CD8 T cells in old mice contribute to the innate immune response to Mycobacterium

Transcription

1 IAI Accepts, published online ahead of print on 26 May 2009 Infect. Immun. doi: /iai Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 2 CD8 T cells in old mice contribute to the innate immune response to Mycobacterium tuberculosis via IL-12p70 dependent and antigen independent production of IFN-γ. 3 4 Running Title: Innate IFN-γ production by CD8 T cells from old mice Bridget Vesosky 1, Erin K. Rottinghaus 1, Craig Davis 1, and Joanne Turner 1, 2 * 1 Center for Microbial Interface Biology, and 2 Division of Infectious Disease, Department of Internal Medicine, The Ohio State University, Columbus, Ohio *Corresponding author: Mailing address: The Ohio State University, BRT 1010, 460 West 12 th Avenue, Columbus, OH, Phone: (614) Fax: (614) joanne.turner@osumc.edu

2 ABSTRACT Elderly individuals have increased morbidity and mortality associated with infectious diseases, due in part to the progressive age associated decline in immune function. Despite this, the old mouse model of Mtb infection has revealed a CD8 and IFN-γ dependent early resistance to infection. In this study we investigated the mechanism by which CD8 T cells from old mice contributed to the early immune response to Mtb. Following a low dose aerosol infection with Mtb, CD8 T cells were identified as a dominant source of IFN-γ expression in the lungs of old mice early after infection, before the typical onset of antigen specific immunity. In addition, Mtb induced IFN-γ production by CD8 T cells isolated from naïve old mice was MHC Class I independent but dependent on IL-12p70, confirming an innate role of CD8 T cells during Mtb infection. Moreover, the ability of CD8 T cells from old mice to produce increased innate IFN-γ in response to Mtb infection was defined as a unique function of CD8 T cells from old mice and not the aged lung environment. Finally, we have identified increased expression of SET as one possible mechanism by which CD8 T cells from old mice produce enhanced IFN-γ. Additional characterization of the signaling events that lead to enhanced innate IFN-γ production by CD8 T cells in old mice may lead to novel strategies to further enhance or perpetuate beneficial immune responses in the elderly.

3 46 INTRODUCTION The world s elderly population is rapidly expanding and is predicted to reach 1.5 billion by the year 2050, a 28% increase since 2000 (35). The largest concentration of elderly individuals (projected to be 78% by the year 2050) is in developing countries (35) where many infectious diseases, including tuberculosis (23), are endemic. Given the increased susceptibility of the elderly to infectious diseases, this rapid rise in the elderly population poses a significant threat to global health care. Research focused on characterizing the immune response of the elderly to pathogens that cause substantial morbidity and mortality in elderly individuals is an area that has been significantly neglected however, such studies would have a considerable impact on the prevention and treatment of infectious diseases in the elderly. As an individual ages, significant immunological changes occur that contribute to the enhanced morbidity and mortality associated with infectious diseases in the elderly. After puberty, thymic atrophy leads to a progressive decrease in the output of naïve T cells and decreased diversity in the T cell repertoire (8). Consequently, the periphery of an elderly individual is dominated by antigen experienced or memory T cells, leading to a significant impairment of immune responses to new antigenic challenges (37). In addition to the immune defects in the T cell compartment, significant deficiencies have been reported in B cells (28), NK cells (21, 22), macrophages (15, 20), and dendritic cells (19, 29). Despite the abundant evidence that the immune system of the aged is significantly altered, there is mounting data suggesting that some components of the

4 immune system, particularly CD8 T cells, remain functionally intact or are enhanced in old age (11, 27, 36). The characterization of immune responses in the elderly, specifically to pathogens, may lead to more effective vaccination and therapeutic strategies in this highly vulnerable and expanding population We have previously demonstrated that although old mice fail to contain a chronic infection with Mycobacterium tuberculosis (Mtb) (24, 33), during the first two weeks following infection old mice display an enhanced resistance to infection that is IFN-γ and CD8 dependent (33, 34). In addition, characterization of in vitro responses of naïve CD8 T cells from old mice to Th1 cytokines identified an IL-12 driven mechanism of enhanced IFN-γ production by these cells (36). Since IL-12 is detected early in the lungs of Mtb infected old mice (36), we hypothesized that this same mechanism of innate IFN-γ production was also biologically relevant during Mtb infection. The aim of this present study was to define and characterize the mechanism by which CD8 T cells contribute to innate immune responses following Mtb infection. Using both in vivo and in vitro models of Mtb infection in old mice we demonstrated that CD8 T cells were a major source of innate IFN-γ. In addition, in vitro assays using purified pulmonary CD8 and CD11c cells from naïve mice conclusively demonstrated that Mtb driven IFN-γ production by CD8 T cells from old mice was dependent on IL-12p70 and independent of MHC/TCR interactions. Finally, we provide evidence that suggests that one mechanism by which CD8 T cells from old mice are capable of enhanced innate IFNγ production is through the regulation of IL-12 signaling.

