that the contraction is not due to decreased IL-7 signaling (another prosurvival marker) or preferential differentiation of CD127lo KLRG-1hi short-lived effector cells (Figure 1C) (31, 45). Notably, Vdr2/2 mice exhibited similar alterations in CTL immunity regardless of their diet; Vdr2/2 mice fed either the WT diet or a high calcium and lactose rescue diet that is required to

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FIGURE 1 Antigen-specific CD8 T cell responses in the PBMCs of WT and Vdr2/2 C57Bl/6 mice after infection with LCMV. (A) Absolute numbers of DbGP33-specific, DbNP396-specific, DbGP276-specific, and CD44hi CD8 T cells in PBMCs longitudinally over the course of LCMV infection. (B) Histogram plots of granzyme B expression in antigen-specific CD8 T cells at 8 d postinfection. The numbers within histograms and bar graphs show the MFI of granzyme B expression. (C) Flow cytometry plots of CD127, KLRG-1, and CD62L expression in DbGP33-specific CD8 T cells 8 d postinfection. Data are from 1 representative experiment of 3 independent repeats. Plotted values are means 6 SEMs for WT (n = 3–5) and Vdr2/2 (n = 3–5) mice. An unpaired t test was used to compare differences in means between WT and Vdr2/2 groups. Significantly different from WT: *P # 0.05, **P # 0.01. CD, cluster of differentiation; GP, glycoprotein; KLRG-1, killer cell lectin-like receptor G1; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; NP, nucleoprotein; PBMC, peripheral blood mononuclear cell; Vdr, vitamin D receptor; WT, wild-type.

prevent bone disorders related to hypocalcemia in the absence of Vdr both showed increased expansion (P = 0.02 and 0.02 compared with WT, respectively) (Supplemental Figure 3A), enhanced contraction (P # 0.09) (Supplemental Figure 3B), lower GzmB expression (Supplemental Figure 3C), and decreased Bcl-2 expression (P # 0.05; data not shown). Thus, together these data demonstrate that irrespective of calcium/ lactose amounts in the diet, 1,25(OH)2D signaling deficiency leads to aberrant effector differentiation and increased death of antigen-specific CTLs through decreased expression of prosurvival protein Bcl-2. Limited breadth of memory CD8 T cell pool in the absence of 1,25(OH)2 vitamin D signals. Differences in the antigenspecific effector CD8 T cell repertoire, the effector differentiation status, and the extent of contraction under conditions of differential 1,25(OH)2D signaling prompted us to analyze the resulting memory CD8 T cell pool. As done for effector responses, we used a panel of 7 LCMV epitope peptides to conduct a detailed epitope-specific analysis of the diversity of the memory pool generated in WTor Vdrdeficient mice after LCMV infection. Consistent with increased contraction observed in the absence of 1,25(OH)2D signals (Figure 3), we found that the dominant epitope specificities GP33, NP396, and GP276 underwent enhanced death but were still detectable in the memory pool at 150 d postinfection (Figure 4A). As observed at the peak of effector responses, a lower SimpsonÕs diversity index indicated that the subdominant epitopes remained underrepresented in the memory population of Vdr2/2 mice (Figure 4A). We also assessed the phenotypic and functional properties of memory cells generated in the absence of 1,25(OH)2D signals. In

contrast to an evidently reduced diversity of the memory CD8 T cell pool, the polyfunctionality of memory cells generated in the absence of 1,25(OH)2D signals was largely unaffected (Figure 4B). Largely similar amounts of IL-2 coproduction in Vdr2/2 and WT mice were also reflected in similar proportions of central (CD127+KLRG-12CD62L+) and effector memory (CD127+KLRG-1+CD62L2) CD8 T cell subsets (Figure 4C)—2 broad classifications of memory subsets (46)—on the basis of their differential abilities to produce IL-2 and migrate to lymph nodes vs. peripheral sites. Nonetheless, memory CD8 T cells exhibited preferential localization in the lymph nodes of Vdr2/2 mice (Supplemental Figure 4B). Collectively, these data demonstrate that in addition to promoting the development of a robust and broad effector CD8 T cell pool and memory cell survival, 1,25(OH)2D signals also exert an important role in promoting the development of a broad memory CTL repertoire that is appropriately distributed in both lymphoid and nonlymphoid sites. 1,25(OH)2 vitamin D signals promote CTL responses in a CD8 T cell–extrinsic and –intrinsic manner during priming. We next sought to gain insight into the mechanism by which vitamin D signals regulate pathogen-specific effector and memory T cell responses. Previous studies established that both effector and memory properties are ‘‘programmed’’ during the priming and expansion phases of T cell responses (24, 40). The extent of contraction is also programmed during early stages of expansion (24, 47). Thus, we next compared early stages of WT CD8 T cell priming and activation in WT and Vdr2/2 mice. Analysis of WT donor P14 cells at 2.5 d after infection in WT Vitamin D regulation of CTL immunity to pathogens

