The role of IL-22–producing CD4+ T cells in intracellular pathogen infections is poorly characterized. IL-22–producing CD4+ T cells may express some effector molecules on the membrane, and therefore synergize or contribute to antimicrobial effector function. This hypothesis cannot be tested by conventional approaches manipulating a single IL-22 cytokine at genetic and protein levels, and IL-22+ T cells cannot be purified for evaluation due to secretion nature of cytokines. In this study, we surprisingly found that upon activation, CD4+ T cells in Mycobacterium tuberculosis-infected macaques or humans could evolve into T effector cells bearing membrane-bound IL-22 after de novo IL-22 production. Membrane-bound IL-22+ CD4+ T effector cells appeared to mature in vivo and sustain membrane distribution in highly inflammatory environments during active M. tuberculosis infection. Near-field scanning optical microscopy/quantum dot-based nanoscale molecular imaging revealed that membrane-bound IL-22, like CD3, distributed in membrane and engaged as ∼100–200 nm nanoclusters or ∼300–600 nm nanodomains for potential interaction with IL-22R. Importantly, purified membrane-bound IL-22+ CD4+ T cells inhibited intracellular M. tuberculosis replication in macrophages. Our findings suggest that IL-22–producing T cells can evolve to retain IL-22 on membrane for prolonged IL-22 t1/2 and to exert efficient cell–cell interaction for anti-M. tuberculosis effector function.

Naive CD4+ T cells can evolve to at least four distinct cell populations, Th1, Th2, Th17, and induced regulatory T cells, in response to Ag (1, 2). Th17 cell lineage differentiation is regulated by a master transcription factor, retinoid-related orphan receptor γt (1, 2). Murine Th17 cells produce and secret the signature cytokines IL-17, IL-17F, and IL-22 (1, 2). IL-22 is an IL-10 family cytokine, and is mainly produced by T cells and a subset of NK cells (36). Some studies suggest that IL-22 plays a role in development of inflammatory diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel diseases (711). Other studies implicate that IL-22 may protect against cell injuries (12, 13), and act on IL-22R to facilitate innate immunity against extracellular bacterial and fungi infections (1417). It is important to note that immune function of Th17 cells or IL-22+ T cells has been investigated through immune manipulation or targeting of IL-17 or IL-22. Given the possibility that lineage differentiation/maturation and in vivo turnover of Th1, Th2, and Th17 cell populations including IL-22+ T effectors are quite plastic and dynamic during infection (1), each of these T effector subsets may represent a heterogeneous cell population producing the IL-17/IL-22 signature cytokine and other effector molecules. From this immunological standpoint, targeting a single IL-17 or IL-22 cytokine in experiments may insufficiently define the function of Th17 T effector cell populations. Use of the IL-17/IL-22 cytokine-expressing Th17 effector cells in experimental investigation may provide additional information regarding the cellular effector function of these cells. However, current biological techniques cannot allow purification or selection of the IL-17/IL-22–producing T effector cell population due to the secretion nature of cytokines, including IL-17/IL-22.

Tuberculosis (TB) remains one of the leading causes for morbidity and mortality worldwide among infectious diseases, with annual 8 million new cases and 2 million deaths (18). Although CD4+ T cells and Th1 cytokines are protective against Mycobacterium tuberculosis infection, full anti-TB immunity appears to involve other undefined immune components (19). Understanding roles of other T cell subsets producing a signature cytokine and other effector molecules should help to elucidate anti-TB immunity and facilitate development of new vaccine and immunotherapeutics against TB. We have recently found that macaque IL-22 transcripts are remarkably increased in lung and lymphoid tissues in severe TB (20), a finding similarly seen in a human study (21). We also demonstrated that whereas most T effector cells secreting conventional cytokines often cannot be directly detected without in vitro Ag restimulation, IL-22 protein produced by macaque T cells could be visualized in situ even in lung tissues containing TB granuloma (22, 23). Moreover, macaque IL-22+ T cells that have matured during M. tuberculosis infection can readily be detected in blood and tissue lymphocytes by intracellular cytokine staining without in vitro Ag restimulation (23). However, immune function of IL-22–producing CD4+ T cells in M. tuberculosis infection remains unknown.

Our finding that IL-22+ T effector cells can be readily detected in situ in TB allowed us to hypothesize that IL-22+ T effector cells or IL-22 play a role in M. tuberculosis infection and that IL-22 intracellular trafficking/secretion may differ somehow from most other T cell-producing ILs (24, 25). In testing this hypothesis, we surprisingly found that IL-22 produced by CD4+ T cells could evolve to retain in cell membrane, and engage as nanoclusters or nanodomains after de novo production in M. tuberculosis infection. Notably, membrane-bound IL-22+ CD4+ T cells were able to inhibit intracellular M. tuberculosis replication in macrophages.

Adult rhesus macaques were used in this study. Macaques were naive prior to M. tuberculosis infection on a basis of tuberculin skin test, IFN-γ ELISPOT assays, and thoracic radiographs. M. tuberculosis-infected macaques were housed at the Biologic Research Resources Annex BSL3 nonhuman primate facilities in University of Illinois and sacrificed after 2 mo of infection. All animal experimental procedures and protocols were approved by the University of Illinois Animal Care Committee.

Each monkey was infected with 500 CFU M. tuberculosis Erdman (the standard challenge stock from Food and Drug Administration) by the bronchoscope-guided injection of the inoculum into the right caudal lobe, as previously described (23, 26). This was done in the procedure room at the Annex BSL3 nonhuman primate facilities at University of Illinois. The inoculum used for infection was diluted and plated on 7H11 agar plates (BD Pharmingen) to further confirm the bacterial CFU dose for inoculation.

PBMC were isolated from freshly collected EDTA blood of macaques or humans by Ficoll-Paque Plus (Amersham, Piscataway, NJ) density gradient centrifugation, as described previously (27). For isolation of lymphocytes from lung and spleen tissues, lung and spleen tissues were minced with sharp scissors and squeezed with sterile copper mesh in Petri dish and collected with RPMI 1640, followed by filtering through 40-μm cell strainers. Mesenteric lymph nodes were carefully teased apart with 18-gauge needle in Petri dish with RPMI 1640 to form single-cell suspension. Collected cell suspensions from lung, spleen, and mesenteric lymph nodes were further purified by Ficoll-Paque Plus density gradient centrifugation to collect pure lymphocytes. The freshly isolated lymphocytes from blood, lung, spleen, and mesenteric lymph nodes were stained by trypan blue to examine the viability and numerate cell counts. The lymphocytes were finally suspended into 10% FCS–RPMI 1640 media with a concentration of 107 cells/ml for further study.

