Loss of function in virus-specific lung effector T cells is independent of infection

Transcription

1 Loss of function in virus-specific lung effector T cells is independent of infection Subhashini Arimilli, Ellen M. Palmer, and Martha A. Alexander-Miller 1 Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA Abstract: Recently, several studies, including those with respiratory syncytial virus, mouse pneumovirus, and simian virus 5, have reported that virus-specific CD8 effector cells entering the lung as a result of respiratory infection undergo significant loss of function. The impaired function in these cells has been proposed to be the result of infection-induced changes in the lung. Although virus-specific effects may contribute to regulation of T cells in the lung, the findings from this study provide evidence that the basal lung environment is sufficient to promote loss of function in effector cells. Loss of function occurs within 48 h of entry into the lung and is most evident in cells residing in the lung parenchyma. These findings suggest an additional paradigm for the immunoregulation of effector cells that enter the lung as a result of virus infection. J. Leukoc. Biol. 83: ; Key Words: CTL regulation CD8 respiratory tract INTRODUCTION A large number of viral infections of clinical significance are contracted via the respiratory route. Thus, an effective immune response at this site is of critical importance. Following infection of the respiratory tract, antigen is carried to the draining lymph nodes, where pathogen-specific CD8 effector T cells are generated. These cells then traffic to the lung, arriving, in many models, at Days 7 8 following virus infection [1 3]. The presence of functional CD8 effector cells is often crucial for viral clearance from this tissue. Upon entry, these cells exert potent effector function, including the release of IFN- and TNF- as well as the lysis of infected cells. Surprisingly however, there is a growing body of evidence, including our studies with the paramyxovirus simian virus 5 (SV5) [4] and those of others using respiratory syncytial virus (RSV) [5, 6], influenza (FLUAV/PR8/34) [6], and mouse pneumovirus [7], that over the course of infection, function in CD8 effector T cells in the lungs becomes increasingly impaired. These findings suggested that negative modulation of effector cell function in the lung may be more the rule than the exception. The interpretation of these findings by us, as well as others, has been that the lung environment induced in response to infection triggers the loss of function in effector T cells, potentially a mechanism used by the virus to limit clearance or the host to limit tissue damage. However, an alternative hypothesis is that the lung environment is inherently immunosuppressive and thus, responsible for the loss of function in cells. This hypothesis is in line with the known presence of a variety of immunosuppressive mediators in the lung. These mediators, which include factors such as NO, PGs, and suppressive cytokines, can be detected at low levels, even in the absence of infection [8, 9], and are produced by a number of cell types, including alveolar macrophages [10], epithelial cells [10], and T regulatory cells (Tregs) [11]. The immunomodulatory effects of these mediators have been studied intensely. For example, NO is known to lead to inhibition of dendritic cell maturation [12], proinflammatory cytokine production [10], and T cell proliferation [13]. The lung is also a rich source of PGE 2, a mediator that has been reported to enhance IL-10 production [14]. The immunosuppressive effects of IL-10 are far-reaching, including the down-regulation of costimulatory molecule expression by APCs [15, 16], IFN- production by T cells [17], and CD25 expression [18, 19]. Additionally, TGF-, which is also constitutively present in the lungs, has been shown to suppress T cell proliferation [20] and function [16, 21]. Whether the loss of function in SV5-specific CD8 effector cells that entered the lung following virus infection, previously reported by us, was a result of virus-induced changes in the lung or immunoregulation inherent in this tissue was unknown. Thus, in the current study, we analyzed the requirement for SV5 infection for loss of function to occur. Surprisingly, we found that loss of function was evident in lung effector cells regardless of whether infection was present. These data strongly support the ability of the normal lung environment to modulate function in effector T cells. Importantly, changes in CD8 T cell function were selective for cells in the lung, as effectors in the spleen retained full activity. Within the lung, function in effector cells was dictated by location, as nonresponsiveness was mainly restricted to cells in the parenchyma as opposed to the airway. These findings have significant 1 Correspondence: Department of Microbiology and Immunology, Room 5053, Hanes Building, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. marthaam@ wfubmc.edu Received April 9, 2007; revised November 1, 2007; accepted November 15, doi: /jlb Journal of Leukocyte Biology Volume 83, March /08/ Society for Leukocyte Biology

2 importance for our understanding of the lung environment and its ability to regulate effector T cells that enter in response to infection. MATERIALS AND METHODS Mice, cell lines, Listeria monocytogenes, and recombinant viruses Six- to 8-week-old female BALB/c mice were purchased from the Frederick Cancer Research and Development Center (Frederick, MD, USA). Transgenic (Tg) mice expressing the L9.6 Listeria p60-specific TCR [22] and Thy1.1 were a kind gift of Dr. Eric Pamer (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). All research performed on mice in this study complied with federal and institutional guidelines set forth by the Wake Forest University Animal Care and Use Committee (Winston-Salem, NC, USA). The I10-specific CD8 CTL line was generated by weekly restimulation of splenocytes obtained from a mouse infected with the vaccinia virus expressing the HIV gp160 protein with 10 9 M peptide-pulsed splenocytes [23]. Short-term cultures from p60 TCR Tg mice for adoptive transfer were generated by two rounds of in vitro stimulation of splenocytes with p60 peptide-pulsed splenocytes (10 9 M). All in vitro-derived CTL cultures were maintained in 24-well plates containing 2 ml RPMI-1640 medium supplemented with 2 mm L-glutamine, 0.1 mm sodium pyruvate, nonessential amino acids (NEAA), 100 U/mL penicillin, 100 g/ml streptomycin, 2-ME (0.05 mm), 10% FBS, and 10% T-stim (Collaborative Biomedical