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FIGURE 2 Direct ex vivo cytokine production in splenic CD8 T cells of WT and Vdr2/2 mice infected with LCMV and killed 8 d postinfection. Splenocytes were stimulated with the indicated peptides to measure intracellular IFN-g, TNF-a, and IL-2 production. (A) Representative flow cytometry plots of percentages of IFN-g+ cells gated on CD8+ T cells. SimpsonÕs diversity index was calculated as an unbiased indicator of the breadth of the CD8 T cell immune response. (B) IFN-g+ CD8 T cells were analyzed. Percentages of TNF-a+ of IFN-g+, percentages of IL-2+ of IFN-g+, and MFI of IFN-g+ CD8 T cells are shown as bar graphs. Plotted values are means 6 SEMs for WT (n = 3–5) and Vdr2/2 (n = 3–5) mice. Data are from 1 representative experiment of at least 3 independent repeats. An unpaired t test was used to compare differences in means between WT and Vdr2/2 groups. *Significantly different from WT, P # 0.05. CD, cluster of differentiation; GP, glycoprotein; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; NP, nucleoprotein; Vdr, vitamin D receptor; WT, wild-type.

and Vdr2/2 recipients showed significantly increased retention of WT P14 donor cells in the inguinal lymph nodes of Vdr2/2 mice than in spleen or liver (Figure 5A). Preferential localization of P14 donor cells in the lymph nodes was associated with increased expression of CD69 in Vdr2/2 mice (Figure 5A), which typically suppresses the expression of receptor S1P1 necessary for egress of activated T cells from the lymph nodes (48). Notably, antigenspecific CD8 T cells preferentially localized in the lymph nodes early during infection and also throughout later effector (8 d) and memory (150 d) stages (Supplemental Figure 4), despite similar cell surface expression of the lymph node homing molecule CD62L (Figures 1C and 4C). These observations of altered localization indicated dysregulated priming and altered ‘‘programming’’ of antigen-specific CD8 T cells in Vdr2/2 mice. Forward scatter is a measure of cell size, which increases upon T cell activation to sustain rapid cell division associated with logarithmic CTL expansion. Further indications of suboptimal priming in the absence of vitamin D signals were reflected in the decreased blast size of P14 donor cells in Vdr2/2 mice along with modestly decreased expression of the effector molecule GzmB as early as 2.5 d after infection (Figure 5A). The expression of IL-2 receptor a [IL2Ra (CD25)] is increased early after TCR stimulation and drives GzmB expression (30, 43). However, lesser GzmB upregulation in the absence of 1,25(OH)2D signals occurred independently of CD25 expression (Figure 5A). In vivo experiments presented in Figure 5A suggested that vitamin D signals are required in a CD8 T cell–extrinsic manner for optimal priming. We confirmed this by using a minimalistic in vitro system where WT P14 cells were stimulated with either WT or Vdr2/2 antigen-presenting cells (APCs) loaded with GP33 peptide. Smaller blast size (as indicated by lower forward scatter) 2078

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of WT CD8 T cells upon in vitro stimulation with Vdr2/2 APCs (Figure 5B) compared with WT APCs is consistent with our in vivo observations of reduced blast size and dysregulated CD8 T cell activation in the Vdr2/2 environment and further supports a CD8 T cell–extrinsic role of 1,25(OH)2D signals during priming. We also investigated whether direct expression of Vdr on antigen-specific CD8 T cells was required for optimal activation. For this, we used the approach of directly stimulating naive WT or Vdr2/2 CD8 T cells by using anti-CD3 and anti-CD28 antibodies, thus bypassing the requirement of APCs. The smaller blast size of Vdr2/2 CD8 T cells upon direct stimulation with anti-CD3/CD28 compared with WT CD8 T cells (Figure 5C) demonstrates a CD8 T cell–intrinsic role of Vdr during priming as well. Together, these data establish CD8 T cell extrinsic and intrinsic requirements of 1,25(OH)2D signals in driving optimal effector T cell priming, localization, and programming of robust, broadly reactive T cell immunity.