PBMC that isolated freshly from the blood of M. tuberculosis-infected macaques or purified protein derivative (PPD)+ or PPD human donors were infected with M. tuberculosis in 96-well plates in multiplicity of infection (MOI) = 1 for 18 h. Then cells were washed with 2% FBS/PBS and stained without treatment with membrane permeabilization reagents with FITC CD4 (L200; BD Pharmingen), PE/Cy7 CD3 (SP34-2; BD Pharmingen), IL-22 biotinylated or Pacific blue (PB) (goat anti-human; R&D Systems), and PE IFN-γ (4S.B3; BD Pharmingen) Abs for 30 min. After washing, cells were incubated with streptavidin PB (Invitrogen), followed by washing with 2% FBS/PBS. These cells were then fixed with 2% formalin/PBS and analyzed by flow cytometry. IL-22R expression in macrophages was analyzed by flow cytometry using anti–IL-22R (305405; R&D Systems).

Cells that isolated freshly from the spleens or lungs of monkeys after 2-mo infection were stimulated with 15-mer overlapping peptides spanning Ag85B and ESAT6 (GenScript), anti-CD28 (CD28.2; BD Pharmingen), and anti-CD49d (9F10; BD Pharmingen) for 1 h. After thoroughly washing, these cells were used for Ab staining without treatment with membrane permeabilization reagents. Cells were incubated with FITC CD4 (L200; BD Pharmingen), PE/Cy7 CD3 (SP34-2; BD Pharmingen), and IL-22 biotinylated or PB (goat anti-human; R&D Systems) Abs for 30 min. After thoroughly washing, cells were incubated with streptavidin PB (Invitrogen). These cells were then washed twice with 2% FBS/PBS, fixed with 2% formalin/PBS, and then analyzed by flow cytometry. To investigate whether secretory form of IL-22 also existed, IL-22 in culture supernatants from activated, enriched CD4+ lymphocytes or IL-22+ CD4+ T cells was examined using the dot blot assay, as we described (28, 29). We used the dot blot assay, as ELISA assay for quantitation of macaque IL-22 was not commercially available. Typically, ∼1 × 107 enriched CD4+ T cells from spleens of infected macaques were stimulated by PPD in culture for 2 or 4 d. Supernatants were collected, either unconcentrated or concentrated 50× using Vivacell concentrator (Vivasciences), as we described (28, 29), and then tested for IL-22 by the dot plot assay (28, 29) using the same anti–IL-22 Ab, as described above. Negative control was culture supernatants from unstimulated PBL of uninfected macaques. The specificity of IL-22 Ab staining was confirmed repeatedly using matched normal serum, control Abs, or control animals. No or very few IL-22–producing CD4 T cells were detectable in the longitudinal control experiments (data not shown). Fixed lymphocytes were run on a CyAn ADP flow cytometer (DakoCytomation, Carpinteria, CA) for analysis.

Cells that isolated freshly from the spleens or lungs of monkeys after 2-mo infection were stained directly with Abs without additional in vitro treatment of Ag or Abs or membrane permeabilization reagents. Cells were then incubated with FITC CD4 (L200; BD Pharmingen), PE/Cy7 CD3 (SP34-2; BD Pharmingen), and IL-22–biotinylated (goat anti-human; R&D Systems) Abs for 30 min. After thoroughly washing, cells were incubated with streptavidin PB (Invitrogen). These cells then were washed twice with 2% FBS/PBS, fixed with 2% formalin/PBS, and analyzed by flow cytometry.

We found that macaque and human IL-2 shared >98% similarity in amino acid sequence (data not shown), and that the anti-human IL-22 Ab could readily cross-react with soluble macaque IL-22 produced either by Lactococcuslactis (30) or Listeria monocytogenes expression system on dot blot or Western blot assay (data not shown). Consistently, nonconjugated anti-human IL-22 Ab pretreatment of IL-22+ T cells could block the immune staining of membrane-bound IL-22+ T cells by fluorescence IL-22–biotinylated Ab (see above). In addition, matched normal serum or isotype IgG served as negative controls for staining cytokines or surface markers. We did not see nonspecific immune staining of IL-22 as evaluated by flow cytometry, confocal imaging, or near-field scanning optical microscopy (NSOM)/quantum dot (QD)-based molecular imaging (see below). Furthermore, to ensure the specific immune staining of membrane-bound IL-22 in M. tuberculosis infection, PBMC were obtained biweekly for 8 wk from three healthy uninfected and five simian HIV–infected cynomolgus macaques, and assessed for IL-22–producing T cells using the immune staining assay, as described above. No or very few membrane-bound IL-22+ CD4+ T cells (<0.02% of CD3) were detectable in the longitudinal control experiments (data not shown).

Cells that isolated from the spleens of monkeys after 8-wk infection of M. tuberculosis were fixed with 2% formalin/PBS first. Our previous works have shown that short-term 2% formalin/PBS would greatly reduce unspecific activation during Ab staining, but would not induce significant artificial cellular nanostructures (3133). These cells then were washed extensively with 2% FBS/PBS and ready for Ab staining. Cells were incubated with mouse anti-human CD4 (L200; BD Pharmingen) and goat anti-human IL-22–biotinylated (R&D Systems) Ab for 30 min. After washing with 2% FBS/PBS, these cells were stained with streptavidin-QD655 (Invitrogen) and QD605 goat anti-mouse IgG (Invitrogen). After incubating with all the Abs or fluorochromes, cells were fixed with 2% formalin/PBS and washed thoroughly again using 2% FBS/PBS. Cells then were washed again with dd water twice to remove the salt in the PBS. NSOM (Veeco, Aurora 3.0) imaging was done, as described in our previous work (30).

Cells that isolated from the spleens of monkeys after 8-wk infection of M. tuberculosis were washed extensively with 2% FBS/PBS and incubated with mouse anti-human FITC CD4 (L200; BD Pharmingen) and goat anti-human IL-22–biotinylated (R&D Systems) Ab for 30 min. After washing with 2% FBS/PBS, these cells were stained with streptavidin-QD655 (Invitrogen). After incubating with all the Abs or fluorochromes, cells were fixed with 2% formalin/PBS and washed thoroughly again using 2% FBS/PBS. Cells then were washed again with dd water twice to remove the salt in the PBS. Confocal microscopic imaging was done, as described in our previous work (30).