5 92 MATERIAL AND METHODS Mice. Specific-pathogen free female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA) or through a contract with the National Institute on Aging (Supplied by Charles River Laboratories). Young mice were 2-3 mo of age and old mice were mo of age. 2-6 mo old IL-12p35 -/-, β 2 m -/-, and C57BL/6 wild-type (WT) control female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Repeat experiments were performed using young and old BALB/c mice in some studies with similar results. Cell isolation. Lungs were perfused through the pulmonary artery with phosphatebuffered saline (PBS) containing 50U/ml of heparin (Sigma, St. Louis, MO) and placed into Dulbecco s modified Eagle s medium (DMEM) (500ml; Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (Atlas Biologicals, Ft. Collins, CO), 1% HEPES buffer (1M; Sigma), 1% L-glutamine (200nM; Sigma), 10ml of a 100X MEM nonessential amino acid solution (Sigma), 5ml of a penicillin/streptomycin solution (50,000 U penicillin and 50mg streptomycin; Sigma), and 0.1% β- mercaptoethanol 2-hydroxyethylmercaptan (50mM; Sigma)(complete DMEM). A single cell suspension was obtained using enzymatic digestion as described previously (33). Viable cells were counted using trypan blue exclusion and resuspended at working concentrations in complete DMEM. Single cell suspensions of splenocytes were obtained by pressing individual spleens through a sterile 70µM nylon mesh screen, followed by RBC lysis with ACK lysis buffer (0.15 M NH 4 Cl, 1 mm KHCO 3 ) for 3 minutes. Cells

6 were washed in complete DMEM, counted using trypan blue exclusion and resuspended at working concentrations in complete DMEM Cell purifications. For CD8 +, NK, and CD11c + cell purification, single cell suspensions from individual mice were resuspended in complete DMEM and cultured for 1 hour in 100mm tissue culture dishes (BD Biosciences) at 37 C with 5% CO 2 to allow cell adherence. For CD8 + and NK cell purification, non adherent cells were collected by vigorous pipeting, counted using trypan blue exclusion, and centrifuged. Cell pellets were incubated with 50µl of CD8 DM beads (BD Biosciences) per 10 7 cells for 30 minutes at 4 C. For NK cells, cells were incubated with 50µl of anti-dx5 antibody per 10 7 cells for 15 minutes at 4 C, washed, and incubated with 50µl of anti-pe beads per 10 7 cells for 30 minutes at 4 C. Cells were resuspended in 1ml of complete DMEM and purified on the BD IMagnet per manufacturer s instructions (BD Biosciences). Purified cells were counted using trypan blue exclusion and adjusted to working concentrations in complete DMEM. CD8 + and NK cells were assessed for purity by flow cytometry and typically exceeded 95%. For CD11c + cell purification, adherent cells were washed with warm PBS twice, and incubated with trypsin-edta (Sigma) for 10 minutes at 37 C. Adherent cells were collected, washed in complete DMEM, and counted using trypan blue exclusion. Cells were incubated with 2.5µg of CD11c + biotin (BD Biosciences) for 15 minutes at 4 C and washed in complete DMEM. 50µl of streptavidin beads (BD Biosciences) per 10 7 cells were incubated with the cells for 30 minutes at 4 C and resuspended in 1ml of complete DMEM. CD11c + cells were purified on a BD IMagnet according to the manufacturer s instructions. Purified cells were counted using trypan

7 blue exclusion and adjusted to working concentrations in complete DMEM. Purity was assessed by flow cytometry and was typically greater than 90% In vitro cell culture. Single cell suspensions obtained from whole lungs were cultured in complete DMEM without antibiotics either alone or with Mtb Erdman (MOI 0.5) for hours (RT-PCR) or 48 hours (IFN-γ ELISA). For overlay cultures, CD11c + cells were cultured along with Mtb Erdman and CD8 + cells (1:1:1 ratio of CD8:CD11c:Mtb) for 48 hours. For IL-12 neutralization, anti-il-12 (clone C17.8; BioXCell, West Lebanon, NH) or rat IgG2A isotype control was added at 1µg/ml. Antibody concentrations were optimized previously. For antigen independent assays, CD11c + cells were cultured either alone or with Mtb Erdman (MOI 1) for 72 hours at 37 C with 5% CO 2. Culture supernatants were frozen at -80 C. Prior to use, cell supernatants were thawed and filtered with a 0.2µm microfuge filter (Corning, Lowell, MA). CD8 + cells were cultured with 150µl of filtered CD11c + supernatant for 48 hours at 37 C with 5% CO 2. To assess the induction of IFN-γ production by p35 and p40 containing cytokines, CD8 + cells purified from the spleens of naïve old mice were cultured with media, IL-12p70 (5ng/ml; PeproTech, Rocky Hill, NJ), IL-23 (20ng/ml; R&D Systems, Minneapolis, MN), IL-12p40 (20ng/ml; PeproTech), or IL-12p40p40 (20ng/ml; R&D Systems) for 48 hours. All cultures also contained IL-18 (20ng/ml; R&D Systems) to amplify the amount of detectable IFN-γ. IL-18 alone does not drive IFN-γ production in old CD8 T cells.

8 To assess SET expression levels in vitro, CD8 + cells purified from the spleen were cultured either alone or with a combination of IL-2 (100ng/ml; R&D Systems), IL-12p70 (5ng/ml; PeproTech), and IL-18 (10ng/ml, R&D Systems) for 1, 4, and 7 hours For flow cytometric analysis, CD8 + cells purified from the lungs of individual mice were cultured in 40µM 1,9 dideoxyforskolin (Calbiochem, Gibbstown, NJ) or DMSO control for 30 minutes and then with a combination of IL-2 (100ng/ml), IL-12p70 (5ng/ml), and IL-18 (10ng/ml) or complete DMEM for 4 hours. In vivo bacterial infections. Mtb Erdman (ATCC 35801) was obtained from American Type Culture Collection (Manassas, VA) and grown in Proskauer-Beck liquid medium containing 0.05% Tween 80 (Sigma) to mid-log phase. Bacterial suspensions were frozen in aliquots at -80 C. Mice were aerogenically infected with a low dose of Mtb Erdman ( CFU). Lungs were harvested for CD8 + and NK cell purification at 0, 8, and 12 days post infection. Real-time PCR. Purified CD8 + and NK cells were homogenized in 1ml Trizol (Invitrogen, Carlsbad, CA) and frozen at -80 C. RNA was isolated by chloroform extraction and isopropanol precipitation. RNA was further purified using an RNeasy mini kit (Qiagen, Valencia, CA). RNA was then reverse transcribed with Omniscript RT (Qiagen) and cdna amplified by real-time PCR using a BioRad icycler (Hercules, CA). Taqman gene expression assays for IFN-γ and SET were used for all real-time PCR assays (Applied Biosystems, Foster City, CA). 18s ribosomal RNA gene was used as an

