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* Beirne B. Carter Center for Immunology Research, and Departments of
Microbiology and
Pathology, University of Virginia Health Sciences Center, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After experimental intranasal (i.n.)3 influenza virus infection of mice, virus replication is restricted primarily to the respiratory epithelium (12) and is cleared within 10 days (13). Therefore, influenza virus infection of the respiratory tract should serve as a useful model for the analysis of early events in the activation/differentiation of CD8+ T cells responding to virus infection localized to a discrete peripheral site. Because the activation of naive T cells is believed to occur within organized secondary lymphoid tissues (14), e.g., the draining mediastinal lymph nodes (MLN) in the case of respiratory influenza virus infection, viral Ag must gain access to these lymphoid compartments and be presented to naive CD8+ T cells by professional APCs, e.g., dendritic cells (DC). Indeed, it has been reported that DC isolated from the MLN of influenza virus-infected mice are capable of stimulating virus-specific T cell responses (15). Recently, the migration of respiratory DC from the infected lungs to the draining regional lymph nodes in response to i.n. influenza virus infection has been documented (16).
The analysis of early events in the activation and differentiation of naive CD8+ T cells within secondary lymphoid tissues is hampered by the low frequency of Ag-specific CD8+ T cell precursors within the pool of naive T cells present in normal nonimmune animals (13). This limitation has made it difficult to precisely evaluate the tempo of naive CD8+ T cell activation/proliferation and the accumulation of effector T cells at the site of infection, as well as the relationship between T cell activation and cell division and the acquisition of effector functions. This limitation of naive T cell frequency has been overcome by the analysis of the response of naive TCR transgenic T cells to antigenic stimulation in vivo after adoptive transfer of the T cells into naive recipient mice (17, 18, 19). This approach has been successfully used to analyze the response of influenza-specific CD4+ T cells in vivo during pulmonary influenza virus infection (20).
To evaluate early steps in the activation, differentiation, and migration of CD8+ T cells responding to pulmonary influenza virus infection, we have analyzed the response of adoptively transferred CD8+ TCR transgenic clone 4 T cells in recipient mice to i.n. infection with the A/PR/8/34 (PR/8) influenza virus strain. This TCR transgenic T cell population, which is directed to a dominant epitope displayed by the PR/8 hemagglutinin (HA) (21), has been previously used to analyze aspects of CD8+ T cell tolerance or autoimmunity (21, 22, 23, 24), tumor development (25), and CD8+ T cell mediated lung injury during infection (26).
In this report, we have examined the early phase of the CD8+ T cell response of transferred clone 4 T cells to influenza virus infection (days 2–7). We demonstrate that the induction of the CD8+ T cell response occurs primarily in the draining MLN. In the MLN, activated CD8+ T cells acquire effector activities. Analysis of the kinetics of the T cell response suggests that effector CD8+ T cells generated in the MLN not only migrate to the infected lungs but also seed other secondary lymphoid tissues, e.g., the spleen and nondraining peripheral lymph nodes (NDLN). Finally, we provide evidence that CD8+ T cell encounter with viral Ag in the draining MLN may be limited despite Ag persistence in the lymph nodes more than 5 days after infection. The implications of these findings are discussed.
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Materials and Methods |
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Clone 4 TCR transgenic mice (H-2d; Thy-1.2) were a generous gift from Dr. R. W. Dutton (Trudeau Institute, Saranac Lake, NY) (27). These mice were bred and housed in a pathogen-free environment and used at 8–11 wk of age for experiments. Thy-1.1+/+-BALB/c mice were a kind gift from Dr. R. I. Enelow (Yale University, New Haven, CT). These mice were bred and housed in a pathogen-free environment and used at 10–13 wk of age for experiments.
Adoptive transfer experiments
Naive CD8+ T cells were purified from spleens of clone 4 mice by negative selection column separation techniques (R&D Systems, Minneapolis, MN) according to manufacturer’s instructions. Within the purified population, >95% of CD8+ T cells exhibited a naive phenotype with respect to CD69, CD62L, and CD25 expression. Purified CD8+ T cells were labeled with 10 µM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes, Eugene, OR) for 10 min at room temperature. Labeled cells were washed with equal volumes of FBS (Atlanta Biologicals, Norcross, GA) and Iscove’s media (Invitrogen, Carlsbad, CA), then again with serum-free IMEM. A total of 8.5 x 106 CFSE-labeled CD8+ T cells were i.v. injected into the tail vein of individual Thy-1.1+/+-BALB/c recipients. Recipient mice were infected with influenza virus 24 h later.
Infection of mice
Influenza A/PR/8/34 (H1N1) was grown in day 10 chicken embryo allantoic cavities. Mouse-adapted influenza A/HK/1/68 (H3N2) was passaged several times in mouse lungs before a final preparation was grown in day 10 chicken embryo allantoic cavities. Mice were lightly anesthetized by Metofane inhalation (Janssen Pharmaceutica, Toronto, ON) and sublethally infected by i.n. inoculation (in 50 µl) with either 350 egg infectious doses (EID) of influenza A/PR/8/34 or 24 EID of control influenza A/HK/1/68.