Products, Bedford, MA, USA) as an IL-2 source. Recombinant viruses were constructed as described previously [24]. Viruses were purified by centrifugation (25,000 rpm, 6 h, SW28 rotor) through a 20% glycerol cushion, resuspended in DMEM with 0.75% BSA, and titrated on CV-1 cells as described previously [25]. L. monocytogenes (strain 10403S) was grown overnight at 30 C in brain heart infusion broth to stationary phase. Bacteria were washed twice prior to injection. Immunizations Mice were immunized as described previously [3]. Briefly, mice were anesthetized with Avertin (2,2,2-tribromoethanol) by i.p. injection. Virus ( pfu) was delivered intranasally in a volume of 50 l. Virus was diluted in PBS. Mock-infected mice received PBS alone. Adoptively transferred cells I10-specific cells or p60 Tg cells (both on Day 5 following in vitro stimulation) were passed over a Histopaque (Sigma Chemical Co., St. Louis, MO, USA) gradient to enrich for live cells. Subsequently, cells were delivered intratracheally (i.t.) or i.v. via the tail vain into naive or SV5-infected BALB/c recipients. Infected mice had received virus 8 days prior. In vivo effectors for transfer were generated as follows: Thy1.1 cells from p60 TCR Tg mice were positively selected on Miltenyi columns. Following selection, cells were i.v.-injected into the naïve BALB/c mice. The following day, mice were inoculated i.v. with L. monocytogenes, and 7 days later, spleen and lung cells were collected. A sample of the cells was taken, and the presence of activated p60-specific, IFN- -producing effector cells was determined by intracellular cytokine staining (ICCS). Thy1.1 cells were enriched from the remainder of the sample, and cells were adoptively transferred into naïve BALB/c ( i.v. and i.t.). Isolation of effector cells Mice were killed, and the trachea was surgically exposed to perform bronchoalveolar lavage (BAL). The trachea was cannulated with a syringe, and a 1-ml aliquot of PBS was used to rinse the lower respiratory tract to collect lymphocytes from the airway. Parenchymal lymphocytes were isolated by digestion of lavaged lungs with collagenase D (Sigma Chemical Co.) as described previously [3]. Spleens were isolated from adoptively transferred mice, and single cell suspensions were prepared. Analysis of adoptively transferred CD8 T cells Cells isolated from mice adoptively transferred with the I10-specific line were stained with the I10 tetramer [provided by National Institutes of Health (NIH) Tetramer Core Facility at Emory University, Atlanta, GA, USA] and anti- CD8 -PerCpCy5.5 (clone , BD PharMingen, San Diego, CA, USA). Cells isolated from mice adoptively transferred with p60 Tg cells were stained with Thy1.1-PE (OX-7) and anti-cd8 -PerCpCy5.5 (BD PharMingen). Staining was performed for 30 min on ice. Following three washes with PBS containing 1% FCS (FACS buffer) to remove unbound antibody, samples were acquired on a BD Biosciences FACSCalibur flow cytometer, and data were analyzed using CellQuest software (BD Immunocytometry Systems, San Jose, CA, USA). For cytokine analysis, lymphocytes isolated from the lung or spleens were cultured for 5 h in 96-well flat-bottom plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at a concentration of cells/well in a volume of 200 l complete medium (RPMI, 10% FCS, M 2-ME, 1% each HEPES, L-glutamine, pen/strep, sodium pyruvate, and NEAA) containing l/ml monensin (GolgiPlug, BD PharMingen). Stimulation was carried out in the presence of I10 or p60 peptide (1 M) as appropriate. Following culture, the cells were harvested, washed, and stained for expression of CD8 and Thy1.1 by addition of antibodies for 30 min on ice. After washing, cytokine production was assessed by ICCS using anti-ifn- and anti-tnf- antibodies (BD PharMingen), using the Cytofix/Cytoperm kit, according to the manufacturer s instructions (BD PharMingen). Samples were acquired on a BD Biosciences FACSCalibur flow cytometer, and data were analyzed using CellQuest software (BD Immunocytometry Systems). For proliferation analyses, Thy 1.1 TCR Tg p60-specific effectors were i.t.-transferred into BALB/c mice. On Day 3 post-transfer, cells were isolated from the lungs, labeled with CFSE, and plated at cells/well of a 96-well round-bottom plate in the presence of irradiated BALB/c splenocytes pulsed with 10 9 M peptide. T-stim (10%) was added as a source of IL-2. On Day 3 of culture, cells were harvested and stimulated in the presence of 1 M p60 peptide for 5 h. Cells were then stained with Thy1.1-specific antibodies followed by fixation, permeabilization, and staining with anti-ifn- antibody. Proliferation and IFN- production were analyzed by flow cytometry. Detection of apoptotic cells Apoptosis in lung cells at Days 7 and 12 postinfection with SV5 was determined by staining for active caspase 3. Lung cells were isolated by digestion with collagenase D (Sigma Chemical Co.), as described previously [3]. For analysis of SV5 M-specific effectors, lung cells were stained with M-tetramer and anti-cd8 antibody or were restimulated with M peptide and subsequently stained with anti-cd8 antibody. For analysis of Thy 1.1 p60-specific effectors, cells were adoptively transferred by i.t. administration. After 48 h, lung cells were isolated and restimulated with p60 peptide. Cells were then stained with anti-thy1.1 and anti-cd8 antibodies. Effector cells were then fixed and permeabilized using the Cytofix/Cytoperm kit, according to the manufacturer s instructions (BD PharMingen), followed by staining with active caspase 3-specific antibody and anti-ifn- antibody where indicated (BD PharMingen). Stained samples were run on a BD Biosciences FACSCalibur flow cytometer, and data were analyzed using CellQuest software (BD Immunocytometry Systems). RESULTS The normal lung environment induces loss of function in T cells We have previously reported the observation that following intranasal infection with SV5, an increasing percentage of virus-specific cells present in the lung loses function over time, such that by Day 12, 50% of the effectors are nonfunctional, and by Day 40 postinfection, nearly 85% of the cells are incapable of producing IFN- in response to peptide stimulation [4]. Loss of function extended to the ability of cells to secrete lytic granules [4], and thus, these effectors have lost the Arimilli et al. The normal lung induces effector nonresponsiveness 565