9 endogenous normalizer and the Delta Delta cycle threshold method was used for relative quantification of mrna expression Flow Cytometry. BD Phosflow Protocol III for mouse splenocytes or thymocytes was used to measure intracellular pstat4 and IFN-γ in CD8 T cells from the lungs of individual mice. Lung cells were concomitantly stained with µg IFN-γ PE-Cy7 (Clone XMG1.2), pstat4 Tyr693 Alexa fluor 488 (Clone 38/pSTAT4), CD3ε PE (Clone 145-2C11), and CD8 Alexa Fluor 647 (Clone ) or isotype control antibodies (BD Biosciences). Samples were run on a LSRII flow cytometer and data analyzed using FACSDiva software (BD Biosciences). Lymphocytes were gated based on their characteristic forward and side scatter profiles. CD8 T cells were subsequently identified by the presence of specific fluorescently labeled antibodies to CD8 in combination with CD3ε. IFN-γ and pstat4 were measured within this population of CD8 T cells and quadrant gates set according to staining of isotype control antibodies. ELISA. 48 hour cell culture supernatants were thawed and the concentration of IFN-γ protein measured by enzyme-linked immunosorbent assay (ELISA). Purified anti-ifn-γ (Clone R4-6A2) and biotin anti-ifn-γ (Clone XMG1.2) were used for capture and detection antibodies respectively (BD Biosciences). Statistical analysis. Statistical significance was determined using Prism 4 software (GraphPad Software, San Diego, CA). The unpaired two-tailed Student s t test was used

10 for comparison of young and old mice and the One-way ANOVA with Tukey s post test was used for multigroup comparisons

11 RESULTS Mtb infection drives IFN-γ expression in pulmonary CD8 T cells in old mice During Mtb infection, old mice express an elevated level of IFN-γ mrna throughout the first 8-12 days (36) and we have hypothesized that resident CD8 T cells within the aged lung are the source of IFN-γ. To determine the in vivo cellular source of IFN-γ within the lungs of old mice during Mtb infection, young and old mice were infected with a low dose aerosol of Mtb and at various time points post infection, CD8 T cells were purified from the lungs and IFN-γ mrna was quantified using RT-PCR directly ex vivo, without further stimulation. Baseline IFN-γ mrna within purified CD8 T cells was increased in naïve old mice relative to young (day 0) suggesting that these cells may be primed to rapidly synthesize IFN-γ (Figure 1A). In response to Mtb infection, old mice had an additional and significant increase in IFN-γ mrna within CD8 T cells as infection progressed (Figure 1A). In contrast, pulmonary CD8 T cells from young mice did not generate IFN-γ mrna in response to Mtb infection throughout the 12 day period studied. Therefore, CD8 T cells within the lungs of old mice have the capacity to synthesize IFNγ in response to infection with Mtb. NK cells, like CD8 T cells from old mice, can also be an early source of IFN-γ in response to infection (10, 13), and therefore we purified NK cells from the lungs of old and young mice during early Mtb infection and determined IFN-γ mrna levels within this specific cell subset. In contrast to CD8 T cells, there was no significant increase in the levels of IFN-γ mrna within NK cells from the lungs of old mice in response to Mtb

12 infection (Figure 1B). NK cells purified from the lungs of Mtb infected young mice expressed a significant increase in IFN-γ mrna levels after 8 days of infection, which dissipated by day 12, indicating an early functional role for NK cells in the lungs of young mice. We therefore formally demonstrate that CD8 T cells are an early source of IFN-γ mrna in the lungs of old mice during Mtb infection and also show that the dominant cellular source of innate IFN-γ during Mtb infection in vivo may differ between young and old mice. Pulmonary CD8 T cells isolated from naïve old mice produce innate IFN-γ following in vitro stimulation with Mtb To determine the mechanism by which CD8 T cell derived IFN-γ from old mice contributed to early immune responses to Mtb infection, we established an in vitro infection model. Initially, to demonstrate that lung cells from old mice produced innate IFN-γ when cultured in vitro with Mtb, lung cells were isolated from naïve young and old mice and stimulated in vitro with Mtb for 48 hours. Following in vitro Mtb stimulation, lung cells from old mice produced significantly more IFN-γ protein than their young counterparts (Figure 2A). To then demonstrate that CD8 T cells were the source of innate IFN-γ in the whole lung cultures, lung cells were isolated from naïve young and old mice and cultured with or without Mtb for hours. In order to obtain sufficient numbers of cells for RT-PCR, lung cells were pooled by age. CD8 T cells were purified and IFN-γ mrna quantified using RT-PCR. After 24 hours of stimulation with Mtb, the expression of IFN-γ mrna in CD8 T cells isolated from lung cell cultures of old mice