The ≈5-μm–thick frozen lung sections were prepared, as we recently described (34), from optimal cutting temperature compound (OCT)-embedded lung tissues from healthy macaques or M. tuberculosis-infected macaques. Tissue sections were first incubated overnight in a wet box with polyclonal rabbit anti-human IL-22 (N terminus; Capralogics) and monoclonal mouse anti-human CD3 (F7.2.38; DakoCytomation) or isotype control IgG or normal rabbit serum. After thoroughly washed with PBS, tissue sections were fixed with 2% formalin/PBS and washed thoroughly with PBS. Tissue sections were then incubated with FITC-conjugated donkey anti-rabbit IgG (BioLegend) and Cy3-conjugated goat anti-mouse IgG (BioLegend), followed by thorough PBS washing. Next, tissue sections were fixed gently with 2% formalin/PBS again and washed thoroughly with dd water to remove the salt in PBS. Finally, tissue sections were mounted on slides using fluorescence mounting medium with DAPI for confocal microscope (Zeiss; LSM 510, ×63 NA) imaging.

The tissue preparation and the primary Ab staining are the same for the confocal microscopic imaging. After primary Ab staining, tissue sections were washed and incubated with QD655-conjugated goat anti-mouse secondary Ab and QD605-conjugated goat anti-rabbit secondary Ab. These tissue sections were then washed thoroughly, fixed with 2% formalin/PBS again, and washed thoroughly with dd water to remove the salt in PBS. Then these tissue sections were imaged with dual-color NSOM/QD-based nanoscale imaging system (Veeco, Aurora 3.0).

PBMC were isolated from the blood of M. tuberculosis-infected macaques, and monocytes were obtained by adherence purification on plastic plates (Falcon), as described previously (27). The plates were washed after 2 h of adherence, and monocytes were detached by cold 2% FBS/PBS. The nonadherent cell fraction containing T and B cells was stimulated with PPD in the presence of anti-CD3/anti-CD28 Abs (BD Pharmingen) to generate large numbers of membrane-bound IL-22+ CD4+ T cells. After overnight culture, the stimulated T cells were stained with anti–IL-22 PE (R&D Systems), followed by anti-PE magnetic beads (Miltenyi Biotec). The stained live cells were then loaded to the purification column following instructions from the manufacturer. The passing fraction was collected as IL-22 (negative) cells that did not bear membrane-bound IL-22; IL-22+ T cells held by anti-PE magnetic beads were then released by releasing buffer (Miltenyi Biotec). The isolated IL-22+ T cells were stained again with anti-CD4 FITC (BD Pharmingen), followed by anti-FITC magnetic microbeads for secondary purification.

Autologous monocytes (5 × 104/well) were cultured in round-bottom 96-well plates with 10% FBS–RPMI 1640 medium in the presence of human rIL-4 (BD Pharmingen) and GM-CSF (Sigma-Aldrich) for 4 d, as previously described (35). Supernatants were then removed, and M. tuberculosis inoculum was added at a MOI = 1. After 3 h of incubation at 37°C, supernatants were aspirated and each well was washed three times to remove noningested mycobacteria. Enriched IL-22+ CD4+ T cells (5 × 105) were incubated with anti–IL-22 neutralizing Ab (10 μg/ml, 142928; R&D Systems) or mouse IgG (10 μg/ml) and cultured in the presence of each of them, as indicated in 16Results. Autologous, bead-enriched CD20+ B cells, CD25CD4+ T cells, or IL-22 CD4+ T cells (5 × 105 for each subset) served as a control. For the noncontact setting in Transwell culture system (Corning) experiments, enriched IL-22+ CD4+ T cells (5 × 105) were placed in the upper chamber of Transwell culture system without physical contact with M. tuberculosis-infected macrophages (5 × 104) that were cultured in the lower chamber. Therefore, noncontact IL-22+ CD4+ T cells were not able to contact macrophages, but secretory IL-22 could trans-pass through the microholes and reach macrophages in the lower chamber. Secretory IL-22 was detectable in the concentrated culture medium. For the contact setting, enriched IL-22+ CD4+ T cells and infected macrophages were cocultured in the lower chamber of Transwell culture system. After culturing in 5% CO2 at 37°C for 4 d, wells were aspirated, and 100 μl lysis buffer (0.067% SDS in Middlebrook 7H9) was added to each well. Plates were incubated at 37°C for 10 min, followed by neutralization of SDS with 300 μl PBS with 20% BSA. Lysates from each well were pooled, and two 10-fold serial dilutions of lysate in 7H9 medium were made. Aliquots of each dilution of lysate and supernatant were plated onto Middlebrook 7H10 agar and incubated for 3 wk in 5% CO2 at 37°C until colonies were large enough to be counted. GM-CSF/human IL-4 treatment alone only induced background level of IL-22R expression, but subsequent M. tuberculosis infection of macrophages or immature DC induced apparent IL-22R expression (data not shown).

Statistical analysis was done using Student t test, as previously described (30). A p value <0.05 was considered statistically significant.

Although IL-22, like most other cytokines, should be secretory (21, 36), cell membrane-bound IL-22 after de novo production may enjoy longer t1/2 and exert better effector effects on IL-22R+ target cells via surface–molecule interactions between a IL-22+ T cell and a IL-22R+ cell. The notion for membrane-bound IL-22 appears consistent with our recent observation indicating that IL-22+ T cells matured during M. tuberculosis infection could directly be detected in situ even in lung tissues or directly by intracellular IL-22 staining (22, 23). We therefore hypothesized that TB-induced IL-22+ T cells could have their IL-22 to retain in cell membrane after de novo production, and thus readily be detected by surface immune staining. To test this hypothesis, PBMC from M. tuberculosis-infected macaques were cultured with M. tuberculosis for activation, and assessed for cell membrane-bound IL-22+ T effector cells after de novo IL-22 production using cell surface cytokine staining that skipped the treatment with cell membrane permeabilization reagents. Interestingly, significant numbers of CD4+ T effector cells bearing membrane-bound IL-22 were detected in PBMC from M. tuberculosis-infected macaques, but not from uninfected ones (Fig. 1A, 1C). In contrast, very few membrane-bound IFN-γ+ T cells were detectable by direct cell surface staining in PBMC from the same macaques (Fig. 1A, 1C). Consistently, appreciable numbers of CD4+ T cells bearing membrane-bound IL-22, but not IFN-γ, were also detected by direct cell surface staining after M. tuberculosis stimulation of PBMC from latently M. tuberculosis-infected humans (Fig. 1B, 1D). We did not observe any coexpression of Th1 cytokines by IL-22+ CD4+ T cells (Fig. 1E). The membrane-bound IL-22 could also be seen by fluorescence membrane imaging (Fig. 1F), although secretory form of IL-22 also existed in culture supernatants of activated CD4+ T effectors (Fig. 1G). These results therefore suggested that significant numbers of CD4+ T cells in Mycobacterium-infected macaques and humans could differentiate into effector cells armed with membrane-bound IL-22 in response to M. tuberculosis stimulation in vitro.