3 capacity to clear virus by the lytic and cytokine pathways. Of note, although function at the population level is decreasing between Days 7 and 12 postinfection, the absolute number of virus-specific cells is increasing (approximately fourfold) [3]. These findings left us with a number of unanswered questions with regard to the mechanism responsible for the loss of function in effector cells in the lung. The question arose as to whether infection with SV5 produced a lung environment that would result in the loss of function of any effector cells present in this tissue. To address this question, we used an adoptive transfer approach, wherein effector cells specific for an irrelevant antigen were transferred into SV5-infected or mockinfected recipient mice. We realize that effector cell entry into the lung would normally occur as a result of infection. However, the contribution of the normal lung environment versus the infection-induced environment can only be tested by comparing mock-infected with SV5-infected animals. As shown below, effector cells enter the lung in both instances. As our initial approach, we used an established CTL line specific for the gp160 I10 epitope presented in the context of the H-2D d molecule. On Day 5 following routine stimulation, cells were i.t.-transferred into recipient BALB/c mice that were uninfected or had been infected with SV5 8 days Fig. 1. Loss of function in effector T cells in the lung is not dependent on SV5 infection. BALB/c mice were infected intranasally with 10 6 PFU wild-type recombinant (r)sv5. On Day 8 postinfection, SV5-infected or naïve mice received I10-specific cells by i.t. instillation. At the time of transfer, a portion of CTL was simulated with I10 peptide (pep), and IFN- and TNF- production was determined (A). Forty-eight hours following transfer, the lung lymphocytes were isolated and stained with I10 tetramer (Tet) or stimulated with 1 M I10 peptide, and IFN- and TNF- production was assessed (B). As a negative control, cells were cultured in the absence of peptide. The numbers represent the percentages of CD8 T cells that were tetramer-positive, IFN- -positive, or TNF- -positive. The data shown are representative of four independent experiments with three mice each. prior. At the time of transfer, 96% of cells produced IFN-, and 98% produced TNF- (Fig. 1A). Forty-eight hours posttransfer, lungs were isolated, and the percent of functional cells was determined. In SV5-infected mice, transferred I10-specific cells present in lungs were 25% functional, as measured by comparing the percent of CD8 cells that stained positive for the I10 tetramer (28%) with the percent that produced IFN- following stimulation with the I10 peptide (7%; Fig. 1B, left column). A similar loss was apparent in the ability to produce TNF- (Fig. 1B). Surprisingly, analysis of the cells transferred into the mock-infected mice revealed that these cells still underwent significant impairment of function (43% functional; Fig. 1B, right column). The finding that transfer of cells into naïve animals resulted in significant loss of function was unexpected and suggested that the baseline lung environment was capable of mediating this effect. Thus, in contrast to previously proposed models, these data suggest that immunoregulation could occur in the absence of any infection-related changes in the lung. We realized that it was possible that the loss of function was specific to the established T cell line transferred. Thus, a second effector population was examined. Splenocytes from Listeria p60-specific TCR Tg Thy1.1 mice underwent two rounds of weekly stimulation. At this time, 76% of cells from these short-term cultures produced IFN-, and 89% expressed TNF- in response to peptide stimulation (Fig. 2A). Cells from these cultures were i.t.-transferred in naïve, Thy1.2 -recipient mice, and 48 h later lungs were isolated and function in p60-specific cells was determined. In agreement with the results obtained with the I10-specific line, we observed a significant loss of function in cells present in the lungs, as now only 47% of Thy1.1 p60-specific cells were capable of producing IFN- in response to peptide stimulation (Fig. 2B). As with the I10-specific line, TNF- production was also impaired in these cells (49% decrease in function; Fig. 2B). The presence of some nonfunctional cells in the transferred population left open the possibility that nonfunctional cells in this population were selectively homing to the lung. However, we believe that this is not the case for the following reasons. First, with two additional rounds of stimulation, function in the p60-specific cells increased to 98%. Following transfer of this highly functional population, a similar loss of function was apparent in cells isolated from the lungs 48 h following transfer (data not shown). Second, analysis of CCR5 expression, a receptor known to be involved in effector cell trafficking to the lung [26], revealed no correlation between expression of this molecule and presence in the lung or whether cells were functional (data not shown). Loss of function in adoptively transferred effectors was restricted to cells in the lung We had shown previously that following SV5 infection, the loss of function in virus-specific cells was present in lung but not spleen or lymph node resident cells [4]. To ensure that this was the case for the adoptively transferred effectors, p60-specific cells were administered via i.v. injection. This route was chosen based on preliminary studies, showing that it resulted in an increased number of p60-specific cells in the spleen compared with i.t. instillation. Thy Journal of Leukocyte Biology Volume 83, March

4 p60-specific, short-term cultured cells ( ) were injected i.v. into naïve BALB/c mice. At the time of transfer, 87% of cells were functional, as determined by IFN- production and 89% by TNF- production (data not shown). Forty-eight hours post-transfer, lungs and spleens were isolated. Following peptide stimulation, only 32% of effector cells in the lung were functional (Fig. 3). These data show that cells present in the lung following i.v. transfer lost function similar to cells transferred i.t. In contrast to lung resident cells, 81% of cells in the spleen were functional, a percentage similar to that in the pretransfer population. These data suggested that the loss of function was not a passive outcome of in vivo transfer but was an active effect of residence in the lung. A similar retention of function in spleen resident cells was found following transfer of the I10-specific line (data not shown). Fig. 2. Loss of function occurred in short-term, cultured, p60-specific effector cells following entry into the lung. (A) Thy1.1 p60 Tg cells were stimulated twice in vitro to generate highly functional, short-term, cultured effector populations. IFN- and TNF- production in these cells was determined by ICCS following stimulation with p60 peptide (1 M). (B) Thy1.1 p60-specific effector cells ( ) were i.t.