Discussion Understanding the mechanisms regulating CD8 T cell differentiation is an area of active investigation. In this study, we performed a comprehensive analysis of pathogen-specific effector and memory CD8 T cell responses to the intracellular viral and bacterial pathogens LCMV and LM in the presence or absence of 1,25(OH)2D signals. Our data demonstrate that 1,25(OH)2D signals serve to regulate antigen-specific effector and memory CD8 T cell size, repertoire, and survival. Mechanistically, 1,25(OH)2D signaling is needed in both CD8 T cells and non-CD8 immune cells to promote optimal effector and memory T cell programming during early stages of T cell activation. The CD8 T cell–intrinsic role of 1,25(OH)2D signals

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FIGURE 3 Expansion and contraction of antigen-specific CD8 T cell responses in the PBMCs of Vdr2/2 and WT C57Bl/6 mice infected with LCMV. (A) Representative flow cytometry plots of DbGP33-specific, DbNP396-specific, and DbGP276-specific CD8+ T cell frequencies 8 and 36 d postinfection and the percentage of contraction (decrease due to death) of antigen-specific CD8 T cells between those 2 time points. (B) Histogram and bar graph of Bcl-2 expression in antigen-specific CD8 T cells at 13 d postinfection. The numbers within the histograms and bar graphs show MFIs of Bcl-2 expression. Plotted values are means 6 SEMs for WT (n = 3–5) and Vdr2/2 (n = 3–5) mice. Data are from 1 representative experiment of 4 independent repeats. An unpaired t test was used to compare differences in means between WT and Vdr2/2 groups. Significantly different from WT: *P # 0.05, **P # 0.01. Bcl-2, B cell lymphoma 2; CD, cluster of differentiation; GP, glycoprotein; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; NP, nucleoprotein; PBMC, peripheral blood mononuclear cell; Vdr, vitamin D receptor; WT, wild-type.

during priming is consistent with Vdr upregulation in human T cells upon activation (18). These studies represent the first systematic analysis, to our knowledge, of how infection-driven CD8 T cell differentiation events are regulated by 1,25(OH)2D signaling by using the wellestablished Vdr gene ablation murine model of infection with prototypic pathogens. Vdr2/2 mice were used as the appropriate murine model of vitamin D deficiency because they are unable to transmit vitamin D signals and manifest all clinical symptoms of human vitamin D deficiency such as rickets, osteomalacia, hypocalcemia, and hyperparathyroidism (2). In addition to clearly establishing a link between vitamin D deficiency and CD8 T cell responses to infections, the Vdr2/2 mice also permitted a careful dissection of CD8 T cell–intrinsic and –extrinsic requirement of 1,25(OH)2D signaling in CTL regulation. These studies pave the way for future investigations into when and how intracrine, paracrine, and endocrine 1,25(OH)2D signals contribute to the regulation of CD8 T cell responses and how varying levels of vitamin D signaling (from deficiency to

nonrachitic insufficiency or dietary supplementation) may regulate immunologic outcome. Because biological effects of 1,25(OH)2D are predominantly mediated through Vdr, results similar to those obtained with Vdr2/2 mice are expected with a WT model of dietary vitamin D deficiency. This is an area of direct public health relevance, considering that vitamin D insufficiency is widespread in the human population (3, 4). Vitamin D is imbibed from 1) food sources (e.g., fatty fish, mushrooms, fortified cereal, and dairy products, etc.), 2) supplements, and 3) sunlight. Although massive public health initiatives of milk fortification have largely led to the disappearance of severe rachitic deficiency, there are large variations in population vitamin D concentrations due to inadequate dietary intake (largely vegetarian/vegan diets), suboptimal exposure to UV-B rays, genetic factors, age, obesity, and other factors (49). With respect to immune cells, one can speculate that local 1,25(OH)2D concentrations may be more severely reduced under conditions of low serum 25(OH)D because of combined reduction in localized production of Vitamin D regulation of CTL immunity to pathogens

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FIGURE 4 Direct ex vivo cytokine production and memory phenotype of antigen-specific CD8 T cells in the splenocytes of WT and Vdr2/2 mice infected with LCMV and killed at 150 d postinfection. (A) Representative flow cytometry plots of percentages of IFN-g+ cells gated on CD8+ T cells. SimpsonÕs diversity index was calculated as an unbiased indicator of the breadth of CD8 T cell immune response. (B) IFN-g+ CD8 T cells were analyzed. Percentages of TNF-a+ of IFN-g+, percentages of IL-2+ of IFN-g+, and MFI of IFN-g+ CD8 T cells are shown as bar graphs. Plotted values are means 6 SEMs for WT (n = 3–5) and Vdr2/2 (n = 3–5) mice. (C) Flow cytometry plots of CD127, KLRG-1, and CD62L expression in DbGP33-specific CD8 T cells from LCMV-infected mice at 150 d postinfection. Data are from 1 representative experiment of at least 3 independent repeats performed at the memory time point (150 d) postinfection. An unpaired t test was used to compare differences in means between WT and Vdr2/2 groups. *Significantly different from WT, P # 0.05. CD, cluster of differentiation; GP, glycoprotein; KLRG-1, killer cell lectin-like receptor G1; KO, knockout; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; NP, nucleoprotein; Vdr, vitamin D receptor; WT, wild-type.