Preparation of tissue lymphocytes
At multiple days after influenza virus infection, mice were sacrificed by cervical dislocation. Lungs were perfused via the right ventricle of the heart with 5–10 ml of PBS containing 10 U/ml heparin (Sigma-Aldrich, St. Louis, MO) to remove blood lymphocytes from the lung vasculature. The lungs, spleen, and lymph nodes (draining MLN and nondraining popliteal, inguinal, mesenteric, and axillary lymph nodes) were dissected and placed into cold IMEM. Spleen and lung cell preparations were made by passing tissue through a steel screen. Lymph node cells were gently prepared by manual tissue disruption. Tissue debris was removed by centrifugation at 300 x g. Cells were counted and resuspended at appropriate concentrations for each particular experiment.
Peptide
Synthetic A/PR/8/34 HA peptide spanning residues 533–541 (HA533–541) (IYSTVASSL) was synthesized by the University of Virginia Biomolecular Research Facility.
Ex vivo Ag sensor assay
To detect the presence of Ag in MLN at various times after infection, MLN were harvested from Thy-1.1+/+-BALB/c mice at multiple days after infection with influenza A/PR/8/34 or 5 days after influenza A/HK/1/68 infection. MLN cells (1 x 106) were incubated in 96-well round-bottom plates (Corning, Corning, NY) with 2 x 105 purified naive CFSE-labeled clone 4 CD8+ T cells in IMEM medium supplemented with 10% FBS, 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, 2 mM L-glutamine, and 0.05% 2-ME. In some experiments, 10 µg/ml anti-H-2Kd Ab (clone SF1-1.1) or control IgG2a Ab (clone MPC-11) was added to the cultures. After 3 days of incubation at 37°C, the cells were labeled with Abs against CD8
and Thy-1.2 and then analyzed for CFSE dilution by flow cytometry.
Ab labeling and flow cytometry
For cell surface labeling experiments, 
2 x 106 cells were incubated with the following Abs in the presence of anti-CD16/32 (clone 2.4G2): anti-CD8 (clone 53-6.7), anti-CD11a (clone M1/70), anti-CD25 (clone PC61), anti-CD49d (clone R1-2), anti-CD62L (clone MEL-14), anti-CD69 (clone H1.2F3), and anti-Thy-1.2 (clone 53-2.1) from BD Biosciences (San Diego, CA). Cells were labeled for 45 min at 4°C in staining buffer (PBS with 1% FBS, 0.02% NaN3). The cells were then fixed and erythrocytes lysed in FACS Lysing Solution (BD Biosciences). For intracellular staining, cells were incubated for 5 h in IMEM supplemented with 10% FBS, 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, 100 U/ml human IL-2, 2 mM L-glutamine 0.05% 2-ME, and 1 µg/ml brefeldin-A (Sigma-Aldrich) in the presence or absence of 1 µM HA533–541 peptide. After incubation, the cells were surface labeled with anti-CD8 and anti-Thy-1.2 as described earlier. Fixed cells were permeabilized in staining buffer supplemented with 0.5% saponin (Sigma-Aldrich). Permeabilized cells were labeled with anti-granzyme B (clone GB12; Caltag Laboratories, Burlingame, CA), anti-IFN-
(clone XMG1.2), or anti-TNF- (clone MP6-XT22) (BD Biosciences). For analysis of CD69 expression by proliferating MLN and splenic T cells, cell suspensions of MLN and spleen tissue were prepared from adoptive transfer recipients after 5 days of infection. Cells (1–2 x 106) were incubated with or without 50 nM HA533–541 peptide for 6 h in IMEM medium supplemented with 10% FBS, 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, 2 mM L-glutamine, and 0.05% 2-ME. Cells were also stimulated with P815 mastocytoma cells (H-2d) infected with A/PR/8/34 for 6 h. Cell surface labeling using Abs against CD8, CD69, and Thy-1.2 was performed as described earlier. Flow cytometry data for each of the experiments were acquired using a BD FACSCaliber (BD Immunocytometry Systems, San Diego, CA) and analyzed using CellQuest software (BD Biosciences).
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Results |
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To analyze the kinetics of viral Ag-specific CD8+ T cell proliferation and recruitment during pulmonary virus infection in the murine model, we used an adoptive transfer approach using influenza A/PR/8/34 (PR/8) virus-specific TCR transgenic donor CD8+ T cells. Purified, naive transgenic clone 4 CD8+ T cells were labeled with the cell division-sensitive fluorescent vital dye CFSE to assess T cell proliferation in vivo by dye dilution in the standard flow cytometry based assay (28). Approximately 8.5 x 106 purified CFSE-labeled Thy-1.2+ clone 4 CD8+ T cells were adoptively transferred into Thy-1 disparate recipient mice (Thy-1.1+/+-BALB/c), allowing early detection and quantitation of donor CD8+ T cells in vivo based on Thy-1.2 isoform expression. One day after cell transfer, recipient mice were inoculated i.n. with a sublethal dose of PR/8 virus. CFSE fluorescence intensity and Thy-1.2 expression were then monitored in the lungs and various lymphoid tissues of infected recipients over 24 h intervals between days 2 and 7 of infection to identify and assess the proliferative expansion and tissue localization of the responding CD8+ T cells.