-transferred into naïve BALB/c mice. Forty-eight hours post-transfer, lung cells were isolated and stimulated with 1 M p60 peptide for 5 h. Following stimulation, cells were costained for Thy1.1 and antibodies to IFN- or TNF-. The numbers represent the percentage of Thy1.1 cells that stained positive for IFN- or TNF-. The data shown are representative of four independent experiments with four mice each. Fig. 3. The loss of function is restricted to adoptively transferred cells that are resident in the lung. p60-specific effector cells ( ) were i.v.-transferred into naïve BALB/c mice. Forty-eight hours post-transfer, lung and spleen cells were isolated and stimulated in the presence of 1 M p60 peptide. At the end of the 5-h culture period, p60-specific cells were identified by staining with anti-thy1.1 and anti-cd8 antibodies. IFN- and TNF- production was assessed by intracellular staining. As a negative control, cells were stimulated in the absence of peptide. The numbers represent the percentages of Thy1.1 cells that show positive cytokine staining under the various conditions. The data shown are representative of four independent experiments, each containing four mice. Arimilli et al. The normal lung induces effector nonresponsiveness 567

5 Functional inactivation occurred in adoptively transferred, in vivo-generated effector cells We were aware that it was possible that the effector cell populations generated in vitro would not be representative of primary effector cells generated in vivo. To address this concern, we i.v.-infected BALB/c mice that had received Thy1.1 p60-specific TCR Tg cells (on Day 1) with L. monocytogenes. On Day 7 postinfection, splenocytes were harvested, and Thy1.1 p60-specific effectors were isolated by magnetic bead separation. A portion of the population was exposed to peptide antigen to determine baseline function in these cells. At the time of transfer, 80% of cells were capable of making IFN- (data not shown). Effector cells were transferred into naïve BALB/c mice and 48 h later, re-isolated from the lung and spleen and stimulated with peptide. As observed with other effector populations, there was a significant decrease in the percentage of cells isolated from the lung versus the spleen that could produce cytokine (IFN- and TNF- ) following stimulation (Fig. 4, and data not shown). Although spleen cells exhibited a similar percentage of cells that could produce cytokine in response to peptide stimulation compared with this population at the time of transfer, in the lung, only 37% of cells were functional. These data strongly suggest that the results obtained with in vitro-generated lines/cultures are representative of effectors generated in vivo in response to infection. Loss of function in lung effector cells was not associated with cells undergoing apoptotic death One possibility to explain the loss of function in lung effectors was that these cells were undergoing apoptosis. In this scenario, cells would be scored as nonfunctional, not because they were being maintained in a nonresponsive state, but because they were disabled as a result of the initiation of the death pathway. To test this possibility, we analyzed the percentage of cells that were undergoing apoptosis as evidenced by the presence of active caspase 3. We used two effector populations to address this question: analysis of SV5-specific cells present as a result of infection, and analysis of adoptively transferred, p60-specific effectors. For M specific effectors, at early times postinfection (Day 7), 90% of the cells were functional, as determined by comparing the percentage of cells positive for M /L d tetramer with IFN- (Fig. 5A). By Day 12, however, only half of the cells produced IFN- in response to stimulation with the M peptide. If the loss of function were a result of the initiation of cell death, we would expect to see a higher proportion of M-tetramerpositive cells undergoing apoptosis in the population isolated from the lungs on Day 12 (where 50% of the M specific cells were nonfunctional) versus the Day 7 population (where 10% of the M specific cells were nonfunctional). Instead, we found that a similar percentage of tetramer cells was positive for active caspase 3 in these two populations (Fig. 5B, representative data, and Fig. 5C, averaged data). A parallel analysis of adoptively transferred, p60-specific TCR Tg cells revealed a similar finding. p60-specific cells were 96% functional at the time of transfer. Following reisolation 48 h later, p60-specific cells, identified by expression of CD8 and Thy1.1 (see gating in Fig. 5D, top left panel), were now 59% functional. Analysis of apoptosis was performed by gating on the Thy1.1 IFN- and Thy1.1 IFN- cells. Although there was some increase in the percentage of nonfunctional cells (IFN- ) that stained positive for active caspase 3 compared with the percentage in functional cells (IFN- ; 29% vs. 20% on average), active caspase 3 cells comprised less than one-third of the nonfunctional cells (Fig. 5, D and E; in Fig. 5D, active caspase 3 staining is shown in the right column and isotype control antibody staining in the left column). These data suggest that the lack of function is not dependent on the apoptotic process. Nonfunctional cells retained proliferative potential Fig. 4. In vivo-generated effector cells are susceptible to the loss of function that results from residence in the lung environment. Thy1.1 cells from p60 TCR Tg mice were i.v.-injected into the naïve BALB/c mice. The following day, mice were inoculated i.v. with Listeria, and 7 days later, spleen cells were collected. Thy1.1 cells from infected animals were transferred into naïve BALB/c mice ( cells i.v. and i.t.). Two days post-transfer, lung and spleen cells were isolated, and IFN- production in Thy1.1 cells was determined by ICCS. IFN- production shown is following gating on Thy1.1 CD8 cells (gating shown in top row). The numbers in the dot-plots represent the percentage of Thy1.1 CD8 cells that are IFN-. The data shown are representative of two independent experiments with four mice each. The loss of all effector functions (lysis, IFN- production, and TNF- production) in the lung resident cells suggested that these cells may be completely incapable of responding to TCR engagement. To determine if this were the case, we assessed the ability of transferred cells to proliferate following residence in the lung. In vitro-activated Thy1.1 p60-specific effectors (98% functional by IFN- production) were i.t.-transferred into naïve BALB/c mice. On Day 3 post-transfer, lung cells were re-isolated and labeled with CFSE. At this time, cells were 52% functional (data not shown), demonstrating loss of function in this population. Labeled cells were cultured for 3 days in the presence of p60 peptide-pulsed BALB/c splenocytes. Cells were then restimulated with peptide, and IFN- production was assessed by ICCS. The proliferative profile of IFN- -producing and -nonproducing cells 568 Journal of Leukocyte Biology Volume 83, March

