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*Tuberculosis
The Journal of Immunology, 2007, 178: 976-985.
Copyright © 2007 by The American Association of Immunologists, Inc.

CD27low CD4 T Lymphocytes That Accumulate in the Mouse Lungs during Mycobacterial Infection Differentiate from CD27high Precursors In Situ, Produce IFN-{gamma}, and Protect the Host against Tuberculosis Infection1

Marina A. Kapina2,*, Galina S. Shepelkova2,*, Vladimir V. Mischenko*, Peter Sayles{dagger}, Polina Bogacheva*, Gary Winslow{ddagger}, Alexander S. Apt* and Irina V. Lyadova3,*

* Department of Immunology, Central Institute for Tuberculosis of the Russian Academy of Medical Sciences, Moscow, Russia; {dagger} Trudeau Institute, Saranac Lake, NY 12983; and {ddagger} Wadsworth Center, New York State Department of Health, Albany, NY 12201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of effector, IFN-{gamma} producing T lymphocytes and their accumulation at sites of infection are critical for host protection against various infectious diseases. The activation and differentiation of naive T lymphocytes into effector memory cells starts in lymphoid tissues, but it is not clear whether the Ag-experienced cells that leave lymph nodes (LN) are mature or if they undergo further changes in the periphery. We have previously shown that CD44highCD62Llow effector CD4 T lymphocytes generated during the course of mycobacterial infection can be segregated into two subsets on the basis of CD27 receptor expression. Only the CD27low subset exhibited a high capacity for IFN-{gamma} secretion, indicating that low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes. We demonstrate now that CD27low IFN-{gamma}-producing CD4 T lymphocytes accumulate in the lungs but are rare in LNs. Several factors contribute to their preferential accumulation. First, CD27low CD4 T lymphocytes present in the LN are highly susceptible to apoptosis. Second, circulating CD27low CD4 T cells do not enter the LN but efficiently migrate to the lungs. Third, CD27high effector CD4 T cells that enter the lungs down-regulate CD27 expression in situ. In genetically heterogeneous mice that exhibit varying susceptibility to tuberculosis, the accumulation of mature CD27low CD4 T cells in the lungs correlates with the degree of protection against infection. Thus, we propose that terminal maturation of effector CD4 T lymphocytes in the periphery provides the host with efficient local defense and avoids potentially harmful actions of inflammatory cytokines in lymphoid organs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Effector T lymphocytes play crucial roles in the protection of the host against various infectious agents. During tuberculosis (TB)4 infection, protection largely depends upon CD4 T lymphocytes and their capacity to produce IFN-{gamma} and to activate infected macrophages (1, 2, 3, 4, 5, 6). Therefore, to develop improved strategies of TB control, it is important to understand how effector T lymphocytes that produce IFN-{gamma} are generated, distributed, and regulated at sites exposed to the pathogen. These sites include lymphoid organs and peripheral tissues, in particular, the lung.

According to currently accepted models of T cell differentiation, effector T lymphocytes are generated from naive precursors in the draining lymph nodes (LNs), where naive cells engage their TCRs, receive costimulatory signals, and proliferate (7, 8). Ultimately, T lymphocytes acquire effector functions and reprogram expression of their homing receptors. In particular, they increase expression of the S1P sphingolipid receptor and down-regulate CD62L and CCR7, which allow the newly generated effector lymphocytes to egress from the LN (9) and migrate to the periphery (10, 11, 12, 13, 14). In the periphery, CD62LlowCCR7low cells represent a pool of effector memory lymphocytes that are able to mount strong and immediate responses (15). It is not clear, however, whether cells leaving the LN are fully mature or whether they undergo additional maturation in peripheral tissues.

Several reports have shown that populations of peripheral effector CD4 T cells are not phenotypically homogeneous (12, 16, 17). We have reported that CD62Llow effector CD4 T lymphocytes that accumulate in the lungs during mycobacterial infection can be segregated into two subsets that express or lack CD27 on their surface (18). These subsets differ in their functional activity; the CD27low CD4 T cells contain many more IFN-{gamma}-producing cells and express higher levels of IFN-{gamma} mRNA at the single cell level than CD27high cells. Thus, low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes.

CD27, a member of TNF receptor superfamily, is constitutively expressed by naive T lymphocytes and is rapidly up-regulated following cell stimulation via TCR/CD3 (19). Signaling through CD27 is essential for the survival of Ag-primed T lymphocytes (20). In the absence of CD27-mediated signals, peripheral pools of CD4 and CD8 effector lymphocytes are dramatically decreased (21). In contrast, hyperstimulation through CD27, which can occur in mice transgenic for the CD27 ligand, CD70, induces profound expansion of T lymphocytes and accumulation of effector cells in the periphery (22, 23). Although CD27 is required for the successful generation of effector T lymphocytes, mature effector cells lose CD27. Loss of CD27 is a characteristic feature of both cytotoxic and IFN-{gamma}-producing CD8 (24, 25) and CD4 (18, 26) T lymphocytes. What drives the down-regulation of CD27 and in which anatomical sites does this event take place remain unclear. To address these questions, we monitored survival, anatomical distribution, and migration patterns of the CD27high and CD27low subsets of CD62Llow effector CD4 T lymphocytes during mycobacterial infection in mice.

We show that CD27low CD4 T lymphocytes are mature and terminally differentiated cells that originate from CD27high precursors. In the LN, CD27low CD4 T cells are rare and susceptible to apoptosis. In the lungs, CD27low lymphocytes represent a major population of CD4 T cells and originate from CD27high CD4 T lymphocytes that down-regulate CD27 expression in situ. Accumulation of CD27low CD4 T cells in the lungs correlates with protection against fatal TB infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Inbred female C57BL/6 mice and female (A/Sn x I/St)F2 hybrid mice 2–3 mo of age were used. All mice were bred under conventional conditions at the Animal Facility of the Central Institute for Tuberculosis (Moscow, Russia), in accordance with Russian Ministry of Health Guideline no. 755 and National Institutes of Health Office of Laboratory Animal Welfare (OLAW) Assurance no. A5502-01. F2 hybrid mice were generated by crossing (A/Sn x I/St)F1 hybrid mice originating from A/JSnYCit (A/Sn) and I/StYCit (I/St) inbred mice. Water and food were provided ad libitum. All experimental procedures were approved by the institutional animal care committee of the Central Institute for Tuberculosis.