After transfer, donor T cells were detectable throughout the lymphoid tissue of recipient mice, including the lung-draining MLN, peripheral NDLN, and spleen. Few donor T cells were detected in the lungs of uninfected recipient mice at any time after cell transfer or in infected mice during the first 4 days of infection (Fig. 1). No evidence of cell division, i.e., CFSE dilution, was observed in any site until day 4 of infection when extensive proliferation was evident exclusively in the MLN (Fig. 1A). This proliferative response was associated with a 10-fold increase in total donor T cells in the draining MLN between days 3 and 4 of infection (Fig. 1B). The evidence of extensive proliferation, i.e., up to 7–8 cell divisions, (Fig. 1A) and donor T cell accumulation in the draining lymph nodes (Fig. 1B) between days 3 and 4 of infection suggests a division rate for these CD8+ T cells of 3–6 h.
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At days 6–7 postinfection, there was extensive accumulation of CFSElow donor CD8+ T cells in the infected lungs (150-fold increase in total cells in the lungs between days 4 and 7; Fig. 1B). A further decrease of CFSE intensity in donor CD8+ T cells in the lungs occurred between days 5 and 7 of infection (Fig. 1A). This may reflect further proliferation of recruited effector CD8+ T cells in the infected lungs (see below), or could also reflect the contribution of donor T cells that had undergone greater than eight divisions in the draining MLN before their migration to the infected lungs during the latter stages of infection. In addition, at days 5–7 we repeatedly observed in both the MLN, and to a lesser extent in the spleen and the NDLN, the presence of a small but significant number of donor T cells which had undergone fewer than six divisions (CFSEmoderate) (Fig. 1A). These proliferating T cells could reflect the delayed proliferation of local CFSEhigh T cells resident in the lymph nodes and/or spleen. Alternatively, they may represent the recruitment of recirculating naive donor T cells to the MLN, with the subsequent dissemination of the early activated T cells to these other lymphoid tissues.
Activation and adhesion marker expression by responding CD8+ T cells
To evaluate the phenotypic evolution of the donor CD8+ T cells during their activation and proliferation in response to virus infection, we analyzed the expression of several cell surface markers linked to T lymphocyte activation (CD69, CD25) and T lymphocyte adhesion/homing (CD62L, CD11a, CD49d) on the donor CD8+ T cells in the lymph nodes, spleen, and the lungs at various times after infection (Fig. 2). CD69 is an early marker of T cell activation whose expression is dependent on TCR engagement (31, 32, 33, 34, 35). In the draining MLN, we observed up-regulation of CD69 on a fraction (14%) of the CFSEhigh undivided donor T cells at day 2 of infection, with the majority of the as yet undivided donor CD8+ T cells expressing CD69 by day 3 of infection (Fig. 2A). CD62L (L-selectin), the T cell lymph node homing receptor (36, 37, 38), is rapidly shed from the surface of T cells shortly after activation (39, 40). CD62L expression progressively fell on the MLN resident undivided donor CD8+ T cells between days 2 and 3 of infection in parallel with the increase in CD69 expression (Fig. 2A). CD62L expression remained low on the majority of the CFSEmoderate/low dividing donor T cell population through days 4–5. The -chain of the high affinity IL-2R (CD25) was also up-regulated initially in undivided CFSEhigh donor T cells within the draining MLN, and CD25 expression was sustained at a high level up to day 5 of infection among the dividing T cell population (Fig. 2A). The LFA-1 integrin -chain, CD11a, and the VLA-4 integrin -chain, CD49d, were expressed at low levels on undivided (CFSEhigh) donor T cells and were increased in expression on dividing cells (Fig. 2A). These integrins are important for the interaction of activated T cells with vascular endothelium at sites of inflammation (41, 42, 43) and have been directly implicated in T cell recruitment to the lung (44, 45, 46, 47, 48, 49).
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When donor cells first appeared in significant numbers in the lungs at day 5, the T cells were uniformly CFSElow, suggesting that they had been derived from precursor cells undergoing greater than five divisions (Fig. 2C). These T cells exhibited the phenotype characteristic of activated effector CD8+ T cells (CD62Llow, CD25high, CD11ahigh, CD49dhigh; Fig. 2C) that was shared by comparable CFSElow T cells in the MLN and spleen (Fig. 2, A and C). Furthermore, the high expression levels of CD11a and CD49d may contribute to the enhanced migration efficiency of activated T cells into the lung compared with naive T cells (Figs. 1 and 2).
Effector activity of activated proliferating CD8+ T cells
The previously mentioned results suggested that during i.n. influenza virus infection, the draining MLN were the primary source of activated CD8+ T cells that homed to the infected lungs as well as the spleen, at least through day 5 of infection. Because the production of effector cytokines in response to antigenic stimulation and cell-associated killing activity is a hallmark of activated CTL with antiviral effector activity (1, 2), we examined the relationship between proliferation status (extent of cell division) and the expression of soluble effector molecules by proliferating donor CD8+ T cells in the draining MLN. To this end, we adoptively transferred naive CFSE-labeled clone 4 CD8+ T cells into Thy-1 congenic recipient mice as performed previously in Figs. 1 and 2. On day 4 following i.n. PR/8 virus infection, MLN cell suspensions were cultured for 5 h in vitro with or without the synthetic cognate PR/8 HA533–541 peptide. At the end of brief in vitro culture, the CD8+ T cells were examined by flow cytometry for intracellular expression of IFN-, TNF-, and granzyme B.