FIGURE 1.

Upon activation, CD4+ T cells in M. tuberculosis-infected macaques and humans differentiated into T effector cells bearing membrane-bound IL-22 after de novo IL-22 production. Percentage numbers of CD4+ T cells bearing membrane-bound IL-22 or CD4+ T cells expressing membrane-bound IFN-γ are shown in the upper right quadruple of flow cytometry histograms. Data were gated on CD4 T cells. All cells were surface stained directly without treatment with membrane permeabilization reagents. No membrane-bound IL-22 were detected for negative controls (see 1Materials and Methods). Membrane-bound IL-22 did not appear to be specific for M. tuberculosis infection as CD4+ T cells from Listeria-vaccinated macaques could also express membrane-bound IL-22 upon activation by CD3/CD28 Ab (data not shown). A, Representative flow cytometry histograms show that M. tuberculosis stimulation of PBMC from M. tuberculosis-infected macaques induced membrane-bound IL-22+ CD4+ T effector cells, whereas CD4+ T cells in PBMC from uninfected macaques contained very few membrane-bound IL-22+ T effectors. PBMC from infected macaques were isolated from the blood collected 2 mo after M. tuberculosis infection. Only 0.18% of CD4+ T cells expressing membrane-bound IFN-γ was detectable in M. tuberculosis-infected PBMC. PBMC of macaques were infected with M. tuberculosis in 96-well plates in MOI = 1 for 18 h prior to analysis. B, Representative flow cytometry histograms show that M. tuberculosis stimulation of PBMC from PPD+ human donors induced 12% of CD4+ T cells bearing membrane-bound IL-22. Only ∼0.3% of CD4 T cells bearing membrane-bound IL-22 were detected in PBMC isolated from PPD donors. Also, only ∼0.01% of CD4 T cells expressing membrane-bound IFN-γ could be detected in blood lymphocytes of PPD+ donors. Human PBMC were similarly infected with M. tuberculosis, as described above, or stimulated overnight with anti-CD3/anti-CD28 in presence of PPD and stained without cell permeabilization treatment. C, Bar graph data show that M. tuberculosis-infected macaques exhibited significantly greater numbers of CD4+ T cells bearing membrane-bound IL-22 than uninfected macaques after M. tuberculosis stimulation in vitro (***p < 0.001, n = 3). Data are mean percentage numbers among CD4+ T cells with error bars of SEM derived from three rhesus macaques. D, Bar graphic data show that PPD+ human donors exhibited significantly greater numbers of CD4+ T cells bearing membrane-bound IL-22 than PPD donors after M. tuberculosis stimulation in vitro (*p < 0.05, n = 3). Data are mean percentage numbers among CD4+ T cells with error bars of SEM derived from three different donors. E, A representative flow cytometry data showing that IL-22–producing CD4+ T cells do not coexpress TFN-α (CD4-gated). No coexpression of IFN-γ was found either (data not shown). Cells were isolated from peripheral blood of M. tuberculosis-infected macaques. Similar data were observed in five different macaques. F, A representative confocal microscopic image shows that IL-22 forms the capping domain (as marked by white arrowheads) and colocalizes in part with CD4 on T cell membrane. Cells were isolated from the blood of M. tuberculosis-infected macaques, and were stained directly with anti–IL-22 and anti-CD4 Abs without membrane permeabilization reagents. G, IL-22 in concentrated culture supernatant (left) from activated, enriched CD4+ T cells could be detected by the dot blot assay. CD4+ T cells from spleens of M. tuberculosis-infected macaques were enriched by deleting CD8+ cells using immune-magnetic beads, as described above. Approximately 1 × 107 enriched CD4+ T cells were stimulated by PPD in culture for 2 d. Supernatants were collected, either unconcentrated or concentrated 50× using Vivacell concentrator (Vivasciences) (28, 29), and then tested for IL-22 by the dot plot assay, as we described (28, 29). Negative control (right) was culture supernatant from unstimulated PBL of uninfected macaques.

FIGURE 1.