Bacteria and infections

Mice were infected with mid-log-phase Mycobacterium tuberculosis strain H37Rv Pasteur or Mycobacterium bovis bacillus Calmette-Guérin (BCG). The method for establishment and evaluation of clump-free mid-log-phase mycobacteria has been described (27, 28). Depending on the experiment, mycobacteria were given intratracheally (i.t.) (103 CFU per mouse in 50 µl of PBS; Ref. 29), s.c. (1–5 x 105 CFU per mouse in 200 µl of PBS), or i.v. (5 x 105 CFU per mouse in 200 µl of PBS). To assess mycobacterial load in LNs, spleens, and lungs, 0.1 ml of serial 10-fold dilutions of organ homogenates were plated onto Dubos Agar, and colonies were enumerated after 18–20 days.

Flow cytometry

Suspensions of lung cells were prepared using an enzyme digestion method (27). Lungs were perfused with 0.02% EDTA-PBS, incubated in RPMI 1640 containing collagenase/DNase, and cell suspensions were washed. LN and spleen cell suspensions were prepared by mild homogenization. Cells were stained with FITC-anti-CD44, PE-anti-CD27, PerCP-anti-CD4, allophycocyanin-anti-CD62L (BD Pharmingen), and biotin-conjugated anti-CD27 mAbs (eBioscience). To measure apoptosis, cells were stained with annexin V and 7-aminoactinomycin D (7-AAD; BD Pharmingen). Cells were analyzed using a BD Biosciences FACSCalibur with CellQuest (BD Biosciences) and FlowJo (Tree Star) software. In most experiments, 20,000 CD4+ events were analyzed.

For intracellular cytokine staining, 1.5 x 106 cells were cultured in 24-well plates in the presence of mycobacteria sonicate (8 µg/ml) and GolgiPlug (1 µl/ml; BD Pharmingen). After 10 h of culture, cells were stained using the Cytofix/Cytoperm kit (BD Pharmingen) and analyzed.

Adoptive transfer experiments

CD4 T lymphocytes were purified from the spleen by negative selection using a mouse CD4+ T cell isolation kit (Miltenyi Biotec). The purity of the CD4 T lymphocytes was always >95%. To isolate CD27low and CD27high cells, purified CD4 T lymphocytes were stained with PE-anti-CD27 mAbs (BD Pharmingen), incubated with anti-PE MultiSort MicroBeads (Miltenyi Biotec), washed, and separated by sequential passage through two LS MACS columns (Miltenyi Biotec). For the isolation of CD62Lhigh, CD62LlowCD27high, and CD62LlowCD27low cells, CD4 T cells were incubated with FITC-anti-CD62L mAbs, magnetically labeled with anti-FITC MultiSort MicroBeads (Miltenyi Biotec), and sorted into CD62Lhigh and CD62Llow subsets. The CD62Lhigh cells were largely CD27high (>95%). The CD62Llow cells comprised both CD27high and CD27low cells, which were further separated using PE-anti-CD27 mAbs and anti-PE MultiSort MicroBeads (Miltenyi Biotec), as described above. The purity of the separated cells was confirmed by flow cytometry after each step of cell purification. The final purity of the cell populations was ~98% for CD62LlowCD27high cells and ~95% for CD62LlowCD27low cells.

CFSE labeling and adoptive transfer

Spleens were isolated from infected mice and subsets of spleen CD4 T lymphocytes were purified using magnetic cell separation. The purified T cells (10 x 106/ml) were incubated with CFSE (0.5 µM for 10 min at room temperature) and washed thoroughly with PBS containing 10% FCS. The CFSE staining was controlled by flow cytometry and cells were injected i.v. into recipient mice (4 x 106 cells per mouse).

In vitro cultures

Subsets of CD4 T cells were isolated from the spleens of mice challenged with M. tuberculosis. Cells were cultured in 24-well plates (3–5 x 105 cells per well) in the presence of APCs (1.5 x 106 spleen cells per well), mycobacterial sonicate (8 µg/ml), and rIL-2 (1 ng/ml). The APCs were spleen cells isolated from noninfected B6 mice and treated with mitomycin C (Sigma-Aldrich). To allow exclusion from the flow cytometry analysis, APCs were labeled with CFSE. Cultured cells were harvested every 4–5 days, stained with mAbs, 7-AAD, and annexin V, and analyzed.

In vivo labeling of LN cells

Mice were euthanized by i.p. injection of hexenal (200 µl per mouse; 10 mg/ml), the skin in the axillary space was incised, brachial LNs were found, and 30–40 µl of 0.4 mM CFSE was injected under the LN capsule. The incision was closed and the mice were monitored.

Statistical analysis

Differences between the means of experimental groups were analyzed using the Kruskal-Wallis test with Dunn’s posttest. Differences were considered significant where p < 0.05. The correlation between body weight loss and the frequency of CD4 T cell subsets in the lungs of F2 hybrid mice was assessed using nonparametric (Spearman) correlation analysis (InStat software; GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD27low effector CD4 T cells accumulate in the lungs

Our previous observations indicated that the effector CD62Llow CD4 T lymphocytes that persist in the lungs during mycobacterial infection are composed of CD27high and CD27low subsets and that functionally mature cells with a high capacity for IFN-{gamma} secretion belong to the CD27low subset (18). In the present study we examined how CD27high and CD27low subsets of effector CD62Llow CD4 T lymphocytes are distributed between secondary lymphoid organs and lungs during the early stages of mycobacterial infection.