As Fig. 3 demonstrates, we detected spontaneous IFN- synthesis from a fraction (36%) of the proliferating (CFSEmoderate/low) donor CD8+ T cells in the MLN at day 4 postinfection. This low level of spontaneous IFN- synthesis was restricted primarily to the dividing T cell population, as <10% of the undivided (CFSEhigh) T cells produced the cytokine. After acute in vitro antigenic stimulation, the vast majority (>80%) of the dividing CD8+ T cells produced high levels of IFN-, and a significant fraction (42%) of the undivided (CFSEhigh) T cells produced the cytokine in response to peptide stimulation. In contrast, there was minimal TNF- production by the donor CD8+ T cells in the absence of antigenic stimulation, but both dividing and undivided cells produced this cytokine in response to antigenic stimulation at frequencies comparable to that of IFN- (Fig. 3). The cytolytic granule-associated protease granzyme B was detected solely in the proliferating T cells. There was a trend toward higher levels of granzyme B expression with increasing cell division (Fig. 3), although the level of expression of this lytic molecule was not enhanced by specific acute in vitro antigenic stimulation. Overall, these data suggest that activated CD8+ T cells within the draining MLN are fully capable of expressing effector activity upon contact with Ag.
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The previously discussed results suggested that the proliferating CD8+ T cells in the draining MLN were the primary source of fully differentiated effector CD8+ T cells which emigrated from the lymph nodes and localized to the infected lungs and spleen. Supporting this concept, we found that the expression of most activation and adhesion/homing markers was comparable for lung and spleen homing effector CD8+ T cells (Fig. 2). There was, however, one notable exception. CD69, a marker of early T cell activation/acute TCR engagement, was expressed on the majority of CD8+ T cells detected in the infected lungs on day 5 of infection (Fig. 2C). This finding is consistent with the concept that exposure of these effector T cells to viral Ag in the infected lungs would result in TCR engagement and acute up-regulation of CD69 on these T cells. By contrast, at day 5 of infection the vast majority of CFSElow (greater than six cell divisions) CD8+ T cells present in the spleen did not express CD69 (Fig. 2B).
Analysis of CD69 expression on donor CD8+ T cells in the MLN at days 4–5 of infection revealed a much more complex pattern (Fig. 2A). At this site, high CD69 expression was evident on the undivided (CFSEhigh) donor T cell subpopulation, but expression fell progressively over successive cell divisions (day 4). Cells that had undergone greater than five divisions (day 5) demonstrated the CD69– phenotype comparable to effector CD8+ T cells in the spleen where viral Ag appears to be limited early after infection (Fig. 2, A and B). CD69 expression on T cells is linked to TCR engagement by Ag (31, 32, 33, 34, 35). In the current circumstance, CD69 levels fell progressively with succeeding cell divisions, suggesting that the majority of activated CD8+ T cells were no longer accessible to a reservoir of viral Ag still accessible to the undivided (CFSEhigh) CD8+ T cells present in the MLN at this time.
To more directly establish a link between CD69 expression and specific antigenic stimulation, we conducted adoptive transfers of naive donor CD8+ T cells into control mice subsequently infected i.n. with an antigenically distinct (H3N2) influenza A/HK/1/68 virus whose HA is not recognized by the TCR transgenic clone 4 T cells. At day 4 postinfection, lymphoid tissues were harvested and donor CD8+ T cells resident in these sites were examined for expression of CD69, CD62L, and CD25. As Table I demonstrates, donor CD8+ T cells harvested from the MLN, spleen, and NDLN exhibited a naive CD69– phenotype shared by donor clone 4 CD8+ T cells harvested from uninfected recipient mice. Thus, the inflammatory stimulus produced by infection with a control influenza virus strain did not result in nonspecific CD69 expression on the CD8+ T cell population.
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Because in vitro Ag exposure of the CD69–, CFSEmoderate/low T cells in the MLN resulted in the rapid expression of this early T cell activation marker, it seems likely that at days 4–5 of infection, these T cells were not in direct contact with Ag in vivo. However, as indicated above (Fig. 2A), we noted a direct correlation between CFSE content and the level of CD69 expression during this time period. This suggested that undivided T cells and T cells early in their proliferative cycle (fewer than three to four divisions) may still have been in contact with Ag still present in the MLN at day 5 of infection.