6 Fig. 5. The loss of function in lung effector cells is not a marker for cells undergoing apoptotic death. Loss of function in CD8 T cells was assessed in BALB/c mice on Days 7 and 12 following intranasal infection with 10 6 PFU wild-type rsv5 postinfection. Lymphocytes were stained with M /L d MHC-I tetramer or stimulated with 1 M M peptide and stained for intracellular IFN-. As a negative control, cells were stimulated in the absence of peptide. The percent functional values were obtained by dividing the percentage of CD8 cells that produced IFN- by the percentage that was tetramer (A). Comparison of the percentage of active caspase 3-positive cells in the M-tetramer population showed apoptosis was similar at Days 7 and 12 (B, representative data; C, averages SEM). Apoptosis was also analyzed in Thy 1.1 p60 effector cells following i.t. transfer into BALB/c mice. Forty-eight hours post-transfer, lung cells were isolated, restimulated with 1 M peptide, and stained for the expression of Thy1.1, CD8, IFN-, and active caspase 3. Representative analyses of active caspase 3 in IFN- and IFN- cells (following gating on Thy1.1 and CD8) are shown in D and averaged data in E. Data shown are representative of four independent experiments. was determined. The data in Figure 6, A (representative) and B (averaged), show that cells proliferated independent of whether they were capable of producing IFN-. Although the percent of cells that were in the divided gate was somewhat higher in the IFN- versus IFN- population, a significant portion of IFN- cells retained the ability to proliferate in an antigen-dependent manner. These data suggest that nonfunctional cells were capable of responding on some level to TCR engagement. Loss of function in lung resident effector cells occurred between Days 1 and 2 following entry into the lung From the above analyses, we knew that a significant portion of cells lost function within 48 h of entry into the lungs. However, the time required for this effect to occur and whether the loss of function was maximal at 48 h were unknown. Thus, a kinetic analysis was performed to assess function in p60-specific cells between 16 h and 4 days following adoptive transfer (Fig. 7). At the time of transfer, 70% of cells produced IFN-, and 80% produced TNF-. The data in Figure 7 show that at an early time post-transfer (16 h), the percentage of cells that produced IFN- or TNF- was similar to that of the transferred population. However, by 48 h post-transfer, there was an 50% reduction in the percentage of cells that produced cytokines following stimulation, and this level of nonresponsiveness was maintained throughout the 4-day analysis period. The loss of function is primarily a property of tissue resident lung effector cells During viral infection of the respiratory tract, effector cells are found at two distinct anatomical sites within the lung: in the parenchyma and at the airway surface. We were interested in whether the lack of responsiveness in our model was specific to cells at one of these anatomical sites or whether it was a general property of all effector cells present within the lung. We made use of the effector populations tested above, SV5 effectors infiltrating the lung following virus infection, and adoptively transferred cells from the established I10-specific CTL line. SV5-specific effectors were isolated from the airway (by BAL) or tissue (by digestion with collagenase) on Days 7 and 12 postinfection. At both time-points, 5% of the total M specific cells in the lung were found in the lavage. As above, isolated cells were stimulated in the presence of the M peptide, and the number of functional cells was determined by comparing the percent of cells that produced IFN- with the percent that bound tetramer. Arimilli et al. The normal lung induces effector nonresponsiveness 569

7 We have previously reported the finding that CD8 virusspecific effector cells in the lungs of SV5-infected mice become increasingly nonresponsive over time [4]. This nonresponsiveness encompasses all effector functions assessed, i.e., IFN- production, TNF- production, and secretion of lytic granules [4]. One trivial possibility to explain the loss of function in lung resident effectors was that the cells were delayed in the kinetics with which they produced cytokines following stimulation. However, allowing for longer periods of stimulation prior to intracellular cytokine analysis does not result in detectable function in these cells (data not shown). Interestingly, the cells are not ignorant of peptide encounter as they undergo peptide-dependent proliferation, suggesting that there is, at minimum, a partial signal initiated as a result of TCR engagement. Loss of function is not unique to the setting of SV5 infection but has also been reported for CD8 T cells entering the lung following infection with LCMV [5, 6] or mouse pneumovirus [7]. Further, a recent study demonstrated that the loss of function in T cells was not antigen-specific; i.e., RSV-specific cells nonspecifically recruited to the lung as a result of RSV or Fig. 6. Cells that are incapable of producing IFN- retain the ability to undergo antigen-dependent proliferation. Thy 1.1 TCR Tg p60-specific, in vitro-generated effectors ( ) were transferred into BALB/c mice. At the time of transfer, these cells were highly functional (98%). On Day 3 posttransfer, cells were isolated from the lungs, and function was determined using a sample of the cells. Following re-isolation, 52% of cells produced IFN- in response to peptide stimulation. The remaining lung cells were labeled with CFSE and cultured in the presence of peptide-pulsed BALB/c splenocytes for 3 days. Cells were then restimulated with peptide for 5 h, and IFN- production was assessed by ICCS. The data shown are following gating on CD8 and Thy1.1. IFN- and IFN- cells were assessed for proliferation by monitoring CFSE expression. (A) Representative data. (B) Averaged data from six individual mice assayed over two experiments. (B) Percentages