Because the distribution and phenotypic characteristics of CD4 effector cells in peripheral tissues may be mycobacterium dependent, mice were challenged with attenuated M. bovis BCG or virulent M. tuberculosis H37Rv via both i.t. and s.c. routes. Bacteria inoculated by the i.t. route multiplied in lungs, spleens, and mediastinal LNs (Fig. 1A). Following s.c. injection, mycobacteria were found in lymphoid organs (brachial LN (BLN) and spleen), while lungs largely remained free from the pathogen (100% of the mice challenged with BCG and 85% of the mice challenged with virulent M. tuberculosis did not show any CFU growth in their lungs; Fig. 1A). Thus, two different routes of mycobacterium delivery resulted in the localization of the mycobacteria to different sites.


Figure 1
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  		FIGURE 1. Following different routes of mycobacteria delivery, IFN-{gamma}-producing CD27low effector CD4 T lymphocytes accumulate in the lungs. Mice were challenged with BCG or M. tuberculosis (M. tub.) via the i.t. or s.c. route. Four weeks later mycobacterial loads and the activation of CD4 T lymphocytes were assessed. A, Mycobacteria load. Data are representative of three independent experiments (n = 3–5 per group per experiment). Following s.c. injection of M. tuberculosis, mycobacteria growth was detected in one of five mice (two of twelve mice in three experiments; sensitivity of assay, 15 CFU). These mice are represented separately and marked with an asterisk. B, Identification of effector CD4 T lymphocytes and their subsets. Analyses of CD4 T cells isolated from mice challenged s.c. with BCG are shown. Similar subsets were observed when cells from unchallenged or M. tuberculosis-infected mice were analyzed (data not shown). C, Frequency of IFN-{gamma} producing cells in lungs and LNs. Cells were isolated from BLNs and lungs of mice challenged i.t. or s.c. with M. tuberculosis (week 4). After overnight culture in the presence of mycobacteria sonicate (Myc. sonicate) or medium, the cells were stained for intracellular IFN-{gamma} and the percentage of CD4 T lymphocytes producing IFN-{gamma} was determined. Simultaneously, aliquots of LN and lung cell suspensions were plated to determine the mycobacterial load (sensitivity of assay, 15 CFU per lung).

 
We next followed the accumulation of effector CD4 T lymphocytes and their subsets in LNs, spleens, and lungs 1, 2, and 4 wk postinfection. In all organs, three major subsets of CD4 T lymphocytes could be identified on the basis of CD27 and CD62L expression: CD62LhighCD27high ("naive/memory"), CD62LlowCD27high ("CD27high effector cells"), and CD62LlowCD27low ("CD27low effector cells") (Fig. 1B). As reported previously (18, 30), the level of CD62L expression was inversely correlated with CD44 expression (data not shown).

In the LNs of uninfected mice, ~80% of the CD4 T cells were of the naive CD44lowCD62Lhigh phenotype (Table I). Among the effector CD44highCD62Llow CD4 cells, the CD27high subset predominated (18.5 vs 4.7%). Following i.t. or s.c challenge, the frequency of CD27high and CD27low effector CD4 T lymphocytes did not increase. However, the absolute numbers of all CD4 T cell subsets, including naive, CD27high, and CD27low effector lymphocytes increased significantly (4.0-, 3.2-, and 5.3-fold, respectively, in the BLNs of mice challenged via the s.c. route; Table I). Thus, mycobacterial infection promoted a strong local inflammatory reaction but did not alter the ratio of the major CD4 lymphocyte subsets in the LNs.


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  		Table I. CD27low effector cells accumulate in the lungs but not in the LN of infected micea

 
In contrast to the LNs, the lungs of uninfected mice contained lower frequencies of CD44lowCD62Lhigh cells and more CD27low effector CD4 T cells (Table I). In mice challenged with M. tuberculosis i.t., the frequencies of CD27low effector cells reached nearly 80%. A significant accumulation of the CD27low subset in the lungs was also observed in mice challenged s.c., which was unexpected because these lungs were not found to harbor mycobacteria at the time of analysis. The frequency of CD27low cells also increased in the spleen following infection, but to a lesser extent than in the lungs (Table I). The data demonstrate that CD27low effector CD4 T lymphocytes accumulate preferentially in the lungs, but not in the LN, during TB infection.

Even though our data revealed an accumulation of CD27low CD4 T lymphocytes in the lungs, the specificity of these cells was not known. To evaluate the distribution of CD4 T cells specific for mycobacteria, we next analyzed cells from LNs and lungs for their capacity to produce IFN-{gamma} in response to in vitro stimulation with mycobacterial Ags. Cells were recovered from mice that had been challenged with M. tuberculosis i.t. or s.c. The frequency of IFN-{gamma}-producing CD4 T lymphocytes in the LNs was lower than in the lungs (Fig. 1C). This did not depend on mycobacteria load, because following s.c. challenge the BLNs contained significantly more mycobacteria than the lungs. As reported previously (18), in both LNs and lungs nearly all of the IFN-{gamma}-producing cells were CD27low (data not shown). Thus, Ag-specific CD27low IFN-{gamma}-secreting CD4 T lymphocytes preferentially accumulated in the lungs even when this tissue was not infected. In contrast, CD27low IFN-{gamma} secreting CD4 T lymphocytes were present at low frequencies in the LNs.

In lymphoid tissues CD27low CD4 T lymphocytes are proapoptotic

To address possible mechanisms responsible for the low frequency of CD27low effector CD4 T cells in the LNs, we first evaluated apoptosis in LN, spleen, and lung CD4 T cells. Cells isolated from uninfected mice or mice challenged with M. tuberculosis were stained with annexin V, and annexin V binding was assessed in each of the three subsets: CD62Lhigh naive/memory lymphocytes and CD62LlowCD27high and CD62LlowCD27low effector cells. In the LNs, spleens, and lungs of uninfected mice, annexin V staining was lowest in the CD62Lhigh cells, intermediate in the CD27high effector cells, and highest in the CD27low effector lymphocytes (Fig. 2A). In CD62Lhigh CD4 T lymphocytes the frequency of apoptotic cells was roughly similar in all organs (20–30%; Fig. 2A, Subset 1). In contrast, CD62LlowCD27low effector cells were highly apoptotic in the LN but significantly less so in the lung (87 vs 48%; Fig. 2A, Subset 3).