Upon activation, CD4+ T cells in M. tuberculosis-infected macaques and humans differentiated into T effector cells bearing membrane-bound IL-22 after de novo IL-22 production. Percentage numbers of CD4+ T cells bearing membrane-bound IL-22 or CD4+ T cells expressing membrane-bound IFN-γ are shown in the upper right quadruple of flow cytometry histograms. Data were gated on CD4 T cells. All cells were surface stained directly without treatment with membrane permeabilization reagents. No membrane-bound IL-22 were detected for negative controls (see 1Materials and Methods). Membrane-bound IL-22 did not appear to be specific for M. tuberculosis infection as CD4+ T cells from Listeria-vaccinated macaques could also express membrane-bound IL-22 upon activation by CD3/CD28 Ab (data not shown). A, Representative flow cytometry histograms show that M. tuberculosis stimulation of PBMC from M. tuberculosis-infected macaques induced membrane-bound IL-22+ CD4+ T effector cells, whereas CD4+ T cells in PBMC from uninfected macaques contained very few membrane-bound IL-22+ T effectors. PBMC from infected macaques were isolated from the blood collected 2 mo after M. tuberculosis infection. Only 0.18% of CD4+ T cells expressing membrane-bound IFN-γ was detectable in M. tuberculosis-infected PBMC. PBMC of macaques were infected with M. tuberculosis in 96-well plates in MOI = 1 for 18 h prior to analysis. B, Representative flow cytometry histograms show that M. tuberculosis stimulation of PBMC from PPD+ human donors induced 12% of CD4+ T cells bearing membrane-bound IL-22. Only ∼0.3% of CD4 T cells bearing membrane-bound IL-22 were detected in PBMC isolated from PPD donors. Also, only ∼0.01% of CD4 T cells expressing membrane-bound IFN-γ could be detected in blood lymphocytes of PPD+ donors. Human PBMC were similarly infected with M. tuberculosis, as described above, or stimulated overnight with anti-CD3/anti-CD28 in presence of PPD and stained without cell permeabilization treatment. C, Bar graph data show that M. tuberculosis-infected macaques exhibited significantly greater numbers of CD4+ T cells bearing membrane-bound IL-22 than uninfected macaques after M. tuberculosis stimulation in vitro (***p < 0.001, n = 3). Data are mean percentage numbers among CD4+ T cells with error bars of SEM derived from three rhesus macaques. D, Bar graphic data show that PPD+ human donors exhibited significantly greater numbers of CD4+ T cells bearing membrane-bound IL-22 than PPD donors after M. tuberculosis stimulation in vitro (*p < 0.05, n = 3). Data are mean percentage numbers among CD4+ T cells with error bars of SEM derived from three different donors. E, A representative flow cytometry data showing that IL-22–producing CD4+ T cells do not coexpress TFN-α (CD4-gated). No coexpression of IFN-γ was found either (data not shown). Cells were isolated from peripheral blood of M. tuberculosis-infected macaques. Similar data were observed in five different macaques. F, A representative confocal microscopic image shows that IL-22 forms the capping domain (as marked by white arrowheads) and colocalizes in part with CD4 on T cell membrane. Cells were isolated from the blood of M. tuberculosis-infected macaques, and were stained directly with anti–IL-22 and anti-CD4 Abs without membrane permeabilization reagents. G, IL-22 in concentrated culture supernatant (left) from activated, enriched CD4+ T cells could be detected by the dot blot assay. CD4+ T cells from spleens of M. tuberculosis-infected macaques were enriched by deleting CD8+ cells using immune-magnetic beads, as described above. Approximately 1 × 107 enriched CD4+ T cells were stimulated by PPD in culture for 2 d. Supernatants were collected, either unconcentrated or concentrated 50× using Vivacell concentrator (Vivasciences) (28, 29), and then tested for IL-22 by the dot plot assay, as we described (28, 29). Negative control (right) was culture supernatant from unstimulated PBL of uninfected macaques.

Close modal

We then asked a question as to whether membrane-bound IL-22+ T effector cells could mature and sustain in vivo under highly inflammatory environments during active M. tuberculosis infection. To address this straightforward question, macaques were infected with M. tuberculosis by bronchoscope-guided inoculation, as previously described (35), and assessed directly for membrane-bound IL-22+ CD4+ T cells using surface immune staining without in vitro stimulation and membrane permeabilization. Surprisingly, appreciable percentage numbers (0.82 ± 0.09%) of spleen CD4+ T cells bearing membrane-bound IL-22 could be detected without in vitro culture and membrane permeabilization treatment (Fig. 2). A short-term in vitro restimulation of spleen lymphocytes by pooled ESAT-6 and Ag85B peptides resulted in the increased percentage numbers (3.03 ± 0.66%) of membrane-bound IL-22+ CD4+ T cells, suggesting that IL-22 binds to and retains in membrane after de novo production in newly generated IL-22+ T effector cells (Fig. 2). Consistently, the mature CD4+ T effector cells bearing membrane-bound IL-22 appeared to be driven by bacterial Ag loads in vivo because greater percentages (3.91 ± 1.66%) of these effector T cells could be detected directly without Ag restimulation in lymphocytes isolated from M. tuberculosis-infected lung tissues of the same macaques in comparisons with blood and spleen lymphocytes (Fig. 2) (37). The number of membrane-bound IL-22+ T cells from the lung was only subtly increased after the in vitro Ag stimulation (4.67 ± 1.88%) (Fig. 2), suggesting that most of these lung IL-22+ T cells matured enough in vivo and sustained their capability to retain IL-22 on membrane without rapid degradation after de novo production in highly inflammatory environments during M. tuberculosis infection.

FIGURE 2.

Membrane-bound IL-22+ CD4+ T cells matured in vivo and sustained in highly inflammatory environments during active M. tuberculosis infection. All cells used for experiments were not treated with cell permeabilization reagents during staining. For experiments without in vitro Ag restimulation, lymphocytes isolated from the spleens or lungs of M. tuberculosis-infected macaques (2 mo postinfection) were stained directly with anti–IL-22 Abs conjugating fluorochromes. Matched normal serum or isotype control Ab did not confer detectable staining of IL-22 as analyzed by flow cytometry (data not shown). Also, control PBMC from simian HIV–infected macaques displayed undetectable staining of membrane-bound IL-22+ CD4+ T cells (data not shown). Data were gated on CD4+ T cells. A, Left panel, A representative flow cytometry histogram showing that 1.1% of CD4+ T cells expressing membrane-bound IL-22 could be directly detected without any in vitro Ag stimulation in lymphocytes isolated from the spleens of M. tuberculosis-infected macaques. Right panel, A representative flow cytometry histogram showing that, after in vitro Ag stimulation, 3.0% of CD4+ T cells expressing membrane-bound IL-22 could be detected in lymphocytes isolated from the spleens of M. tuberculosis-infected macaques. Percentage numbers of membrane-bound IL-22+ CD4+ T cells are shown in the upper right quadruple of flow cytometry histograms. For in vitro Ag stimulation, lymphocytes isolated from spleens or lungs were stimulated with pooled ESAT-6 and Ag85B peptides in the presence of CD28/CD49d, and then stained directly with Abs without membrane permeabilization. B, Bar graphic data show that lymphocytes from the lung tissues contained much higher percentages of membrane-bound IL-22+ CD4+ T cells than those from the spleens when surface stained without Ag stimulation (*p < 0.05). After Ag stimulation, no significant increases (NS) in membrane-bound IL-22+ CD4+ T cells were detected in lung lymphocytes, although increases in these effector cells could be seen in spleen lymphocytes after in vitro Ag stimulation (**p < 0.01). Data are mean percentage numbers among CD4+ T cells with error bars of SEM derived from four rhesus macaques. In general, <2% of unstimulated CD4+ T cells in the blood from M. tuberculosis-infected macaques expressed membrane-bound IL-22. The studied macaques had ∼1000 CFU/10 ml lung tissue lysate, but had no or very few CFU measurable in the spleen tissue lysate. Also, these macaques showed severe TB lesions only in lung tissues, but mostly no gross lesions in the spleen tissues.