To determine the presence and extent of persistence of viral Ag in the draining MLN, we prepared MLN cell suspensions from Thy-1.1+/+-BALB/c mice at different days after i.n. PR/8 virus infection. These cells were cocultured with purified naive CFSE-labeled clone 4 CD8+ T cells for 3 days, and T cell proliferation was determined by dye dilution. Controls for this analysis included the culture of labeled naive T cells alone or with MLN cell suspensions from mice infected with the antigenically distinct influenza A/HK/1/68 virus strain. As Fig. 5A demonstrates, day 5 postinfection MLN cell suspensions from PR/8-infected mice were able to support proliferation of naive CD8+ T cells. Viral Ag persistence in the draining lymph nodes appeared to be of limited duration, as the proliferative response of the labeled CD8+ T cells rapidly declined to almost background levels when cocultured with whole MLN cell suspensions harvested at successive days up to day 12 of infection (Fig. 5A). The Ag specificity of the naive T cell proliferative response was indicated by the absence of significant proliferation by naive-labeled CD8+ T cells cultured alone or with MLN cell suspensions from lymph node donors infected with the control influenza A/HK/1/68 virus strain (Fig. 5A).
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Discussion |
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After adoptive transfer of the TCR transgenic CD8+ T cells, donor cells were located throughout the lymphoid tissues of recipient mice (Fig. 1), as was expected for naive CD62L+ T cells (50). Donor T cells located in the MLN demonstrated activation by day 2 postinfection, as CD69 was rapidly up-regulated on donor T cells before their division within the MLN (Fig. 2A). CD25 expression and CD62L shedding was also rapidly induced on activating CD8+ T cells before their division in the MLN (Fig. 2A). Donor T cells divided rapidly between days 3–4 after infection exclusively in the MLN. This exclusive proliferation of T cells in the MLN early after infection correlates with the preferential accumulation of migrating airway DC to the draining regional lymph nodes during influenza virus infection, which occurs within 24 h after i.n. inoculation (16). Proliferation of naive clone 4 T cells in vitro is first evident 2 days after stimulation by cognate Ag (data not shown), suggesting naive T cells require 2 days for the cell division machinery to be fully activated to complete a cell division. Induction of T cell division in vivo was relatively delayed, however, due to the time required for airway DC to both deliver viral Ag from the lungs and initiate contact with T cells in the draining lymph nodes. As previously reported for TCR transgenic CD4+ T cells responding in vivo to influenza virus infection (20), division of the T cells in the MLN was asynchronous. Hence, we were able to detect T cells over a range of divisions at days 3–4 of infection by the laddering of CFSE intensities. We have preliminary evidence suggesting that the asynchronous T cell proliferation may reflect the continual asynchronous recruitment of circulating naive T cells into the MLN (H. Yoon and T.J. Braciale, unpublished observations). As such, T cells with fewer divisions likely migrated into the MLN slightly later and therefore activated more recently than earlier immigrant T cells in the MLN that have undergone several additional divisions.
In contrast to the proliferation of T cells in the MLN, there was no evidence of division of resident CD8+ T cells in any other tissues examined up to the fourth day after infection. We detected a small number of transferred, nondividing donor T cells resident in the lung over the first 4 days after infection (Fig. 1A). These resident lung CD8+ T cells were not stimulated to divide in situ during infection of the lung and remained CFSEhigh despite extensive virus replication in the lungs (data not shown). This suggests that the environment of the infected lung is unsuitable for naive T cell activation. This finding may be related to a variety of factors, including DC maturation state, site(s) of naive T cell localization, and local cytokine milieu in the infected lungs. Only after the activation and multiple divisions of cells in the MLN did we detect significant numbers of donor T cells accumulating in a synchronous manner in the lung by day 5 postinfection.
Dividing CD8+ T cells were also found to leave the MLN and not only populate the lungs, but also the spleen and peripheral lymph nodes. The activated CD8+ T cells that populated the spleen at day 5 of infection differed in cell surface phenotype from the migrating T cells trafficking to the lungs at day 5 of infection. In particular, CD69 was not expressed on the majority of migrant CD8+ T cells detected in the spleen at day 5, whereas the majority of corresponding T cells in the lungs were CD69+. Acute CD69 up-regulation on T cells is linked to TCR engagement by Ag (Refs. 30, 31, 32, 33, 34 and Fig. 4). When antigenic stimulation is withdrawn, CD69 expression subsequently declines (31, 35). It is therefore likely that the absence of TCR stimulation by Ag in the spleen (vs the presence of viral Ag in the infected day 5 lungs) accounts for the difference in CD69 expression between activated CD8+ T cells migrating to these sites.
We observed that the absolute number of responding CD8+ T cells in the lungs continued to increase up to day 7 of infection, whereas the division driven expansion of donor CD8+ T cells in the draining MLN and the accumulation of CFSElow migrant CD8+ T cells in the spleen leveled off between days 5–7 of infection. This continued accumulation of activated CD8+ T cells in the lungs likely reflects the ongoing recruitment of activated T cells from the MLN to the infected lungs. We cannot, however, readily explain the absence of a similar continued accumulation on MLN-derived T cells in the spleen. It is possible that activated CD8+ T cells continue to traffic to the spleen, but in the absence of continued antigenic stimulation at this site, activated T cells in the spleen die from neglect at a rate comparable to the rate of accumulation. An alternate, perhaps more intriguing explanation for the selective continued accumulation of these activated T cells in the lungs is that the CD8+ T cells undergo one or more rounds of division (perhaps in response to viral Ag) after migration to the infected lungs. The progressive loss of CFSE intensity of donor Thy-1.2+ CD8+ T cells in the lungs between days 5 and 7 (Fig. 1A) is consistent with this interpretation.