13 was 100 fold higher than non-stimulated CD8 T cells and, as predicted from our in vivo infections, substantially higher than the expression in young CD8 T cells (Figure 2B). As in our in vivo model, NK cells purified from lung cell cultures of young mice had higher expression levels of IFN-γ mrna in response to in vitro stimulation with Mtb when compared to NK cells isolated from the lungs of old mice (Figure 2C). Taken together, these data confirm that, in old mice, CD8 T cells are an early and significant source of innate IFN-γ. Furthermore, we have validated an in vitro model that reliably reproduces our in vivo findings allowing for the elucidation of the mechanism leading to CD8 T cell derived IFN-γ production in old mice. Increased IFN-γ production is an intrinsic property of CD8 T cells from old mice To determine the role of the aged lung microenvironment in driving CD8 T cell IFN-γ secretion we developed an in vitro overlay assay using CD11c cells isolated from the lungs of naïve young and old mice infected with Mtb in vitro. CD8 and CD11c cells were sequentially purified from the lungs of naïve young and old mice. Due to the limited number of both CD8 and CD11c cells obtained from the lungs of naïve mice, cells from individual mice were pooled by age. CD11c cells were infected with Mtb and incubated with purified CD8 cells for 48 hours. Initial experiments cultured young CD8 T cells with young CD11c cells and old CD8 T cells with old CD11c cells (Figure 3A). IFN-γ was measured in the cell culture supernatant by ELISA. Figure 3A shows that CD8 T cells purified from the lungs of naïve young mice produced very little IFN-γ in response to young Mtb infected CD11c cells. In contrast, IFN-γ production was abundant from CD8

14 T cells isolated from the lungs of naïve old mice following stimulation with Mtb infected CD11c cells from old mice. These data provide evidence that stimulation with Mtb infected CD11c cells is sufficient to drive IFN-γ production by CD8 T cells from old mice To elucidate whether the capacity of CD8 T cells to secrete IFN-γ was a specific property of CD8 T cells from old mice we established culture conditions in which the source of CD11c cells was from young or old mice. As anticipated, CD8 T cells from young mice failed to produce substantial quantities of IFN-γ in response to Mtb infected CD11c cells from young or old mice (Figure 3B). In contrast, CD8 T cells from old mice were fully capable of secreting IFN-γ in response to Mtb infected CD11c cells from either old or young mice (Figure 3B). Therefore, the increased capacity of CD8 T cells from old mice to secrete IFN-γ in response to Mtb infection is a consequence of altered CD8 T cell function in old age and not altered macrophage function. Mtb induced IFN-γ production by CD8 T cells is antigen independent The capacity of CD8 T cells isolated from the lungs of non-infected old mice to produce IFN-γ in response to Mtb stimulation (Figure 3) suggests that CD8 T cells from old mice produced IFN-γ in an antigen independent manner. To verify this, supernatants were collected from Mtb infected CD11c cells, filtered through a 0.2µm filter to remove any cell associated particulates, and cultured with purified CD8 T cells from young or old mice. CD8 T cells purified from the lungs of naïve old mice produced IFN-γ when

15 stimulated with filtered supernatant derived from Mtb infected CD11c cells from old mice (Figure 4A), indicating that a soluble product was sufficient to stimulate CD8 T cells from old mice to secrete IFN-γ. As expected, CD8 T cells purified from the lungs of naïve young mice did not produce IFN-γ in response to filtered supernatants from Mtb infected CD11c cells (Figure 4A). To conclusively demonstrate that antigen presentation was not necessary for Mtb induced IFN-γ production by CD8 T cells from the lungs of naïve old mice, β 2 m knockout mice were used as the source of CD11c cells. β2m is an essential component of MHC Class I molecules and therefore, mice with homozygous disruptions in the β2m gene lack functional MHC Class I on the cell surface (14). Figure 4B shows that pulmonary CD8 T cells isolated from naïve old mice cultured with Mtb infected CD11c cells from young β 2 m knockout mice were fully capable of producing IFN-γ. Taken together these data demonstrate that Mtb induced IFN-γ production by CD8 T cells from old mice is an antigen independent event, mediated by a soluble factor produced by Mtb infected CD11c cells. IL-12 is essential for Mtb induced IFN-γ production by CD8 T cells CD8 T cells can be stimulated to secrete IFN-γ in the presence of IL-12, IL-18, and IL-2 (2) and, as we have shown, for old mice by IL-12 alone (26, 36). We therefore anticipated that the stimulatory soluble factor produced by Mtb infected CD11c cells would be IL-12. Lung cell suspensions were isolated from naïve young and old mice and cultured with

16 Mtb in the presence of a neutralizing antibody to IL-12p40. Lung cells from naïve old mice produced significantly more IFN-γ than their young counterparts and this was ablated with the inclusion of a neutralizing antibody to IL-12p40 (Figure 5A). Furthermore, IFN-γ production by CD8 T cells from old mice in the presence of Mtb infected CD11c cells was also ablated by a neutralizing antibody to IL-12p40 (Figure 5B). These data indicate that Mtb driven IL-12p40 production is necessary for the stimulation of CD8 T cells from old mice to secrete IFN-γ. Biologically active IL-12p70 is composed of two subunits, p40 and p35. While the p35 subunit is found only in biologically active IL-12, the p40 subunit is found as monomers, homodimers, and in biologically active IL-12 and IL-23. Therefore, in addition to IL- 12p70, the neutralizing antibody to IL-12p40 neutralizes IL-23 and also any biological activity of p40. To formally demonstrate that IL-12p70, and not any other known p35 or p40 containing cytokine, was sufficient to directly stimulate CD8 T cells from old mice to secrete IFN-γ, CD8 T cells were purified from the spleens of naïve old mice and cultured with commercially available p35 and p40 containing cytokines. We have previously demonstrated that stimulation of CD8 T cells from old mice with IL-12p70 alone induces low but detectable quantities of IFN-γ (26, 36). For these studies, IL-18 was included in all wells to amplify the quantity of IFN-γ produced by old CD8 T cells as measured by IFN-γ ELISA. IL-12p70 but not IL-23, IL-12p40, or IL-12p40p40 was sufficient to directly stimulate CD8 T cells from old mice to secrete IFN-γ (Figure 5C).