Figure 2
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  		FIGURE 2. CD27low cells are highly susceptible to apoptosis in lymphoid tissues. Cells were isolated from BLN, spleen, and lungs of control mice (A) or mice challenged with M. tuberculosis 4 wk prior (B). The cells were stained with FITC-annexin V, PE-anti-CD27, PerCP-anti-CD4, and allophycocyanin-anti-CD62L, gated on CD4 T lymphocytes, and analyzed. A, Gating of the three subsets of CD4 T lymphocytes and annexin V (Ann V) staining in each subset. Numbers above the column show the corresponding subset. B, Binding of annexin V by CD27high and CD27low CD4 lymphocytes isolated from mice infected with M. tuberculosis. BLNs were from mice challenged s.c.; lungs and spleens were from mice challenged i.t. All organs contained mycobacteria. Numbers outside the dot plots show the proportion of apoptotic cells as a percentage of all CD27low CD4 T lymphocytes (mean ± SD, n = 6; two independent experiments). The gates for annexin V-positive cells were placed based on the level of fluorescence in the "fluorescence minus one" control (cells stained with anti-CD4, anti-CD27, and anti-CD62L mAbs without addition of annexin V).

 
In infected mice, a similar pattern of CD4 T cell apoptosis was observed. The frequencies of annexin V-positive cells were highest in the LNs (83%), intermediate in the spleens (58%), and lowest in the lungs (36%; Fig. 2B). From these data we concluded that in different tissues CD27low cells exhibit different capacities for apoptosis and that the higher degree of CD27low CD4 T cell apoptosis in the LNs may account for their lower frequency in this tissue.

Both CD27high and CD27low CD4 T cells migrate to the lungs

We next examined whether CD27high and CD27low effector CD4 cells differed in their capacity for migration to lymphoid and lung tissues. To address this question, we performed adoptive transfer studies. Donor mice were i.v. infected with M. tuberculosis, spleen CD4 cells were magnetically sorted into CD27high (containing both CD62Llow and CD62Lhigh cells), CD62LlowCD27high, and CD62LlowCD27low subsets, labeled with CFSE, and transferred into recipient mice that had been infected i.v. with M. tuberculosis 3 wk before (Fig. 3). The i.v. route was used to ensure that the bacteria disseminated to all organs, including LNs, spleens, and lungs. Twenty hours following the transfer of CD27high CD4 T cells, donor lymphocytes were detected in all organs, including lungs, spleens, and LNs (Fig. 3A, group 1). In contrast, following the transfer of CD62LlowCD27high or CD62LlowCD27low CD4 T lymphocytes, donor cells were detected in lungs and spleens but not in the LNs (<0.1%; Fig. 3, B and C, group 2 and group 3), supporting a well-established observation that the expression of CD62L is a prerequisite for cell migration to the LNs (13). In addition, CD27low effector CD4 T cells migrated to the lungs more efficiently than CD27high effector cells. (Fig. 3, B and C).


Figure 3
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  		FIGURE 3. CD27high cells accumulate in the lung and down-regulate CD27 in vivo. Donor mice were challenged i.v. with M. tuberculosis. Three weeks later spleen cells were isolated and CD27high, CD62LlowCD27high, and CD62LlowCD27low CD4+ T lymphocytes were purified using magnetic cell separation. The cells were labeled with CFSE and adoptively transferred into syngenic recipients (4 x 106 cells per mouse) that had been infected with M. tuberculosis 3 wk prior to the transfer. Twenty hours following the transfer, LN, spleen (Spl), and lung cells were isolated from recipient mice and analyzed for the content and phenotype of donor CFSE+ cells. A–C, Surface phenotype of donor cells prior to the transfer and the percentage of CFSE+ donor cells recovered from LNs, spleens, and lungs of recipient mice following the transfer of CD27high (A), CD62LlowCD27high (B), or CD62LlowCD27low (C) CD4 T lymphocytes. Data are mean ± SD derived from two independent experiments (n = 4 for A and B; n = 2 for C). D–F, Surface phenotype of donor cells isolated from LNs, spleens, and lungs of recipient mice 20 h following the transfer of the same subsets as in A–C.

 
We next analyzed whether the donor cells changed the expression of CD62L and CD27 molecules in vivo. Group 1 recipient mice received CD27high donor cells that initially were CD62Lhigh and CD62Llow (Fig. 3A). We intentionally did not separate these populations to allow the different donor cells to migrate to different organs in the same recipient mouse. Following transfer, only naive CD27highCD62Lhigh donor cells were detected in the LNs of recipient mice. In contrast, in lungs three subsets of donor cells were detected: CD62LhighCD27high, CD62LlowCD27high, and CD62LlowCD27low (Fig. 3D). Given that CD62LlowCD27low lymphocytes were not present during transfer, these results indicated that down-regulation of CD27 occurred in vivo. In total, up to 30% of the donor cells detected in the lung had down-regulated CD27 expression. Group 2 recipient mice received CD62LlowCD27high CD4 T cells. In these mice, the proportion of donor cells that expressed the CD27low phenotype in the lungs was even higher than in group 1, reaching 50–60% (Fig. 3E), suggesting that CD27low lymphocytes detected in the lungs originated from CD62LlowCD27high donor cells. In group 3 recipients, who received CD62LlowCD27low cells, the phenotype of donor cells was unaltered (Fig. 3F).

Formally, we could not rule out the possibility that accumulation of CD27low donor CD4 T lymphocytes in the lungs of mice that had received CD27high cells resulted from highly efficient homing of the CD27low cells that were present at a low frequency (<2%) in the sorted CD27high population. However, we consider this possibility unlikely because, following the transfer of CD62LhighCD27high donor cells that contained a similar frequency of CD27low lymphocytes, the donor CD27low populations in the lungs was very low (<5%; data not shown). Thus, we conclude that CD27low CD4 T lymphocytes accumulating in the lungs of mice that received CD27high cells originated from CD62LlowCD27high precursors that down-regulated CD27 in vivo. Given that CD27low cells of donor origin could be detected only in the lungs, we propose that down-regulation of CD27 occurred within the lungs.