Our findings on the response of transgenic CD8+ T cells to pulmonary influenza virus infection directly parallel results reported on the primary CD4+ T cell response to influenza with respect to the tempo of activation and tissue distribution of naive and effector cells (20). However, in contrast to the reported results for influenza virus-specific transgenic CD4+ T cells (20), in which direct proliferative expansion of resident T cells in the spleen in response to infection was suggested, we have been able to identify the draining MLN as the likely primary or at least initial cellular source of activated CD8+ T cells in the spleen. We have also similarly demonstrated the early dissemination of acute effectors from the draining lymph nodes into multiple NDLN. We believe the CD8+ T cells migrating from the MLN likely also contribute substantially to the virus-specific populations detected in peripheral lymph nodes much later after influenza virus infection (11). The dissemination of such activated T cells throughout peripheral lymphoid tissue could ultimately contribute to the pool of central memory CD8+ T cells generated in response to primary pulmonary influenza virus infection.
Important CD8+ T cell effector functions in response to viral infections include the secretion of cytokines and proapoptotic granzymes (2, 51). To determine whether acquisition of these effector functions was initiated before T cell migration to the lung, we examined the effector status of CD8+ T cells in the draining MLN of virus-infected mice. Our results indicate that CD8+ T cells rapidly acquire the ability to produce cytokines and granzyme B during their activation in the MLN (Fig. 3). We coupled our protein analyses with CFSE labeling to further explore the relationship between effector molecule expression and cell division. Granzyme B production was very tightly associated with cell division (Fig. 3). T cells initiated granzyme B expression after the first division, with increasing levels of the effector protease made following each successive division. In addition, granzyme B production was evident without in vitro stimulation, suggesting that accumulation of the cytolytic enzyme (i.e., within granules) is part of a developmental program following T cell activation. Several previous studies have suggested that cytokine expression depends on division (52, 53, 54, 55). Indeed, after in vitro stimulation with specific peptide, T cells that had divided produced high levels of both IFN- and TNF- (Fig. 3), with similar frequencies of divided T cells producing either cytokine (80–81%) or granzyme B (75%). However, we detected a significant fraction of undivided T cells that also produced both cytokines after in vitro peptide stimulation, consistent with reports of cytokine production by activated T cells before the onset of cell division (55, 56, 57, 58). It remains possible that such cytokine producing cells have entered the cell cycle but not yet completed a cell division.
The expression dynamics of CD69 in the MLN provided a dramatic example of the environmental changes that CD8+ T cells experienced during their divisions there. CD69 up-regulation was detected on a fraction (14%) of donor CFSEhigh CD8+ T cells in the MLN at day 2 of infection, with the majority of CFSEhigh cells expressing CD69 on day 3 before cell division (Fig. 2A). With the onset of cell division between day 3 and day 4, CD69 expression was retained at high levels on at least 50% of undivided CFSEhigh cells and was likewise high on early dividing cells (one to two divisions). At the same time, however, cells that had undergone three or more divisions demonstrated a gradient of decreasing CD69 expression with increasing cell division. This culminated at day 5, when the majority of CD8+ T cells that had undergone six or more divisions were CD69low/–. Nevertheless, the few remaining cells that had undergone fewer that six divisions displayed the inverse relationship between the number of cell divisions and CD69 intensity, i.e., a direct correlation between CFSE and CD69 intensities. Because the CFSElowCD69– T cells in the MLN at days 4 and 5 rapidly up-regulated CD69 in response to in vitro antigenic stimulation (Fig. 4), these proliferating T cells were not refractory to Ag-induced CD69 expression. This observation suggests that these activated T cells were proliferating in the absence of continued exposure to Ag. Consistent with this view, a number of recent reports have demonstrated the capacity of CD8+ T cells to undergo multiple rounds of division in an Ag-independent manner (59, 60, 61).
We also detected the presence of viral Ag for >5 days in the draining MLN following virus infection (Fig. 5). This finding of Ag persistence in the MLN reveals an interesting dichotomy. The CD69low/– phenotype of the multiply divided T cells in the MLN at days 4 and 5 implies that these CD8+ T cells are not exposed to Ag. Yet, because undivided and early division CD8+ T cells display a CD69high phenotype, these T cells are most likely exposed to viral Ag in the MLN. To explain this dichotomy and account for the CD69low/– phenotype of the CD8+ T cells undergoing Ag-independent proliferation, we suggest that after one to two divisions, activated CD8+ T cells are no longer in physical contact with viral Ag-presenting APC. A recent report (62) investigating the anatomy of Ag presentation to T cells within lymph nodes supports this concept. In that model, Ag-bearing DC in protein-immunized mice were found to be sequestered in a specific area of the draining lymph nodes. These DC were preferentially localized near high endothelial venules in the outer paracortex, which placed Ag presentation immediately proximal to the T cell entry site for the lymph node. T cells were later found scattered deeper in the Ag-free paracortex at a time when T cells had undergone several divisions. Such a mechanism of Ag partitioning away from proliferating T cells after one to two divisions may occur within the draining lymph nodes of influenza virus-infected mice and account for the difference in CD69 expression between CFSEhigh and CFSElow donor CD8+ T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas J. Braciale, Beirne B. Carter Center for Immunology Research, University of Virginia Health Sciences Center, Medical Research Building 4, Health Sciences Center Box 801386, Charlottesville, VA 22908. E-mail address: tjb2r{at}virginia.edu
3 Abbreviations used in this paper: i.n., intranasal; MLN, mediastinal lymph node; NDLN, nondraining lymph node; HA, hemagglutinin; DC, dendritic cell; EID, egg infectious dose.