FIGURE 2.

Membrane-bound IL-22+ CD4+ T cells matured in vivo and sustained in highly inflammatory environments during active M. tuberculosis infection. All cells used for experiments were not treated with cell permeabilization reagents during staining. For experiments without in vitro Ag restimulation, lymphocytes isolated from the spleens or lungs of M. tuberculosis-infected macaques (2 mo postinfection) were stained directly with anti–IL-22 Abs conjugating fluorochromes. Matched normal serum or isotype control Ab did not confer detectable staining of IL-22 as analyzed by flow cytometry (data not shown). Also, control PBMC from simian HIV–infected macaques displayed undetectable staining of membrane-bound IL-22+ CD4+ T cells (data not shown). Data were gated on CD4+ T cells. A, Left panel, A representative flow cytometry histogram showing that 1.1% of CD4+ T cells expressing membrane-bound IL-22 could be directly detected without any in vitro Ag stimulation in lymphocytes isolated from the spleens of M. tuberculosis-infected macaques. Right panel, A representative flow cytometry histogram showing that, after in vitro Ag stimulation, 3.0% of CD4+ T cells expressing membrane-bound IL-22 could be detected in lymphocytes isolated from the spleens of M. tuberculosis-infected macaques. Percentage numbers of membrane-bound IL-22+ CD4+ T cells are shown in the upper right quadruple of flow cytometry histograms. For in vitro Ag stimulation, lymphocytes isolated from spleens or lungs were stimulated with pooled ESAT-6 and Ag85B peptides in the presence of CD28/CD49d, and then stained directly with Abs without membrane permeabilization. B, Bar graphic data show that lymphocytes from the lung tissues contained much higher percentages of membrane-bound IL-22+ CD4+ T cells than those from the spleens when surface stained without Ag stimulation (*p < 0.05). After Ag stimulation, no significant increases (NS) in membrane-bound IL-22+ CD4+ T cells were detected in lung lymphocytes, although increases in these effector cells could be seen in spleen lymphocytes after in vitro Ag stimulation (**p < 0.01). Data are mean percentage numbers among CD4+ T cells with error bars of SEM derived from four rhesus macaques. In general, <2% of unstimulated CD4+ T cells in the blood from M. tuberculosis-infected macaques expressed membrane-bound IL-22. The studied macaques had ∼1000 CFU/10 ml lung tissue lysate, but had no or very few CFU measurable in the spleen tissue lysate. Also, these macaques showed severe TB lesions only in lung tissues, but mostly no gross lesions in the spleen tissues.

Close modal

Studies from us and others suggest that during cellular signaling/activation, cell membrane ligands or receptors undergo instructed engagements forming clusters or capping domains to enhance ligand–receptor interaction between the two contact cells and to sustain cellular activation (32, 38, 39). Thus, we hypothesized that membrane-bound IL-22 molecules might engage themselves, forming unique nanostructures for putative interaction with IL-22R on responsive cells. To test this hypothesis, we employed our expertise of confocal imaging and NSOM/QD-based nanoscale molecular imaging (30, 32, 39) to visualize membrane-bound nature and nanostructures of IL-22 molecules. Confocal microscopy showed that IL-22 on the surface of spleen CD4+ T effectors localized as large capping domains (Fig. 3A), but could not confer high-resolution nanoscale imaging of molecular details. In contrast, our novel NSOM/QD-based nanoscale imaging system, which exclusively images cell surface membrane molecules, but not intracellular contents of intact cells (30, 32, 39), revealed that IL-22 cytokine proteins engaged themselves as ∼100–200 nm nanoclusters on outer membrane (Fig. 3B). Some of these IL-22 nanoclusters were arrayed to form ∼300–600 nm nanodomains (Fig. 3B, 3C). The formation of these IL-22 nanoclulsters or nanodomains on membrane suggested that large amounts of IL-22 were retained on T cell membrane and actively involved in immune responses during active M. tuberculosis infection. However, these membrane-bound IL-22 nanoclusters were seldom colocalized with CD4 molecules (Fig. 3B, merge).

FIGURE 3.

Membrane-bound IL-22 molecules were engaged as ∼100–200 nm nanoclusters or ∼300–600 nm high-density nanodomains on membrane of IL-22+ CD4+ T effectors. No membrane-bound IL-22 were detected for negative controls (see 1Materials and Methods). A, Confocal microscopic images show that membrane-bound IL-22 formed capping domains on CD4 T cells after in vivo M. tuberculosis infection. Lymphocytes isolated from the spleens of M. tuberculosis-infected macaques (2 mo postinfection) were surface stained with anti–IL-22 Ab, without in vitro Ag stimulation or membrane permeabilization. This only allowed membrane-bound IL-22 to be stained and visualized by confocal microscopy. The white arrowheads indicated membrane-bound IL-22 on CD4+ T cells. Note that membrane-bound IL-22 forms capping domains on CD4+ T cell surface. No membrane-bound IL-22 was observed in PBMC from uninfected macaques (data not shown). Isotype control IgG did not stain cell membrane of PBMC from infected macaques (data not shown). Scale bars, 5 μm. B, Representative NSOM/QD-based nanoscale imaging shows that membrane-bound IL-22 are engaged as ∼100–200 nm nanoclusters or as ∼300–600 nm high-density nanodomains. Upper panels, Show fluorescence imaging of two-color molecular staining of IL-22 (green) and CD4 (red) on outer membrane and representative topography (right) for one representative membrane-bound IL-22+ CD4+ T effector cell. Note that NSOM/QD-based nanoscale imaging only detects fluorescence dots on outer membrane, but not underneath (see text). Few IL-22 molecules colocalize with CD4 in the merged image. Lower panels, Show the enlarged images derived from rescanning the dashed squares in the IL-22 and CD4 images and topography in the upper panels. The scale bars for the upper and lower panels are 3.214 μm and 385 nm, respectively. The staining samples for NSOM/QD imaging were similarly prepared as those for confocal imaging. Integration time for all the images is 30 ms with 400 × 400 scanning lines. C, Upper panel, A representative fluorescent intensity profile of an IL-22 nanocluster. The fluorescent intensity profile (upper) of the cross section of the objects is marked by a line in the fluorescence image. Shown is an IL-22 nanocluster with diameters of 274 nm (full width at half maximum [FWHM]). Lower panel, Histogram graph shows the frequency of different sizes of FWHM of IL-22 nanoclusters on the CD4+ T cells. The green arrowheads marked the FWHM of IL-22 microdomains (>500 nm). D, Upper panel, A representative fluorescence intensity profile of a CD4 nanocluster. The fluorescent intensity profile (upper) of the cross section of the objects is marked by a line in the fluorescence image as well. Shown are CD4 clusters with diameters of 87 nm (FWHM). Lower panel, Histogram graph shows the frequency of different sizes of FWHM of CD4 nanoclusters or nanodomains on the CD4+ T cells.