are based on cells in the divided gate. A representative dataset (Fig. 8A) and averaged data (Fig. 8B) for these analyses are shown. When parenchymal cells were analyzed, on average, % of cells were found to be functional at Day 7, and at this same time, BAL cells were % functional. By Day 12, function in the tissue resident cells had decreased to % (a 40% loss of function). This decrease was highly significant (P.001). Although there was some loss of function observed in BAL cells at Day 12, this population was still highly functional (80 8.4%) compared with parenchymal cells. When these analyses were performed following adoptive transfer of the I10-specific CTL line, a similar pattern was observed. I10-specific cells in the BAL were almost fully functional (94%), whereas only 45% of the tissue resident cells were functional (Fig. 9, A and B). Increased loss of function in tissue resident versus BAL cells was also observed in transferred, p60-specific cells (data not shown). Together, these data demonstrate an anatomical separation in the lung between the responsive and nonresponsive cells. DISCUSSION Fig. 7. Loss of function in lung resident effector cells occurred between Days 1 and 2 following transfer. p60-specific effector cells ( ) were i.v.- transferred into naïve BALB/c mice. At the indicated times post-transfer, lung and spleen cells were isolated and stimulated in the presence of 1 M p60 peptide. At the end of the 5-h culture period, p60-specific cells were identified by staining with anti-thy1.1 antibody. IFN- and TNF- production was assessed by intracellular staining. As a negative control, cells were stimulated in the absence of peptide. The numbers represent the percentages of Thy1.1 cells that show positive cytokine staining under the various conditions. The data shown are the average ( SEM) of four mice in a group. The results are representative of three independent experiments. 570 Journal of Leukocyte Biology Volume 83, March

8 our studies was that modification of the lung environment by SV5 infection was not required for loss of function in effector T cells. Instead, the normal lung environment was sufficient for this effect. This conclusion is based on our finding that the entry of adoptively transferred CD8 effector cells into the lungs of noninfected mice resulted in loss of function, as was observed following entry of these same cells into the lungs of SV5-infected mice. Importantly, this was not an effect simply of adoptive transfer into an in vivo environment, as cells found in the spleen or in the lung airway following transfer retained full function. Further, it was not the result of an atypical response of a single line, as cells with a distinct antigen Fig. 8. The loss of function following SV5 infection is primarily a property of tissue resident effector cells. BALB/c mice were infected intranasally with 10 6 PFU wild-type rsv5, and on Day 7 or 12, postinfection lymphocytes in the BAL and tissue were isolated. (A) Cells were stained with M /L d MHC-I tetramer or stimulated with 1 M M peptide and stained for IFN-. As a negative control, cells were stimulated in the absence of peptide. The numbers represent the percentages of CD8 T cells that were tetramer-positive or IFN- -positive. (B) The percent functional values were obtained by dividing the percentage of cells that produced IFN- by the percentage that was tetramer. The data shown for lung analyses are the average ( SEM) of eight to nine independently analyzed mice over two experiments. The functional data obtained from SV5-specific cells in the spleen (three to four mice) are also shown for comparison. The decrease in function between Days 7 and 12 in tissue resident cells was statistically significant (*, P 0.001). The reduction in function in BAL cells was not statistically significant. influenza PR8 infection still lost function [6]. These results have led to a model, wherein the consequence of infection with these viruses is the generation of a lung environment that induces loss of effector cell function. However, the loss of function reported in a significant number of infectious models suggested the possibility that factors other than infectioninduced changes may contribute to this effect. To determine whether this was indeed the case, we analyzed the retention of function in effectors that entered the lung in the presence versus the absence of infection, using SV5 as our model infection to modify the lung environment. A major finding from Fig. 9. The loss of function in adoptively transferred cells is mainly restricted to tissue resident effector cells. I10-specific CTL ( ) were i.t.-transferred into naïve BALB/c mice. Forty-eight hours following adoptive transfer, BAL and lung parenchymal cells were isolated. I10-specific cells were identified by CD8 and tetramer staining. A portion of the cells was stimulated for 5 h with 1 M peptide to assess IFN- production. (A) The percentage of CD8 T cells that are tetramer-positive or IFN- -positive is indicated. The averaged percent functional values ( SEM) for the BAL and lung tissue shown in B were obtained by dividing the percent of cells positive for IFN- by the percent positive for the I10 tetramer. The difference in function in the cells at these two sites was statistically significant (*, P 0.005). Arimilli et al. The normal lung induces effector nonresponsiveness 571

9 specificity (p60 TCR Tg cells) generated by short-term culture and effectors generated in vivo as a result of infection also lost function following entry into the lung. This finding that the basal lung environment was sufficient for triggering loss of function in effector cells fundamentally impacts our understanding of the regulation of effector cells residing in the lung. We had envisioned that the infectious process resulted in the production of a regulatory factor (from the host or the virus) that impaired function in T cells. However, our current findings suggest that this need not be the case. Instead, the failure to modify the normal lung environment following infection from a suppressive to a supportive milieu may be responsible for the loss of function. Such modification may occur following infection with the A/Japan/ 305/57 strain of influenza, where T cells in the lung appear to retain full function [5]. Although unknown, it is possible that such infections shut-off the production of immunosuppressive mediators or induce the production of inflammatory factors that overcome the negative mediators. Thus, our data direct further studies of functional loss to include self-regulation of the immune response