Received for publication February 26, 2004. Accepted for publication May 5, 2004.
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References |
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by influenza hemagglutinin-specific CD8 effector T cells influences the development of pulmonary immunopathology. Am. J. Pathol. 158:119.4 integrins regulate independent pathways of T lymphocyte recruitment in the pulmonary immune response. J. Immunol. 161:4396.
integrin-expressing T lymphocytes are associated with immunity to pulmonary Mycobacterium tuberculosis infection. J. Immunol. 164:4853. or IL-4. J. Immunol. 166:112.This article has been cited by other articles:
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  J. D. Mintern, S. Bedoui, G. M. Davey, J. M. Moffat, P. C. Doherty, and S. J. Turner Transience of MHC Class I-restricted antigen presentation after influenza A virus infection PNAS, April 21, 2009; 106(16): 6724 - 6729. [Abstract] [Full Text] [PDF]
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 A. E. Gelman, W. Li, S. B. Richardson, B. H. Zinselmeyer, J. Lai, M. Okazaki, C. G. Kornfeld, F. H. Kreisel, S. Sugimoto, J. R. Tietjens, et al. Cutting Edge: Acute Lung Allograft Rejection Is Independent of Secondary Lymphoid Organs J. Immunol., April 1, 2009; 182(7): 3969 - 3973. [Abstract] [Full Text] [PDF]
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E. Ingulli, C. Funatake, E. L. Jacovetty, and M. Zanetti Cutting Edge: Antigen Presentation to CD8 T Cells after Influenza A Virus Infection J. Immunol., January 1, 2009; 182(1): 29 - 33. [Abstract] [Full Text] [PDF]
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V. Robila, M. Ostankovitch, M. L. Altrich-VanLith, A. C. Theos, S. Drover, M. S. Marks, N. Restifo, and V. H. Engelhard MHC Class II Presentation of gp100 Epitopes in Melanoma Cells Requires the Function of Conventional Endosomes and Is Influenced by Melanosomes J. Immunol., December 1, 2008; 181(11): 7843 - 7852. [Abstract] [Full Text] [PDF]
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E. L. Brincks, A. Katewa, T. A. Kucaba, T. S. Griffith, and K. L. Legge CD8 T Cells Utilize TRAIL to Control Influenza Virus Infection J. Immunol., October 1, 2008; 181(7): 4918 - 4925. [Abstract] [Full Text] [PDF]
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M. R. Jenkins, J. Mintern, N. L. La Gruta, K. Kedzierska, P. C. Doherty, and S. J. Turner Cell Cycle-Related Acquisition of Cytotoxic Mediators Defines the Progressive Differentiation to Effector Status for Virus-Specific CD8+ T Cells J. Immunol., September 15, 2008; 181(6): 3818 - 3822. [Abstract] [Full Text] [PDF]
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W. W. Reiley, M. D. Calayag, S. T. Wittmer, J. L. Huntington, J. E. Pearl, J. J. Fountain, C. A. Martino, A. D. Roberts, A. M. Cooper, G. M. Winslow, et al. ESAT-6-specific CD4 T cell responses to aerosol Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes PNAS, August 5, 2008; 105(31): 10961 - 10966. [Abstract] [Full Text] [PDF]
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C. C. Brinkman, S. L. Sheasley-O'Neill, A. R. Ferguson, and V. H. Engelhard Activated CD8 T Cells Redistribute to Antigen-Free Lymph Nodes and Exhibit Effector and Memory Characteristics J. Immunol., August 1, 2008; 181(3): 1814 - 1824. [Abstract] [Full Text] [PDF]
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 J. McGill, N. Van Rooijen, and K. L. Legge Protective influenza-specific CD8 T cell responses require interactions with dendritic cells in the lungs J. Exp. Med., July 7, 2008; 205(7): 1635 - 1646. [Abstract] [Full Text] [PDF]
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D. K. Meyerholz, M. Edsen-Moore, J. McGill, R. A. Coleman, R. T. Cook, and K. L. Legge Chronic Alcohol Consumption Increases the Severity of Murine Influenza Virus Infections J. Immunol., July 1, 2008; 181(1): 641 - 648. [Abstract] [Full Text] [PDF]
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R. Kushwah, H. Cao, and J. Hu Characterization of Pulmonary T Cell Response to Helper-Dependent Adenoviral Vectors following Intranasal Delivery J. Immunol., March 15, 2008; 180(6): 4098 - 4108. [Abstract] [Full Text] [PDF]
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M. A. Fernandez, F. K. Puttur, Y. M. Wang, W. Howden, S. I. Alexander, and C. A. Jones T Regulatory Cells Contribute to the Attenuated Primary CD8+ and CD4+ T Cell Responses to Herpes Simplex Virus Type 2 in Neonatal Mice J. Immunol., February 1, 2008; 180(3): 1556 - 1564. [Abstract] [Full Text] [PDF]