17 We confirmed that bioactive IL-12p70 was required for the stimulation of CD8 T cells from old mice to secrete IFN-γ in response to Mtb infection by using IL-12p35 knockout mice as the source of CD11c cells in our assay. CD8 T cells isolated from the lungs of naïve old mice failed to produce IFN-γ when cultured with Mtb infected CD11c cells purified from the lungs of naïve IL-12p35 knockout mice (Figure 5D). When expressed as percent inhibition, IFN-γ production by CD8 T cells isolated from old mice stimulated with Mtb infected IL-12p35 knockout CD11c cells was inhibited 82 and 100% relative to CD11c cells from young wild type controls (in two separate experiments respectively). These data conclusively demonstrate that Mtb induced innate IFN-γ production by CD8 T cells from naïve old mice is dependent on biologically active IL-12p70. Hyper responsiveness of CD8 T cells from old mice to IL-12p70 is associated with elevated levels of SET, a phosphatase inhibitor We have shown here that the capacity of CD8 T cells to secrete IFN-γ in response to Mtb infection is due to distinct functional properties of CD8 T cells; however the molecular pathways that lead to IL-12p70 hyper reactivity in this unique subset have not been fully elucidated. Recently IFN-γ production by human NK cells was shown to be regulated by SET, an inhibitor of phosphatase PP2A (32). We therefore hypothesized that elevated SET expression is one mechanism by which CD8 T cells from old mice produce increased IFN-γ. To test this hypothesis CD8 T cells were purified from Mtb infected young and old mice at various time points post infection. RNA was isolated and SET mrna expression measured using RT-PCR. SET expression in CD8 T cells from naïve

18 old mice, and day 8 infected old mice, was elevated when compared to levels in CD8 T cells from young mice (Figure 6A). To address the role of SET in more detail we measured SET expression by RT-PCR in CD8 T cells purified from naïve young and old mice that had been stimulated with a Th1 cytokine cocktail of IL-12, IL-18, and IL-2. SET mrna levels in CD8 T cells from old mice stimulated with the Th1 cytokine cocktail peaked at 4 hours and SET mrna levels were nearly three-fold higher than stimulated young CD8 T cells at this time point (Figure 6B). This peak in SET expression correlated with peak IFN-γ mrna expression (Figure 6C). Chemically overriding SET inhibition of PP2A in CD8 T cells from old mice leads to decreased IL-12p70 signaling and IFN-γ production To demonstrate that increased SET in CD8 T cells from old mice, acting via its suppression of PP2A activity, could influence downstream IL-12 induced IFN-γ production, CD8 T cells were purified from the lungs of young and old mice and treated with 1,9 dideoxyforskolin (an activator of PP2A which overcomes SET inhibition). Cells were subsequently cultured with a Th1 cytokine cocktail and STAT4 phosphorylation, indicative of IL-12p70 signaling, in addition to IFN-γ production was determined. Treatment with 1, 9 dideoxyforskolin significantly decreased STAT4 phosphorylation (Figure 7A) and IFN-γ production (Figure 7B) by CD8 T cells from old mice. Taken together, these data suggest that elevated levels of SET may contribute to the elevated levels of IFN-γ produced by CD8 T cells from old mice in response to Mtb infection.

19 DISCUSSION Understanding the immune response of the elderly to relevant pathogens is becoming an urgent health care priority with the projected rapid expansion of this population. It is well known that numerous immunological deficiencies emerge with age, resulting in increased susceptibility of the elderly to infection. One strategy to combat the increased morbidity and mortality associated with infectious agents in the elderly is to identify ways to prevent or reverse the established immunological deficiencies (1, 9). An alternative strategy is to identify components of the aged immune system that respond positively to such infectious agents and determine mechanisms by which successful effector functions can be targeted or manipulated to improve immunological responses in the elderly. Focusing on this alternative strategy, we have defined a mechanism by which a population of pulmonary CD8 T cells in the lungs of old mice mediates early resistance to Mtb infection. Our results indicate that this population of CD8 T cells contributes to the innate immune response of old mice to Mtb infection via antigen independent and IL- 12p70 dependent production of IFN-γ. We have therefore identified a population of CD8 T cells present in old mice that make a unique and effective, albeit transient, contribution to the immune response to a relevant pathogen. Old mice and humans are indeed more susceptible to Mtb infection than their young counterparts; however, our laboratory and others have demonstrated that during the first two weeks of an aerogenic infection with Mtb old mice are more resistant to infection (6, 33, 36). IFN-γ and CD8 T cells were later identified as essential components of the early resistance phenotype in old mice (33, 34) and we now demonstrate that CD8 T cells are a

20 major source of IFN-γ expressed during the innate phase of Mtb infection. The conventional response of CD8 T cells to Mtb is mediated by Mtb specific peptides presented in the context of MHC Class I. Although conventional immune responses may dominate, there is mounting evidence that CD8 T cells are capable of contributing to innate immunity (3-5, 7, 16, 36) as well as evidence suggesting that NK cells contribute to specific acquired immune responses (30). Using Mtb infected CD11c cells isolated from the lungs of naïve β 2 m knockout mice, this study demonstrates that IFN-γ production by CD8 T cells from the lungs of naïve old mice in response to Mtb infection was not dependent on classical MHC Class I antigen presentation. To the best of our knowledge, this is the first direct demonstration of CD8 T cells from naïve mice responding to Mtb infection in an antigen independent manner. Our previous studies characterizing the phenotype and function of pulmonary CD8 T cells in old mice revealed the ability of this population of CD8 T cells to respond to Th1 cytokines by producing IFN-γ (36), here we extend those studies by demonstrating biological relevance to a respiratory pathogen. Mtb induced IFN-γ production by CD8 T cells isolated from the lungs of naïve old mice was ablated in the presence of IL-12p40 neutralizing antibody or when naïve IL-12p35 knockout mice were used as a source of CD11c cells, conclusively demonstrating that IL-12p70 was essential. Taken together, these data suggest that CD8 T cells from old mice are capable of innate IFN-γ production in response to all pathogens that induce IL-12p70. In the case of Mtb infection, this response seems to be beneficial, at least in the short term, but could be detrimental during infections with other infectious agents. In fact, recent data suggests that IFN-γ produced