CD27high cells down-regulate CD27 in the lungs

Although our data suggested that CD27 down-regulation occurred in the lungs, it was possible that donor CD27low CD4 T cells, which appeared in the lungs following adoptive transfer of CD27high CD4 T cells, were cells that had down-regulated CD27 during transit through the LN. To address this possibility, we established a method of in vivo LN cell labeling that allowed us to monitor the migration of CD4 T lymphocytes from the LN to the lungs and other tissues. In this approach, CFSE dye was injected into the BLNs of mice as described in the Materials and Methods. All CD4 T cells recovered from the treated BLNs 30–60 min later had incorporated CFSE (Fig. 4A). In distal organs, including inguinal LNs, spleens, and lungs, CFSE+ cells were not detected or were very rare at this time. Twenty-four hours later, CFSE+ cells appeared in distal LNs, spleens, and lungs and comprised ~1% of the CD4 T cell population in these organs (Fig. 4B). Thus, injection of CFSE into the BLN labeled LN cells and allowed us to monitor their migration to other organs. Unlike in vitro CFSE labeling, the fluorescence intensity of the in vivo-labeled cells was heterogeneous (Fig. 4), so the approach did not allow quantitation of cell division.


Figure 4
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  		FIGURE 4. In vivo labeling of LN cells using CFSE injection. CFSE dye was injected into the BLNs of mice as described in Materials and Methods. Cells were isolated from BLNs, inguinal LNs, spleens, and lungs 1 h (A) or 24 h (B) later and stained with PerCP-anti-CD4 mAbs. Dot plots were generated after gating on live CD4 T lymphocytes.

 
To determine which subsets of CD4 T cells emigrated from infected BLNs and entered the lungs, we challenged mice with BCG s.c. As demonstrated above, such challenge induced mycobacteria growth in BLNs but not in lungs (see Fig. 1A). At the peak of the response (4–6 wks) CFSE was injected into the BLN. Twenty-four hours later the appearance and phenotype of CFSE+ cells in lungs were examined (the experimental approach is summarized in Fig. 5A). As a control, inguinal LNs were also examined. In these LNs, 0.7 ± 0.3% of the CD4 T lymphocytes were CFSE+ (three independent experiments, n = 8; data not shown). These cells were lymphocytes that had recently migrated to the inguinal LNs from the treated BLNs. The majority of the CD4 T lymphocytes residing in the inguinal LNs were CFSE–. The surface phenotype of the CFSE+ ("migrated") and CFSE– ("resident") CD4 T lymphocytes in the inguinal LN was roughly similar, as the majority of both populations of cells were CD62LhighCD27high (Fig. 5, D and E). However, there were some differences between CFSE+ and CFSE– cells. The former contained a lower frequency of CD62Llow cells than were found among the CFSE– lymphocytes (p < 0.05; Fig. 5, D and E). These data suggest that CD4 T lymphocytes down-regulated CD62L in the LN and are in agreement with evidence that down-regulation of CD62L occurs concomitant with cell proliferation in the LN (12).


Figure 5
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  		FIGURE 5. CD4 T lymphocytes down-regulate CD27 following their migration to the lungs. A and B, Experimental design. A, Mice were immunized with BCG s.c. (5 x 105 CFU per mouse). Six weeks later, CFSE was injected into the BLN to stain cells in situ. B, Mice were immunized with BCG s.c. and 4 wk later challenged with M. tuberculosis (103 CFU per mouse given i.t.). Two weeks later CFSE was injected into the BLN. Twenty-four or 72 h after injection of the dye, inguinal LNs and lungs were recovered from mice of both groups, and cells were stained with PerCP-anti-CD4, allophycocyanin-anti-CD62L, and PE-anti-CD27 mAbs and analyzed. C, Gating of CFSE– and CFSE+ populations of lung CD4 T lymphocytes. Similar gates were applied for inguinal LN cells (not shown). CFSE+ lymphocytes represent newly migrated cells, while the majority of "resident" cells are CFSE–. D–G, Surface phenotype of CFSE– and CFSE+ lymphocytes recovered from inguinal LN and lungs of mice immunized with BCG ("BCG" group). H–L, Surface phenotype of CFSE– and CFSE+ lymphocytes recovered from lungs of mice immunized with BCG and challenged with M. tuberculosis ("BCG-Mtb" group). Organs were recovered 24 (H and I) or 72 (K and L) hours postinjection of the dye. Numbers below the dot plots represent mean ± SD for each population of cells (two independent experiments, three or four mice per group per experiment).

 
In the lungs, CFSE+ BLN-derived CD4 T cells comprised 0.6 ± 0.1% of the total CD4 T cells (three independent experiments, n = 8; data not shown). Approximately half of the CFSE+ cells exhibited a CD62LhighCD27high phenotype (Fig. 5F). CD62Llow CFSE+ cells contained equal proportions the CD27high and CD27low subsets, indicating that both subsets were capable of migrating to the lungs from the BLN. This did not imply that both subsets exhibited a similar capacity for lung homing, as the CD27high lymphocytes were found at a higher frequency in the BLN at the time of CFSE administration (the frequencies of CD27high and CD27low effector CD4 T cells in the BLN were 16 and 7%, respectively). Comparison of the CFSE– and CFSE+ lung CD4 T cells revealed that the newly migrated CFSE+ cells contained a lower proportion of CD27low lymphocytes than the resident CFSE– population (24 vs 38%, p < 0.01; compare Fig. 5, F and G), suggesting that down-regulation of CD27 occurred following the migration of the CD4 T lymphocytes to the lungs.