by the host. It is important to note that our finding does not rule out the possibility that virus infection can also impact the function of effector cells in the lung. Many viruses produce immunoregulatory proteins that could alter the immune response [27 29]. In such cases, the basal lung environment and the virus-specific factors could contribute to the regulation of immune cell function. The relative influence of the lung environment versus virus-specific factors is likely to be impacted by the nature of the particular virus, e.g., the production of immunomodulatory proteins as well as the viral load. The lung environment has long been considered to be immunosuppressive, and many factors capable of negatively regulating the immune response can be found in the lung, even in the absence of infection. These include IL-10, TGF-, NO, and PGE 2 [8, 9], all of which have been shown to inhibit T cell activation and/or function. The finding that the normal lung environment is sufficient to trigger loss of function may explain the observation that the percent of cells that are functionally impaired following SV5 or RSV infection increases over time, even long after the virus is cleared [4, 5]. Previously, it was difficult to envision how the regulatory effect proposed to result from infection was maintained over such a long period of time. Our initial studies evaluated the total CD8 effector cell population found in the lungs. However, effector cells in the lungs reside in two anatomically distinct locations: the parenchyma and the airway. Following virus infection, functional effector cells are found at both of these locations, for example [6, 30 32]. Currently, the mechanism dictating the trafficking of cells to these two sites is unknown; however, it has been reported that cells that reside at the airway interface cannot re-enter circulation [31, 32]. In our studies, we found that there was a striking difference in the functional impairment of cells at these two sites. Cells in the airway retained function more efficiently compared with cells residing in the tissue. In seeming contrast to our studies, a report from Vallbracht et al. [6] assessing effector cells present in the lung following RSV infection showed that a greater percentage of cells in the airway versus the tissue lost function when Day 20 versus Day 7 populations were analyzed. This finding led the authors to conclude that cells in the airway were more susceptible to loss of function [6]. However, it is important to note that tissue resident cells in the RSV infection model were highly impaired in function, even at the earliest time examined (Day 7) and similar to the findings presented here, were overall less functional than the BAL cells [6]. The high degree of impairment thus resulted in less change in function over the period analyzed. One possibility that would be consistent with our finding of a modest reduction in function in BAL cells present at Day 12 versus Day 6 postinfection with SV5 and those of Vallbracht et al. [6] is that SV5-specific effectors in the airway can eventually lose function but that this occurs with significantly delayed kinetics compared with the cells in the tissue. It is feasible that the level of a suppressive factor could vary as a result of the type of infection present in the lung (i.e., RSV vs. SV5), and as such, the kinetics with which BAL cells become susceptible to functional impairment would differ. Of note, the authors also observed increased loss of function in models where effector cells were nonspecifically recruited to the lungs by noncognate infections. Thus, it is also possible that the divergence in their results and ours is a result of the viral model studied. This would be consistent with the additional layer of immunoregulation that could be imposed by the virus as noted above. At present, the mediator responsible for the loss of function remains elusive. There are a number of lung factors that are attractive candidates, among them, IL-10, TGF-, NO, and PGE 2. All of these have been shown to have inhibitory effects, directly or indirectly, on T lymphocytes (for review, see ref. [10]). In this regard, preliminary studies suggest that the IL-10 pathway does not contribute to loss of function in our model, nor does the presence of PGE 2 or the inducible NO synthase pathway (our unpublished data). Another potent immunomodulatory mechanism that has received much attention of late is Tregs, the best characterized of which are natural Tregs. These cells are generally identified by the expression of CD4, CD25, and forkhead box P3 and mediate their suppressive effects through contact-dependent and -independent mechanisms [33]. However, these cells do not appear to be involved in the loss of function in our system, as depletion of CD4 cells did not prevent this outcome in lung resident effector cells (data not shown). Interestingly, in our studies of SV5 infection, as well as studies by others using RSV, a subpopulation of cells remained that retained function, even at late times postinfection [4 6]. Further, our kinetic analysis showed that following an initial period during which functional impairment occurred, the percentage of functional cells was relatively stable. This may suggest that not all cells are susceptible to inactivation in the lung environment. Although the basis of this is currently unknown, one hypothesis to explain these findings is that a distinct environment present during the generation of these cells confers resistance to impairment in the lung. This could alter the differentiation state of the cell. Alternatively, as a recent report has shown that virus-specific cells in the lungs are continuously recruited [34], it is possible that the functional cells reflect recent emigrants into the lungs and that continued evaluation of individual cells over time would show 572 Journal of Leukocyte Biology Volume 83, March