FIGURE 3.

Membrane-bound IL-22 molecules were engaged as ∼100–200 nm nanoclusters or ∼300–600 nm high-density nanodomains on membrane of IL-22+ CD4+ T effectors. No membrane-bound IL-22 were detected for negative controls (see 1Materials and Methods). A, Confocal microscopic images show that membrane-bound IL-22 formed capping domains on CD4 T cells after in vivo M. tuberculosis infection. Lymphocytes isolated from the spleens of M. tuberculosis-infected macaques (2 mo postinfection) were surface stained with anti–IL-22 Ab, without in vitro Ag stimulation or membrane permeabilization. This only allowed membrane-bound IL-22 to be stained and visualized by confocal microscopy. The white arrowheads indicated membrane-bound IL-22 on CD4+ T cells. Note that membrane-bound IL-22 forms capping domains on CD4+ T cell surface. No membrane-bound IL-22 was observed in PBMC from uninfected macaques (data not shown). Isotype control IgG did not stain cell membrane of PBMC from infected macaques (data not shown). Scale bars, 5 μm. B, Representative NSOM/QD-based nanoscale imaging shows that membrane-bound IL-22 are engaged as ∼100–200 nm nanoclusters or as ∼300–600 nm high-density nanodomains. Upper panels, Show fluorescence imaging of two-color molecular staining of IL-22 (green) and CD4 (red) on outer membrane and representative topography (right) for one representative membrane-bound IL-22+ CD4+ T effector cell. Note that NSOM/QD-based nanoscale imaging only detects fluorescence dots on outer membrane, but not underneath (see text). Few IL-22 molecules colocalize with CD4 in the merged image. Lower panels, Show the enlarged images derived from rescanning the dashed squares in the IL-22 and CD4 images and topography in the upper panels. The scale bars for the upper and lower panels are 3.214 μm and 385 nm, respectively. The staining samples for NSOM/QD imaging were similarly prepared as those for confocal imaging. Integration time for all the images is 30 ms with 400 × 400 scanning lines. C, Upper panel, A representative fluorescent intensity profile of an IL-22 nanocluster. The fluorescent intensity profile (upper) of the cross section of the objects is marked by a line in the fluorescence image. Shown is an IL-22 nanocluster with diameters of 274 nm (full width at half maximum [FWHM]). Lower panel, Histogram graph shows the frequency of different sizes of FWHM of IL-22 nanoclusters on the CD4+ T cells. The green arrowheads marked the FWHM of IL-22 microdomains (>500 nm). D, Upper panel, A representative fluorescence intensity profile of a CD4 nanocluster. The fluorescent intensity profile (upper) of the cross section of the objects is marked by a line in the fluorescence image as well. Shown are CD4 clusters with diameters of 87 nm (FWHM). Lower panel, Histogram graph shows the frequency of different sizes of FWHM of CD4 nanoclusters or nanodomains on the CD4+ T cells.

Close modal

To examine whether IL-22 was distributed within membrane rather than simply attached to the very surface of membrane, we dissected cross-sectional membrane and intracellular compartments of individual IL-22+ T effector cells in lung tissues containing TB granulomas using in situ confocal microscopy and NSOM/QD imaging techniques. Confocal images showed that IL-22, but not IFN-γ or IL-4, could be reproducibly detected on CD3+ T cells in the sections derived from lung tissues containing TB granulomas (Fig. 4A). Under confocal microscopy, IL-22 and CD3 appeared to exhibit similar membrane distribution in the lung sections (Fig. 4A). Consistently, NSOM/QD-based nanoscale imaging showed that IL-22 cytokine proteins were distributed as nanoclusters/nanodomains in membrane rather than in the cytoplasm of effector cells in the lung sections (Fig. 4B–D). IL-22 indeed shared distribution patterns with membrane CD3 molecules (Fig. 4B). Some IL-22 nanoclusters appeared to colocalize with CD3 nanoclusters (Fig. 4B). Collectively, these results suggest that membrane-bound IL-22 molecules distributed in membrane and engaged as ∼100–200 nm nanoclusters or ∼300–600 nm high-density nanodomains on membrane of IL-22+ CD4+ T effectors in M. tuberculosis infection.

FIGURE 4.

Membrane-bound IL-22 distributed within membrane sharing similar distribution with membrane CD3 molecules of T cells in TB lung tissue sections. A, Representative in situ confocal microscopic images (×63 NA) in the bottom panel show that IL-22 appeared to colocalize with CD3 (marked by yellow arrows) in the membrane of CD3+ T cells in lung tissue sections prepared from the right middle lung lobe containing granulomas. The images at the top panel show that no IL-22 (white arrows) was detectable in control CD3 T cells in the peribronchiolar lymphoid lung section derived from one of three uninfected, healthy macaques. No IFN-γ or IL-4 was detectable in the T cell membrane from the same lung TB tissue sections. Matched normal serum or control isotype IgG did not give rise to any membrane IL-22 staining in the lung TB tissue sections (data not shown). B, Representative in situ NSOM/QD-based nanoscale imaging of membrane-bound IL-22 distributed within membrane sharing similar distribution with membrane CD3 molecules of T cells in TB lung tissue sections. Upper panels, Show in situ fluorescence imaging of two-color molecular staining of CD3 (red) and IL-22 (green) and topography for one representative IL-22+ CD3+ T cell in the lung tissue section. White arrowheads in IL-22/CD3 merge images indicate that IL-22 and CD3 similarly localized in the cell membrane rather than in the cytoplasm. Lower panels, Show the enlarged images derived from rescanning the dashed squares in the CD3, IL-22, merge images, and topography, respectively, in the upper panels. Scale bars for the upper and lower panels are 2.9 μm and 391 nm, respectively. Similar NSOM images were seen from five IL-22+ CD3+ T cells in the lung tissue sections. C, Upper panel, A representative fluorescence intensity profile of a CD3 nanocluster. The fluorescent intensity profile (upper) of the cross section of the objects is marked by a line in the fluorescence image as well. Shown is a CD3 cluster with diameters of 126 nm (full width at half maximum [FWHM]). Lower panel, Histogram graph shows the frequency of different sizes of FWHM of CD3 nanoclusters or nanodomains on the CD3+ T cells. D, Upper panel, A representative fluorescent intensity profile of an IL-22 nanocluster. The fluorescent intensity profile (upper) of the cross section of the objects is marked by a line in the fluorescence image. Shown is an IL-22 cluster with diameters of 143 nm (FWHM). Lower panel, Histogram graph shows the frequency of different sizes of FWHM of IL-22 nanoclusters on CD3+ T cells.