Because the local accumulation of CD27low effector CD4 T cells is promoted by lung tissue infection (see Table I), we next addressed whether the cell population that migrated to lungs infected with mycobacteria contained a higher proportion of CD27low effector cells. To accomplish this, an additional group of mice was added the experiment described in Fig. 5A. As before, one group of mice received s.c. injection of BCG to induce an antimycobacterial response in the BLN ("BCG" group in Fig. 5A). The second group was injected s.c. with BCG and was challenged 4 wk later with M. tuberculosis given i.t. to induce pulmonary TB infection ("BCG-Mtb" group in Fig. 5B). Two weeks following infection with M. tuberculosis, CFSE was injected into the BLNs of all mice and the phenotype of the CFSE+ CD4 T cells appearing in the lungs 24 and 72h later was analyzed. Twenty-four hours postinjection, CFSE+ CD4 T cells detected in the lungs contained CD62Lhigh, CD62LlowCD27high, and CD62LlowCD27low lymphocytes. The proportion of each subset was similar in "BCG" and "BCG-Mtb" mice (compare Fig. 5, F and H), demonstrating that the phenotype of CD4 T lymphocytes entering the lungs was not modified by local infection. In all mice, CFSE+ ("migrated") cells contained significantly fewer CD27low cells than the CFSE– ("resident") lung lymphocytes (compare Fig. 5, H and I). Seventy-two hours postinjection of the dye, the proportion of CFSE+ CD27low cells increased in the lungs of "BCG-Mtb" mice (compare Fig. 5, H and K; p < 0.05) but did not change significantly in "BCG" mice (data not shown).

Several conclusions can be drawn from these experiments. First, all CD4 T cells, including naive/memory CD62Lhigh, effector CD62LlowCD27high, and effector CD62LlowCD27low cells, are capable of entering the lungs. Second, the relative proportions of these subsets among the newly migrated cells is unaffected by lung infection. Third, as CD4 T lymphocytes reside for longer periods in the lungs, the proportion of CD27low cells increases, especially when the lungs are infected. These latter data are consistent with the appearance of CD27low donor cells in the lungs of mice adoptively transferred with CD27high CD4 T lymphocytes (Fig. 3). Taken together, the results suggest that lung CD27low CD4 T lymphocytes differentiate in situ from CD27high precursors.

CD27high CD4 T cells down-regulate CD27 in vitro.

We next addressed whether CD27high CD4 T cells could give rise to CD27low effectors following antigenic stimulation in vitro. In these experiments, CD27high CD4 T lymphocytes were purified from the spleens of M. tuberculosis-infected mice, cultured in vitro in the presence of spleen APCs and mycobacterial sonicate, and phenotyped every 4–5 days. The frequency of CD27low lymphocytes remained low and stable during the first 5–10 days of culture (2–4% of all responding CD4 T cells) but then increased significantly and was 15–20% at day 15 and up to 50% at day 23 (Fig. 6, B and C; the cells were re-stimulated at day 15 after their phenotyping). We also monitored apoptosis of the CD27high and CD27low populations and observed that the proportion of annexin-V positive cells was significantly higher in CD27low cells as compared with CD27high cells (Fig. 6D). To directly compare the survival of CD27high and CD27low CD4 T lymphocytes, in the next set of experiments we isolated both CD27high and CD27low CD4 T lymphocytes, cultured them in parallel, and estimated the absolute numbers of all live lymphocytes and CD27low CD4 T lymphocytes in both cultures at different time points (Fig. 6, E and F). Although the total number of live cells gradually decreased in both cultures, it decreased more rapidly in the "CD27low" cultures (Fig. 6E). The number of CD27low CD4 T cells decreased abruptly in "CD27low" cultures but did not decrease in the "CD27high" cultures (Fig. 6F). In both cultures proliferation was stopped by day 5, i.e., before an increase in the frequency of CD27low cells in "CD27high" cultures was registered (data not shown). Altogether, these data demonstrate that CD27low cells are terminally differentiated cells that originate from CD27high CD4 T lymphocytes. This finding corresponds well to our in vivo and ex vivo observations.


Figure 6
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  		FIGURE 6. CD27high CD4 T cells down-regulate CD27 in vitro. Mice were infected with M. tuberculosis via the i.v. route. Two to three weeks later spleen cells were isolated. CD4+ CD27high and CD4+ CD27low lymphocytes were purified using magnetic cell separation. The purity of separated cells was 98% for CD27high cells and 95–96% for CD27low cells. Purified cells (5 x 105 cells/ml) were cultured in the presence of mitomycin-treated APCs (1.5 x 106 cells/ml), mycobacteria sonicate (10 ng/ml), and recombinant IL-2. The APCs were labeled with CFSE and excluded from the analysis as shown in A. Cultured cells were collected on days 2, 5, 10, 15, and 23, stained with PE-anti-CD27, PerCP-anti-CD4, and allophycocyanin-anti-CD62L mAbs or 7-AAD and allophycocyanin-annexin V and analyzed. A, Cell gating. SSC, Side scatter; FSC, forward scatter. B and C, Flow cytometry (B) and quantification (C) of CD27low cells following the culture of CD27high CD4 T lymphocytes. Arrow in C indicates the day of cell restimulation. D, Binding of annexin V by CD27high and CD27low CD4 T lymphocytes. Cells were recovered on day 14 of the culture and stained with anti-CD4, 7-AAD, and allophycocyanin-annexin V (open histograms) or with anti-CD4 and 7-AAD without annexin V (tinted histograms). E and F, Quantification of all live CD4 T lymphocytes (E) and CD27low CD4 T lymphocytes (F) in "CD27high" and "CD27low" cultures (numbers of cells recovered from one well are shown).