10 that most of the cells would lose function. Clearly effector cells continue to be recruited to the lungs over a period of days following infection (e.g., refs. [2, 3, 35, 36]). Together, these new emigrants along with cells that are potentially resistant to loss of function could mediate viral clearance at early times following infection. At first, the dampening of the CD8 T cell response in the face of ongoing infection would appear to be detrimental to the host, as in many cases, eradication of the pathogen is critical to host survival. Our results are consistent with the possibility that the immunosuppressive nature of the lung can continue even in the face of infection. However, equally important to viral clearance may be limiting damage to the lung tissue. Although lysis of epithelial cells may seem a logical explanation for the immunopathology observed during the course of the immune response, recent data suggest that it is in fact the production of TNF- by CD8 T cells that may be the most damaging [11, 37]. TNF- mediates these effects via triggering epithelial cells to produce chemokines, which in turn, recruit inflammatory cells [38]. In this regard, TNF- production by effector cells is among the functions that were lost in the lung resident cells. Thus, it is tempting to speculate that a major goal of CD8 T cell inactivation in the lung may be to limit its ability to promote the TNF- -mediated inflammatory response. How the loss of function affects pathogen clearance is unknown in the model of SV5 infection, as at this time, an approach to prevent functional impairment in the cells is lacking. Mice do clear SV5; however, as with many paramyxoviruses, SV5 does not grow to high titers in mice. However, given the seeming complete loss of function in affected cells in the lung, i.e., loss of lytic capability, IFN- production, and TNF- production, it is highly unlikely that these cells contribute to clearance. The effect of functional inactivation on virus clearance is likely determined by the number of effector cells generated following infection and the number required for efficient clearance. As such, the impact of loss of function on clearance may differ among viruses. Further, in addition to determining whether the virus can be cleared, the loss of function in these cells may impact the kinetics of clearance; the latter would impact the duration of illness. In summary, these studies suggest a significant addition to the paradigm associated with the loss of function in virusspecific lung effector cells that occurs following respiratory infection. We and others had proposed that the nonresponsiveness detected in these cells was the result of changes in the lung environment induced by infection or alternatively, that it was the direct effect of a viral protein. However, the data reported here suggest that infection-independent effects may be involved. We find that the ability to trigger loss of function is an inherent property of the lung environment. Further, we propose that the retention of maximal function may require modification of this environment. Effector cells responding to infectious agents where such modifications have not occurred, as may be the case for SV5, will be susceptible to negative regulatory effects following entry into the lung. Understanding the mechanism responsible for the loss of function may lead to therapeutics that can prevent this outcome in cases where an increased number of effectors are desired or promote it in cases where pathogenic effector cells are present. ACKNOWLEDGMENTS This work was supported by NIH grants HL (to M. A. A-M.) and F32 AI (to E. M. P.). We are grateful to Dr. Eric Pamer for provision of the Thy1.1 L9.6 Tg mice and Dr. Beth Hiltbold for provision of Listeria. We thank Dr. Beth Hiltbold, Dr. Charles Kroger, and Dr. Griff Parks for helpful critical reading of the manuscript. REFERENCES 1. Hou, S., Doherty, P. C., Zijlstra, M., Jaenisch, R., Katz, J. M. (1992) Delayed clearance of Sendai virus in mice lacking class I MHC- restricted CD8 T cells. J. Immunol. 149, Flynn, K. J., Belz, G. T., Altman, J. D., Ahmed, R., Woodland, D. L., Doherty, P. C. (1998) Virus-specific CD8 T cells in primary and secondary influenza pneumonia. Immunity 8, Gray, P. M., Parks, G. D., Alexander-Miller, M. A. (2003) High avidity CD8 T cells are the initial population elicited following viral infection of the respiratory tract. J. Immunol. 170, Gray, P. M., Arimilli, S., Palmer, E. M., Parks, G. D., Alexander-Miller, M. A. (2005) Altered function in CD8 T cells following paramyxovirus infection of the respiratory tract. J. Virol. 79, Chang, J., Braciale, T. J. (2002) Respiratory syncytial virus infection suppresses lung CD8 T-cell effector activity and peripheral CD8 T-cell memory in the respiratory tract. Nat. Med. 8, Vallbracht, S., Unsold, H., Ehl, S. (2006) Functional impairment of cytotoxic T cells in the lung airways following respiratory virus infections. Eur. J. Immunol. 36, Claassen, E. A., van der Kant, P. A., Rychnavska, Z. S., van Bleek, G. M., Easton, A. J., van der Most, R. G. (2005) Activation and inactivation of antiviral CD8 T cell responses during murine pneumovirus infection. J. Immunol. 175, Bonfield, T. L., Konstan, M. W., Burfeind, P., Panuska, J. R., Hilliard, J. B., Berger, M. (1995) Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin-10, which is downregulated in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 13, Xu, W., Zheng, S., Dweik, R. A., Erzurum, S. C. (2006) Role of epithelial nitric oxide in airway viral infection. Free Radic. Biol. Med. 41, Bingisser, R. M., Holt, P. G. (2001) Immunomodulating mechanisms in the lower respiratory tract: nitric oxide mediated interactions between alveolar macrophages, epithelial cells, and T-cells. Swiss Med. Wkly. 131, Bruder, D., Srikiatkhachorn, A., Enelow, R. I. (2006) Cellular immunity and lung injury in respiratory virus infection. Viral Immunol. 19, Holt, P. G., Oliver, J., Bilyk, N., McMenamin, C., McMenamin, P. G., Kraal, G., Thepen, T. (1993) Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J. Exp. Med. 177, Bingisser, R. M., Tilbrook, P. A., Holt, P. G., Kees, U. R. (1998) Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway. J. Immunol. 160, Huang, M., Sharma, S., Mao, J. T., Dubinett, S. M. (1996) Non-small cell lung cancer-derived soluble mediators and prostaglandin E2 enhance peripheral blood lymphocyte IL-10 transcription and protein production. J. Immunol. 157, Ding, L., Linsley, P. S., Huang, L. Y., Germain, R. N., Shevach, E. M. (1993) IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J. Immunol. 151, Soltys, J., Bonfield, T., Chmiel, J., Berger, M. (2002) Functional IL-10 deficiency in the lung of cystic fibrosis (cftr( / )) and IL-10 knockout mice causes increased expression and function of B7 costimulatory molecules on alveolar macrophages. J. Immunol. 168, Macatonia, S. E., Doherty, T. M., Knight, S. C., O Garra, A. (1993) Differential effect of IL-10 on dendritic cell-induced T cell proliferation and IFN- production. J. Immunol. 150, Groux, H., Bigler, M., de Vries, J. E., Roncarolo, M. G. (1996) Interleukin-10 induces a long-term antigen-specific anergic state in human CD4 T cells. J. Exp. Med. 184, Arimilli et al. The normal lung induces effector nonresponsiveness 573