21 in the lung in response to influenza infection inhibits initial bacterial clearance of secondary bacterial infections (31), which are common and a significant cause of morbidity and mortality in the elderly. This highlights the necessity of understanding the immune responses of the elderly to pathogens Given that increased IFN-γ production in response to Mtb is mediated by IL-12p70 production, two non-mutually exclusive mechanisms to explain the hyper responsive phenotype of old CD8 T cells were evaluated. First, the aged lung environment during Mtb infection could be significantly different from that of a young mouse. We have in fact shown that in vivo, the lungs of Mtb infected old mice contain significantly more IL- 12 than their young counterparts (36). Data presented in this study demonstrated that the amount of IL-12 produced by Mtb infected CD11c cells isolated from either young or old mice was sufficient to drive equivalent amounts of IFN-γ production by CD8 T cells from old mice. These data suggest that while Mtb infected lung cells from old mice may make more IL-12 than young mice, the difference is not biologically relevant and therefore the aged lung environment does not drive the hyper responsive function of these cells. It is important to highlight that although the unique features of the aged lung environment is not biologically relevant to the increased innate IFN-γ production by CD8 T cells, it is likely that the environment in an aged mouse directly contributes to the development of such cells (12, 18). Since the aged lung environment does not contribute to the increased IFN-γ production by CD8 T cells from naïve old mice in response to Mtb, the hyper responsive effector

22 function of these cells must therefore be a consequence of some unique feature or features of old CD8 T cells. Given that CD8 T cells from naïve old mice are functionally similar to NK cells in that both cell types produce innate IFN-γ in response to cytokines (25), we investigated the mechanism by which NK cells produce enhanced IFN-γ in our model. Increased expression of SET was shown to mediate enhanced IFN-γ production by human CD56 bright NK cells (32) via inhibition of PP2A activity through binding the C subunit of the phosphatase (17). Our laboratory has recently demonstrated that CD8 T cells from old mice have enhanced STAT4 phosphorylation in response to IL-12 (26), highlighting a role of altered IL-12 signaling in old CD8 T cells. In this study we demonstrated that SET expression is elevated in CD8 T cells purified from the lungs of Mtb infected old mice and in CD8 T cells stimulated with Th1 cytokines. Furthermore the in vitro expression of SET correlated with IFN-γ production in old CD8 T cells. We also provided evidence that overcoming SET inhibition of PP2A resulted in decreased IL- 12 signaling and IFN-γ production in Th1 cytokine stimulated CD8 T cells from old mice. These data suggest that one possible mechanism by which CD8 T cells from old mice produce increased IFN-γ production is via increased expression of SET. Taken together, our data presented here and in previous studies demonstrate that during the innate phase of an aerogenic Mtb infection resident pulmonary CD11c cells produce IL-12p70 in response to infection. Resident CD8 T cells have increased quantities of the IL-12β2 receptor mrna (26) and increased SET expression and are therefore uniquely able to respond to IL-12p70 by producing increased IFN-γ. Increased IFN-γ production contributes to mycobacterial control in the lung and results in decreased mycobacterial

23 burden in the lungs throughout the first 2 weeks of infection. Additional studies to understand the transient nature of resistance and to characterize the signaling events leading to enhanced IFN-γ production are currently ongoing and will contribute to the development of more effective strategies to combat infectious diseases in the elderly.

24 ACKNOWLEDGEMENTS This work was supported by National Institutes of Health grant AG21097 to JT. ER was supported by a fellowship from PHPID.osu.edu and the Targeted Investment in Excellence of the OAA, President and Research Offices of Ohio State. The authors would like to thank Dr. Rossana Trotta for insightful discussions regarding SET

25 REFERENCES 1. Aspinall, R., and W. Mitchell Reversal of age-associated thymic atrophy: treatments, delivery, and side effects. Exp Gerontol 43: Berg, R. E., C. J. Cordes, and J. Forman Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL-18. Eur J Immunol 32: Berg, R. E., E. Crossley, S. Murray, and J. Forman Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen. J Exp Med 198: Berg, R. E., E. Crossley, S. Murray, and J. Forman Relative contributions of NK and CD8 T cells to IFN-gamma mediated innate immune protection against Listeria monocytogenes. J Immunol 175: Berg, R. E., and J. Forman The role of CD8 T cells in innate immunity and in antigen non-specific protection. Curr Opin Immunol 18: Cooper, A. M., J. E. Callahan, J. P. Griffin, A. D. Roberts, and I. M. Orme Old mice are able to control low-dose aerogenic infections with Mycobacterium tuberculosis. Infect Immun 63: D'Orazio, S. E., M. J. Troese, and M. N. Starnbach Cytosolic localization of Listeria monocytogenes triggers an early IFN-gamma response by CD8+ T cells that correlates with innate resistance to infection. J Immunol 177:

26 Dominguez-Gerpe, L., and M. Rey-Mendez Evolution of the thymus size in response to physiological and random events throughout life. Microsc Res Tech 62: Dorshkind, K., E. Montecino-Rodriguez, and R. A. Signer The ageing immune system: is it ever too old to become young again? Nat Rev Immunol 9: Feng, C. G., M. Kaviratne, A. G. Rothfuchs, A. Cheever, S. Hieny, H. A. Young, T. A. Wynn, and A. Sher NK cell-derived IFN-gamma differentially regulates innate resistance and neutrophil response in T celldeficient hosts infected with Mycobacterium tuberculosis. J Immunol 177: Herndler-Brandstetter, D., E. Veel, G. T. Laschober, G. Pfister, S. Brunner, S. Walcher, W. Parson, G. Lepperdinger, and B. Grubeck-Loebenstein Non-regulatory CD8+CD45RO+CD25+ T-lymphocytes may compensate for the loss of antigen-inexperienced CD8+CD45RA+ T-cells in old age. Biol Chem 389: Jones, S. C., K. Clise-Dwyer, G. Huston, J. Dibble, S. Eaton, L. Haynes, and S. L. Swain Impact of post-thymic cellular longevity on the development of age-associated CD4+ T cell defects. J Immunol 180: Junqueira-Kipnis, A. P., A. Kipnis, A. Jamieson, M. G. Juarrero, A. Diefenbach, D. H. Raulet, J. Turner, and I. M. Orme NK cells respond to pulmonary infection with Mycobacterium tuberculosis, but play a minimal role in protection. J Immunol 171:

27 Koller, B. H., P. Marrack, J. W. Kappler, and O. Smithies Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science 248: Kong, K. F., K. Delroux, X. Wang, F. Qian, A. Arjona, S. E. Malawista, E. Fikrig, and R. R. Montgomery Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly. J Virol 82: Lertmemongkolchai, G., G. Cai, C. A. Hunter, and G. J. Bancroft Bystander activation of CD8+ T cells contributes to the rapid production of IFNgamma in response to bacterial pathogens. J Immunol 166: Li, M., A. Makkinje, and Z. Damuni The myeloid leukemia-associated protein SET is a potent inhibitor of protein phosphatase 2A. J Biol Chem 271: Linton, P. J., S. P. Li, Y. Zhang, B. Bautista, Q. Huynh, and T. Trinh Intrinsic versus environmental influences on T-cell responses in aging. Immunol Rev 205: Moretto, M. M., E. M. Lawlor, and I. A. Khan Aging mice exhibit a functional defect in mucosal dendritic cell response against an intracellular pathogen. J Immunol 181: Murciano, C., E. Villamon, A. Yanez, J. E. O'Connor, D. Gozalbo, and M. L. Gil Impaired immune response to Candida albicans in aged mice. J Med Microbiol 55:

28 Nogusa, S., B. W. Ritz, S. H. Kassim, S. R. Jennings, and E. M. Gardner Characterization of age-related changes in natural killer cells during primary influenza infection in mice. Mech Ageing Dev 129: Ogata, K., E. An, Y. Shioi, K. Nakamura, S. Luo, N. Yokose, S. Minami, and K. Dan Association between natural killer cell activity and infection in immunologically normal elderly people. Clin Exp Immunol 124: Organization, W. H Global tuberculosis control- surveillance, planning, financing, WHO Report 2008 ed 24. Orme, I. M Aging and immunity to tuberculosis: increased susceptibility of old mice reflects a decreased capacity to generate mediator T lymphocytes. J Immunol 138: Papamichail, M., S. A. Perez, A. D. Gritzapis, and C. N. Baxevanis Natural killer lymphocytes: biology, development, and function. Cancer Immunol Immunother 53: Rottinghaus, E. K., B. Vesosky, and J. Turner Interleukin-12 is sufficient to promote antigen-independent interferon-γ production by CD8 T cells in old mice. Immunology:In Press. 27. Schwanninger, A., B. Weinberger, D. Weiskopf, D. Herndler-Brandstetter, S. Reitinger, C. Gassner, H. Schennach, W. Parson, R. Wurzner, and B. Grubeck-Loebenstein Age-related appearance of a CMV-specific highavidity CD8+ T cell clonotype which does not occur in young adults. Immun Ageing 5:14.

29 Shi, Y., T. Yamazaki, Y. Okubo, Y. Uehara, K. Sugane, and K. Agematsu Regulation of aged humoral immune defense against pneumococcal bacteria by IgM memory B cell. J Immunol 175: Stout-Delgado, H. W., X. Yang, W. E. Walker, B. M. Tesar, and D. R. Goldstein Aging impairs IFN regulatory factor 7 up-regulation in plasmacytoid dendritic cells during TLR9 activation. J Immunol 181: Sun, J. C., J. N. Beilke, and L. L. Lanier Adaptive immune features of natural killer cells. Nature 457: Sun, K., and D. W. Metzger Inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection. Nat Med 14: Trotta, R., D. Ciarlariello, J. Dal Col, J. Allard, 2nd, P. Neviani, R. Santhanam, H. Mao, B. Becknell, J. Yu, A. K. Ferketich, B. Thomas, A. Modi, B. W. Blaser, D. Perrotti, and M. A. Caligiuri The PP2A inhibitor SET regulates natural killer cell IFN-gamma production. J Exp Med 204: Turner, J., A. A. Frank, and I. M. Orme Old mice express a transient early resistance to pulmonary tuberculosis that is mediated by CD8 T cells. Infect Immun 70: Turner, J., and I. M. Orme The expression of early resistance to an infection with Mycobacterium tuberculosis by old mice is dependent on IFN type II (IFN-gamma) but not IFN type I. Mech Ageing Dev 125:1-9.