 
The accumulation of lung CD27low effector CD4 T cells correlates with decreased severity of TB infection

We next considered whether the accumulation of CD27low effector CD4 T lymphocytes in the lungs contributed to host defense. To address this question, we examined whether mice that exhibited different susceptibilities to TB infection had different frequencies of effector CD27low CD4 T lymphocytes in their lungs. Previously, we reported that mice of the I/St and A/Sn strains differed in their susceptibility to TB infection (I/St mice were more susceptible) and in the level of IFN-{gamma} secretion in their lungs (27, 28, 29). In this study, we generated (A/Sn x I/St) F2 hybrid mice originating from A/Sn and I/St mice. The F2 mice were challenged with M. tuberculosis and we examined the rate of postinfection body weight loss (a correlate of susceptibility) (28, 31) and the frequencies of CD62Lhigh, CD62LlowCD27high, and CD62LlowCD27low CD4 T cell subsets in their lungs. The analysis was performed at day 23 postinfection, a time when at least some of mice revealed significant wasting.

In agreement with previous reports (32), the F2 hybrid mice segregated with respect to the degree of their susceptibility to TB infection. By day 23 postinfection some mice had lost >30% of their preinfection weight, while many mice gained weight. An analysis of lung CD4 T lymphocytes revealed that the proportion of CD62Lhigh CD4 T lymphocytes was relatively low in all mice (20–30%), indicating that the majority of lung CD4 T cells were effector cells. The frequencies of CD27high and CD27low subsets of effector CD62Llow CD4 T cells varied significantly. The frequency of CD62LlowCD27high cells correlated directly with body weight loss (Spearman correlation coefficient (r) = 0.4820, p < 0.0001, Fig. 7A), and the frequency of CD62LlowCD27low CD4 T cells and body weight loss were inversely correlated (Spearman r = –0.4043, p = 0.0001; Fig. 7B). There was an inverse correlation between body weight loss and absolute numbers of CD62LlowCD27low CD4 T cells (Spearman r = –0.4516, p < 0,0001; data not shown). Thus, the efficient accumulation of CD27low CD4 T cells in the lungs correlated with the protection of mice against fatal TB infection. The different content of CD27low CD4 T cells in the lungs of individual F2 mice may result in or be due to different mycobacterial loads in these mice. Given that Ag (pathogen) promotes down-regulation of CD27 (Ref. 26 and this article) and that the more susceptible mice had lower but not higher content of CD27low cells in their lungs, the first explanation seems to be more likely. We therefore suggest that genetically different mice may have different intrinsic abilities of CD4 T cells to develop into CD27low CD4 T lymphocytes. Experiments are currently ongoing to check this hypothesis.


Figure 7
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  		FIGURE 7. Accumulation of CD27low effector CD4 T lymphocytes in the lungs protects the host against a severe course of TB. (A/Sn x I/St)F2 hybrid female mice were infected with M. tuberculosis (103 CFUs i.t.; 106 mice were analyzed in six independent experiments). To monitor the course of infection, mice were weighed weekly. When some of mice lost >20% of their initial weight (day 23 postchallenge), all mice were sacrificed and lung cells were stained with anti-CD4, anti-CD27, and anti-CD62L mAbs. A and B, Correlation between mice wasting and frequency of CD62LlowCD27high (A) and CD62LlowCD27low (B) CD4 T cells in the lungs.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A current model of effector T cell generation proposes that the activation and differentiation of naive lymphocytes into effector memory cells occur in the LNs. Here, lymphocytes encounter cognate Ag, proliferate, acquire effector functions, and modify the expression of various surface receptors. The latter drives newly formed effector cells to the periphery (8). It remains unclear, however, whether effector lymphocytes that exit lymphoid tissues are fully mature or whether their functional differentiation is completed in the periphery. The vast majority of lymphocytes residing in the lungs are differentiated effector memory cells that exhibit a high capacity for effector cytokine secretion and a typical surface phenotype (CD62Llow, CCR7–, and CD44high) (12, 33, 34, 35, 36). One of the characteristic traits of effector CD4 and CD8 T lymphocytes is their lack of CD27 expression (18, 24, 25). The anatomical distribution of CD27low effector lymphocytes, however, has not yet been addressed in detail. Our data suggest that CD27low CD4 T lymphocytes, major producers of IFN-{gamma} during mycobacterial infection, are much more abundant in the lungs than in the LNs even when mycobacterial infection persists outside the lungs (i.e., following s.c. infection). This is consistent with data indicating that infection is not essential to draw effector cells into the lung tissue and airways (34, 36) and with observations that LNs contain lower frequencies of IFN-{gamma} producing cells relative to peripheral tissues (35, 37). In our studies, the content of fully differentiated CD27low cells in the LN was low, partially due to a higher degree of their apoptosis in the LN compared with the lungs. To our knowledge, this is the first direct evidence that survival of a particular T lymphocyte subset varies in different tissues. Other mechanisms which likely contribute to the preferential accumulation of CD27low CD4 T cells in the lungs are: 1) more efficient migration to the lungs of circulating CD27low cells as compared with circulating CD27high cells; and 2) generation of CD27low cells from CD27high CD4 T lymphocytes in the lung tissue. All of the mechanisms may serve to protect LNs from possible deleterious effects of functionally mature CD27low effector cells.

Using in vivo labeling of BLN cells with CFSE, we were able to monitor the distribution of lymphocytes following their egress from the LN. Both CD62Lhigh and CD62Llow lymphocytes exited the LN. CD62Lhigh CD4 T lymphocytes continued to circulate and homed to other lymphoid tissues and to the lungs. These data appear to be inconsistent with observations made by Roman et al. (12), who reported that only effector cells with low expression of CD62L were recruited into the lung from the LN. The apparent discrepancy between the findings may be due to the differences between experimental systems. Roman et al. (12) followed the migration of adoptively transferred TCR transgenic influenza virus hemagglutinin-specific T cells. The transgenic T cells likely encountered Ag immediately in the recipients that had acute viral infection. In our studies, we monitored migration of a polyclonal LN cell population, which, in addition to mycobacteria-specific lymphocytes, apparently contained cells of irrelevant specificities. The CD62Lhigh CD4 T lymphocytes that entered the lungs may belong to the pool of nonspecific cells that continued to circulate throughout the body. Another possibility is that CD62Lhigh CD4 T lymphocytes that migrated to the lungs were memory cells. Memory CD62Lhigh CD4 T lymphocytes have the capacity for entering the lungs (38). In addition, lung CD4 T lymphocytes freshly isolated from noninfected animals always contain up to 50% CD62Lhigh cells (our unpublished observations).