1,25(OH)2D by multiple immune cell types. Moreover, moderate defects in immune cell function under conditions of vitamin D insufficiency may cumulatively increase with increasing duration of inadequate vitamin D status. Combined with the diverse vitamin D states and clinical associations (16, 17) as well as mouse studies of vitamin D deficiency or supplementation that established an association between inadequate vitamin D and increased risk/severity of several T cell–dependent diseases (14, 50, 51), our studies support a positive outcome of vitamin D supplementation during infection or immunization. With respect to CD8 T cell–extrinsic targets, on the basis of the critical role of dendritic cells (DCs) in priming of CD8 T cells, one can speculate that 1,25(OH)2D may drive the full maturation and antigen presentation function of DCs. Alternatively, appropriate vitamin D signaling may drive DCs to produce the complete array of cytokines and/or chemokines for optimal CTL responses. Vitamin D signals repress transcription of the Il2 gene (52), a key cytokine for T cell expansion and GzmB expression (30, 43, 53). However, our data demonstrating similar amounts of IL-2 production by WT and Vdr2/2 CD8 T cells suggest that, during an active infection, IL-2 concentrations are likely regulated independently of 1,25(OH)2D and other vitamin D–dependent cytokines may be responsible for optimally driving GzmB expression. Our findings of increased 2080

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lymph node localization of antigen-specific CD8 T cells are also suggestive of altered expression of chemokines and chemokine receptors in the absence of 1,25(OH)2D. Increased expression of CD69 suggests a possible regulation of CD69-mediated suppression of S1P1 receptor expression by 1,25(OH)2D to inhibit lymph node egress during early stages of activation. Lack of alterations in the lymph node homing molecule CD62L in the absence of 1,25(OH)2D signals implicates alternate molecular targets such as CCR7 in promoting lymph node localization during later stages of CTL development and merit further investigation. In summary, this comprehensive analysis of CTL immunity to a model acute viral infection uncovered a critical role of vitamin D signals in priming robust effector and memory responses and has laid the foundation for future mechanistic investigations into this relatively unexplored area.

Acknowledgments YY and FMB carried out the experiments, analyzed the data, performed the statistical analysis, prepared the figures, and helped with the manuscript preparation; LAP conducted the experiments and analyzed the data; GPS helped with the data interpretation and manuscript preparation; VK and SS conceptualized the project, designed the experiments, carried out the experiments, supervised the work, analyzed the data, interpreted

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FIGURE 5 In vivo and in vitro priming of CD8 T cells from WT and Vdr2/2 mice. (A) In vivo priming of 106 DbGP33-specific P14 cells in WT and Vdr2/2 mice infected with LCMV and killed at 2.5 d postinfection. Histogram plots for forward scatter, CD25, GzymB, and CD69 expression in donor DbGP33-specific P14 CD8 T cells isolated from spleen are shown; numbers within the histograms represent the MFI of the marker expression. Percentage localizations of donor D bGP33-specific P14 CD8 T cells in lymphoid and nonlymphoid tissues are plotted in bar graphs. (B) WT DbGP33-specific responder P14 CD8 T cells were in vitro stimulated with WT or Vdr2/2 splenocytes loaded with GP33–41 peptide. (C) WT and Vdr2/2 CD8 T cells were in vitro stimulated with plate-bound aCD3 and aCD28. (B, C) Histogram plots of FSC with and without stimulation at day 2 after in vitro stimulation are shown; numbers within histograms represent MFI of FSC. A probability binning algorithm in FlowJo software was used to determine the T(x) metric for estimating the significance of the difference between WT and Vdr2/2 histogram populations. Data are from 1 representative experiment of 2–3 independent repeats. Plotted values are means 6 SEMs for WT (n = 3–5) and Vdr2/2 (n = 3–5) mice. An unpaired t test was used to compare differences in means between WT and Vdr2/2 groups. Significantly different from WT: *P # 0.05, **P # 0.01. CD, cluster of differentiation; FSC, forward scatter; GP, glycoprotein; GzmB, granzyme B; iLN, inguinal lymph node; KO, knockout; LCMV, lymphocytic choriomeningitis virus; LVR, liver; MFI, mean fluorescence intensity; SPL, spleen; Vdr, vitamin D receptor; WT, wild-type.

the results, and wrote the manuscript; and SS has primary responsibility for the final content. All authors read and approved the final manuscript.

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Vitamin D receptor signals regulate effector and memory CD8 T cell responses to infections in mice.

Vitamin D insufficiency is associated with broad-ranging human disease sequelae such as bone disease, cancer, cardiovascular disease, allergy, autoimm...
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