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C.-H. Shen, Q. Ge, O. Talay, H. N. Eisen, A. Garcia-Sastre, and J. Chen Loss of IL-7R and IL-15R Expression Is Associated with Disappearance of Memory T Cells in Respiratory Tract following Influenza Infection J. Immunol., January 1, 2008; 180(1): 171 - 178. [Abstract] [Full Text] [PDF]
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M. R. Jenkins, K. Kedzierska, P. C. Doherty, and S. J. Turner Heterogeneity of Effector Phenotype for Acute Phase and Memory Influenza A Virus-Specific CTL J. Immunol., July 1, 2007; 179(1): 64 - 70. [Abstract] [Full Text] [PDF]
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N. K. Polakos, I. Klein, M. V. Richter, D. M. Zaiss, M. Giannandrea, I. N. Crispe, and D. J. Topham Early Intrahepatic Accumulation of CD8+ T Cells Provides a Source of Effectors for Nonhepatic Immune Responses J. Immunol., July 1, 2007; 179(1): 201 - 210. [Abstract] [Full Text] [PDF]
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H. Yoon, K. L. Legge, S.-s. J. Sung, and T. J. Braciale Sequential Activation of CD8+ T Cells in the Draining Lymph Nodes in Response to Pulmonary Virus Infection J. Immunol., July 1, 2007; 179(1): 391 - 399. [Abstract] [Full Text] [PDF]
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 M. M. Anis, S. A. Fulton, S. M. Reba, C. V. Harding, and W. H. Boom Modulation of Naive CD4+ T-Cell Responses to an Airway Antigen during Pulmonary Mycobacterial Infection Infect. Immun., May 1, 2007; 75(5): 2260 - 2268. [Abstract] [Full Text] [PDF]
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S. L. Sheasley-O'Neill, C. C. Brinkman, A. R. Ferguson, M. C. Dispenza, and V. H. Engelhard Dendritic Cell Immunization Route Determines Integrin Expression and Lymphoid and Nonlymphoid Tissue Distribution of CD8 T Cells J. Immunol., February 1, 2007; 178(3): 1512 - 1522. [Abstract] [Full Text] [PDF]
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M. J. Dobrzanski, J. B. Reome, J. C. Hylind, and K. A. Rewers-Felkins CD8-Mediated Type 1 Antitumor Responses Selectively Modulate Endogenous Differentiated and Nondifferentiated T Cell Localization, Activation, and Function in Progressive Breast Cancer J. Immunol., December 1, 2006; 177(11): 8191 - 8201. [Abstract] [Full Text] [PDF]
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M. E. Wikstrom, E. Batanero, M. Smith, J. A. Thomas, C. von Garnier, P. G. Holt, and P. A. Stumbles Influence of Mucosal Adjuvants on Antigen Passage and CD4+ T Cell Activation during the Primary Response to Airborne Allergen J. Immunol., July 15, 2006; 177(2): 913 - 924. [Abstract] [Full Text] [PDF]
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J. K. Whitmire, N. Benning, and J. L. Whitton Precursor Frequency, Nonlinear Proliferation, and Functional Maturation of Virus-Specific CD4+ T Cells. J. Immunol., March 1, 2006; 176(5): 3028 - 3036. [Abstract] [Full Text] [PDF]
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M. Millrain, D. Scott, C. Addey, H. Dewchand, P. Ellis, I. Ehrmann, M. Mitchell, P. Burgoyne, E. Simpson, and J. Dyson Identification of the Immunodominant HY H2-Dk Epitope and Evaluation of the Role of Direct and Indirect Antigen Presentation in HY Responses J. Immunol., December 1, 2005; 175(11): 7209 - 7217. [Abstract] [Full Text] [PDF]
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 C. C. Clay, D. S. Rodrigues, D. J. Harvey, C. M. Leutenegger, and U. Esser Distinct Chemokine Triggers and In Vivo Migratory Paths of Fluorescein Dye-Labeled T Lymphocytes in Acutely Simian Immunodeficiency Virus SIVmac251-Infected and Uninfected Macaques J. Virol., November 1, 2005; 79(21): 13759 - 13768. [Abstract] [Full Text] [PDF]
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D. R. Drake III, R. M. Ream, C. W. Lawrence, and T. J. Braciale Transient Loss of MHC Class I Tetramer Binding after CD8+ T Cell Activation Reflects Altered T Cell Effector Function J. Immunol., August 1, 2005; 175(3): 1507 - 1515. [Abstract] [Full Text] [PDF]
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C. W. Lawrence, R. M. Ream, and T. J. Braciale Frequency, Specificity, and Sites of Expansion of CD8+ T Cells during Primary Pulmonary Influenza Virus Infection J. Immunol., May 1, 2005; 174(9): 5332 - 5340. [Abstract] [Full Text] [PDF]
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 S. Mrusek, S. Vallbracht, and S. Ehl The impact of splenectomy on antiviral T cell memory in mice Int. Immunol., January 1, 2005; 17(1): 27 - 33. [Abstract] [Full Text] [PDF]
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