In contrast to CD62Lhigh CD4 T lymphocytes, CD62Llow cells homed to lungs and spleens but did not enter LNs, which is consistent with the role of CD62L in directing T cell migration (8, 13, 14). Several of our observations suggest that after migration to the lung, CD62Llow effector CD4 T cells down-regulated CD27 expression. First, following adoptive transfer of CD62Llow CD27high cells into mycobacteria-infected hosts, a substantial proportion of donor cells recovered from the lungs had down-regulated CD27 expression. This was not observed in the LN. Second, T cells that had recently migrated to the lungs from the BLN contained a lower proportion of CD27low cells than the resident lung CD4 T cells; the proportion of CD27low cells in the lungs increased with time. Third, antigenic stimulation of CD27high CD4 T lymphocytes in vitro induced their transition to a CD27low phenotype. Collectively, the data indicate that the CD27high to CD27low transition likely took place in the lungs. Our findings may be related to studies that have indicated that CD27 ligation in the gut induced differentiation of Ag-specific T cells (39).

Our data also suggest a close association between the four processes occurring during CD4 T cell differentiation: down-regulation of CD27, IFN-{gamma} secretion, cell localization at the periphery, and apoptosis. Some of these associations have already been reported. Indeed, other studies have shown that: 1) in CD4 T lymphocytes a loss of CD27 coincided with functional maturation and the acquisition of cytolytic and IFN-{gamma} secreting functions (18, 40); 2) CD27– T lymphocytes were sensitive to apoptosis (20); 3) IFN-{gamma} secretion was associated with cell apoptosis (41, 42); and 4) IFN-{gamma}+ cells were rarely present in lymphoid organs and survived in the lungs longer than in lymphoid organs (43).

How are all these processes regulated and linked at the molecular level? To explain the apoptogenic activity of IFN-{gamma}, two major mechanisms have been suggested. The first is that apoptosis is induced by NO produced by IFN-{gamma}-stimulated macrophages (41). The second involves IFN-{gamma} dependent up-regulation of caspase 8 expression and Fas-mediated apoptosis (42). We propose that apoptosis of IFN-{gamma}-producing CD4 T cells may also result from the lack of CD27 on their surfaces, as CD27-mediated signals result in NF-{kappa}B activation and up-regulation of a variety of anti-apoptotic proteins (19, 20, 44, 45).

It is not clear why IFN-{gamma} production is largely a property of CD27low cells, as signaling through TRAF-binding TNF receptors such as CD27 results in the activation of NF-{kappa}B, which, in turn, induces NF-{kappa}B-dependent ifn{gamma} transcription (45, 46). One possible explanation is that CD27 is a prerequisite for formation of effector T cells but, as they are established, they no longer require signaling through CD27.

The location of IFN-{gamma} producing effector CD4 T lymphocytes at peripheral tissues (Refs. 37 and 43 , and our data from this study) may be explained by the fact that these cells exhibit more efficient homing to peripheral sites than naive and memory lymphocytes. Our data suggest that another mechanism, namely CD27high to CD27low transition of lung CD4 T cells, likely plays a role in the accumulation of IFN-{gamma}-producing CD4 T lymphocytes in the lungs. Why down-regulation of CD27 is promoted in the lung is not yet clear. To date, two major factors have been suggested to induce CD27 down-regulation: ligation of the TCR (26) and ligation of CD27 (47, 48, 49). Our studies suggest the impact of the Ag in CD27 down-regulation, because more LN-derived CD4 T cells converted to a CD27low phenotype in the lungs when lungs harbored mycobacteria. Whether differential expression of CD70, the CD27 ligand, in the lungs and lymphoid tissue may be responsible for down-regulation of CD27 at the periphery is not clear. Tesselaar et al. (50) reported that cells bearing intracellular CD70 were detected in both lungs and draining LNs, while surface expression of CD70 was found on only a small population of lung-infiltrating T cells. We did not see consistent differences in CD70 expression by lung and LN cells, likely due to a very low expression of this marker (our unpublished observation).

In conclusion, our results indicate that functionally mature CD62LlowCD27low CD4 T cells and less differentiated CD62LlowCD27high CD4 T cells have different anatomical distributions. In the LNs CD62LlowCD27low lymphocytes are rare, which likely protects the lymphoid tissue from potentially deleterious action of effector cytokines. At the sites where the effector functions are required, i.e., in the lung tissue during pulmonary TB, an abundance of CD27low lymphocytes is achieved by their preferential migration to the lung and by their local maturation following stimulation with mycobacterial Ags. Once CD27low CD4 T cells are formed, they produce effector cytokines and die, but the pool of effector cells is maintained by the differentiation of newly migrated cells if infection persists. Thus, during persistent TB infection there is a rapid turnover of peripheral effector cells (51). Such turnover allows accumulation of CD27low CD4 T cells in the lungs and simultaneously limits their accumulation when infection is cleared.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Russian Foundation for Basic Research Grant 04-04-49396 (to I.V.L.), U.S. Civilian Research and Development Foundation Grant RUB1-2706-MO-07, (to G.W. and I.V.L.), and, in part, by National Institutes of Health Grant 1-RO1-HL68532-01 (to A.S.A.). Back

2 M.A.K. and G.S.S. contributed equally to the work. Back

3 Address correspondence and reprint requests to Dr. Irina Lyadova, Central Institute for Tuberculosis, Yauza Alley, 2, Moscow 107564, Russia. E-mail address: ivlyadova{at}mail.ru Back

4 Abbreviations used in this paper: TB, tuberculosis; LN, lymph node; BLN, brachial lymph node; i.t., intratracheally. Back

Received for publication August 14, 2006. Accepted for publication October 3, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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