30 United Nations, The The World Population Prospects: The 2006 Revision. The UN Population Database Vesosky, B., D. K. Flaherty, and J. Turner Th1 cytokines facilitate CD8- T-cell-mediated early resistance to infection with Mycobacterium tuberculosis in old mice. Infect Immun 74: Yager, E. J., M. Ahmed, K. Lanzer, T. D. Randall, D. L. Woodland, and M. A. Blackman Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to influenza virus. J Exp Med 205: Downloaded from on June 6, 2018 by guest

31 631 FIGURE LEGENDS FIG. 1. CD8 T cells isolated from the lungs of old mice are a major source of IFN-γ following Mtb infection. Young and old mice were infected aerogenically with Mtb Erdman and lungs harvested at 0, 8, and 12 days post infection (n=3-5). CD8 (A) and NK cells (B) were purified from the lungs of young and old mice using magnetic beads. Purified cells were homogenized in Trizol and frozen at -80 C. RNA was isolated, reverse transcribed, and cdna amplified for IFN-γ by RT-PCR. The relative expression of IFN-γ mrna is shown as mean +/- SEM and is representative of at least 2 individual experiments. Statistical significance was determined using a One-way ANOVA with Tukey s post test (**= p value <0.01, ***= p value <0.001; significance between young and old, += p value <0.05, ++= p value <0.01, +++= p value <0.001; significance between time points for old mice, no statistically significant increases for young mice). FIG. 2. CD8 T cells isolated from the lungs of naïve old mice produce innate IFN-γ in response to in vitro stimulation with Mtb. Lung cells were isolated from naïve young and old mice and stimulated with Mtb Erdman (MOI 0.5) for 48 hours (A) (n= 4-5) or hours (B, C) (n=4-5, pooled). (A) IFN-γ was measured in culture supernatants of individual mice by ELISA. Data are represented as mean +/- SEM and are representative of 2 individual experiments. Statistical significance was determined using a One-way ANOVA with Tukey s post test (**= p value <0.01; significance between young and old, ++= p value <0.01; significance between treatment groups). CD8 (B) and NK (C) cells were purified from lung cultures using magnetic beads. Purified cells were homogenized

32 in Trizol and frozen at -80 C. RNA was isolated, reverse transcribed, and cdna amplified for IFN-γ by RT-PCR. The relative expression of IFN-γ mrna is shown and is representative of at least 2 individual experiments. nd = not detected FIG. 3. Increased IFN-γ production is an intrinsic property of aged CD8 T cells. CD8 and CD11c cells were purified from individual lung cell suspensions using magnetic beads. CD8 and CD11c cells were pooled by age group (n=8). Purified CD11c were infected with Mtb (MOI=1) and cultured with purified CD8 T cells for 48 hours. (A) CD8 T cells from young and old mice were cultured with CD11c cells from the same age group. (B) CD8 T cells from young and old mice were cultured separately with Mtb infected CD11c cells from both young and old mice. (A, B) IFN-γ was quantified in culture supernatants by ELISA. Data are represented as pg/ml of IFN-γ and are representative of 2 individual experiments. nd = not detected. FIG. 4. CD8 derived IFN-γ production is antigen independent. (A-B) Lung cells were isolated from young and old naive mice using gentle enzymatic digestion. (A) CD11c cells were purified from young and old mice, pooled (n=3-10) and cultured for 72 hours with Mtb (MOI=1). Cell supernatants were frozen, thawed, and filtered through a 0.2µm filter. Supernatants were cultured with purified CD8 T cells (n=3-7, pooled) from young and old mice for 48 hours. (B) CD11c cells were purified from young wildtype (n=5), young β 2 mko (n=5), and old (n=10) mice and pooled by group. CD11c cells were infected with Mtb (MOI=1) and cultured with CD8 T cells purified and pooled from old mice (n=10) for 48 hours. (A-B) IFN-γ was quantified in culture supernatants by ELISA.

33 Data are represented as pg/ml of IFN-γ and are representative of at least two individual experiments. nd = not detected FIG. 5. CD8 derived IFN-γ is IL-12 dependent. (A-B, D) Lung cells were isolated from young and old mice using gentle enzymatic digestion. (A) Lung cells from individual mice (n=3-5) were cultured with Mtb (MOI=0.5) for 48 hours in the presence of either Mtb alone or in combination with 1µg/ml anti-il-12 or isotype control antibody. (B) CD8 and CD11c cells were purified from individual lung cell suspensions using magnetic beads. CD8 and CD11c cells were pooled by age group (n=5-7). Purified CD11c cells were infected with Mtb (MOI=1) and cultured with purified CD8 T cells and 1µg/ml anti- IL-12 or isotype control for 48 hours. (C) CD8 cells were purified from the spleens of old mice (n=1-2) and pooled. Purified CD8 cells were cultured with IL-12p70 (5ng/ml), IL-23 (20ng/ml), IL-12p40 (20ng/ml), or IL-12p40p40 (20ng/ml) for 48 hours. All cultures contained IL-18 (20ng/ml). (D) CD11c cells were purified from young wildtype (n=5) and young IL-12p35KO (n=5) mice and pooled by group. CD11c cells were infected with Mtb (MOI=1) and cultured with CD8 cells purified and pooled from old mice (n=10) for 48 hours. (A-D) IFN-γ was quantified in culture supernatants by ELISA. Data are represented as mean +/- SEM (A, C) or pg/ml (B, D) and are representative of at least two individual experiments. Statistical significance was determined using a Oneway ANOVA with Tukey s post test (**= p value <0.01; significance between young and old, ++=p value <0.01, +++= p value <0.001; significance between treatment groups). nd = not detected.