FIGURE 4.

Membrane-bound IL-22 distributed within membrane sharing similar distribution with membrane CD3 molecules of T cells in TB lung tissue sections. A, Representative in situ confocal microscopic images (×63 NA) in the bottom panel show that IL-22 appeared to colocalize with CD3 (marked by yellow arrows) in the membrane of CD3+ T cells in lung tissue sections prepared from the right middle lung lobe containing granulomas. The images at the top panel show that no IL-22 (white arrows) was detectable in control CD3 T cells in the peribronchiolar lymphoid lung section derived from one of three uninfected, healthy macaques. No IFN-γ or IL-4 was detectable in the T cell membrane from the same lung TB tissue sections. Matched normal serum or control isotype IgG did not give rise to any membrane IL-22 staining in the lung TB tissue sections (data not shown). B, Representative in situ NSOM/QD-based nanoscale imaging of membrane-bound IL-22 distributed within membrane sharing similar distribution with membrane CD3 molecules of T cells in TB lung tissue sections. Upper panels, Show in situ fluorescence imaging of two-color molecular staining of CD3 (red) and IL-22 (green) and topography for one representative IL-22+ CD3+ T cell in the lung tissue section. White arrowheads in IL-22/CD3 merge images indicate that IL-22 and CD3 similarly localized in the cell membrane rather than in the cytoplasm. Lower panels, Show the enlarged images derived from rescanning the dashed squares in the CD3, IL-22, merge images, and topography, respectively, in the upper panels. Scale bars for the upper and lower panels are 2.9 μm and 391 nm, respectively. Similar NSOM images were seen from five IL-22+ CD3+ T cells in the lung tissue sections. C, Upper panel, A representative fluorescence intensity profile of a CD3 nanocluster. The fluorescent intensity profile (upper) of the cross section of the objects is marked by a line in the fluorescence image as well. Shown is a CD3 cluster with diameters of 126 nm (full width at half maximum [FWHM]). Lower panel, Histogram graph shows the frequency of different sizes of FWHM of CD3 nanoclusters or nanodomains on the CD3+ T cells. D, Upper panel, A representative fluorescent intensity profile of an IL-22 nanocluster. The fluorescent intensity profile (upper) of the cross section of the objects is marked by a line in the fluorescence image. Shown is an IL-22 cluster with diameters of 143 nm (FWHM). Lower panel, Histogram graph shows the frequency of different sizes of FWHM of IL-22 nanoclusters on CD3+ T cells.

Close modal

The role of IL-22–producing CD4 T effector cells in M. tuberculosis infection remains unknown. Now, our identification of CD4+ T effector cells bearing membrane-bound IL-22 made it possible to isolate IL-22+ CD4+ T cells and to determine whether these cells could have positive or negative impact on intracellular M. tuberculosis replication in IL-22R–expressing macrophages or DC (Fig. 5A). Thus, monocyte-derived macrophages or DCs were infected with M. tuberculosis, and cocultured with autologous membrane-bound IL-22+ CD4+ T cells that were purified from macaque PBL using immunomagnetic beads, as we described previously (35, 39). This procedure enriched membrane-bound IL-22+ CD4+ T cells to the level of 70–96% (Fig. 5B). Purified autologous B cells, CD25CD4+ T cells, and IL-22 CD4+ T cells served as controls, respectively. Interestingly, the purified membrane-bound IL-22+ CD4+ T cells significantly inhibited intracellular M. tuberculosis replication in cultured macrophages (Fig. 5C). Such inhibition of M. tuberculosis replication appeared to be mediated at least in part by membrane-bound IL-22 as anti–IL-22 neutralizing Ab could significantly reverse the inhibition (Fig. 5C) and as the intracellular M. tuberculosis growth was inhibited only by membrane IL-22+ CD4+ T cells contacting with M. tuberculosis-infected macrophages, but not by those noncontacting IL-22+ CD4+ T cells or culture medium containing IL-22 secreted by the effector cells (Fig. 5D). The in vitro work therefore provided novel evidence demonstrating that membrane-bound IL-22+ CD4+ T cells could inhibit intracellular M. tuberculosis replication in macrophages.

FIGURE 5.

Membrane-bound IL-22+ CD4+ T cells inhibited intracellular M. tuberculosis replication in monocyte-derived macrophages. A, Flow cytometry characterization of IL-22R expression induced by M. tuberculosis infection of macrophages. Monocyte-derived macrophages were stained with IL-22R and CD14, and analyzed by flow cytometry. Left panel, A representative flow cytometry histogram shows that only 1.5% of IL-22R–expressing CD14+ macrophages (gated on CD14) were observed in monocyte-derived macrophages without M. tuberculosis infection. Right panel, A representative flow cytometry histogram shows that after in vitro M. tuberculosis infection, ∼18% of CD14+ macrophages (gated on CD14) expressed IL-22R (upper right quadruple). Similar results were seen in other five independent experiments. B, Representative flow cytometry data show that immunomagnetic bead-based purification could enrich IL-22+ CD4+ T cells to the level of 70–96%. Similar results were observed in six other repeated experiments. C, Bar graphic data show that IL-22+ CD4+ T cells inhibited intracellular M. tuberculosis replication in monocyte-derived macrophages. M. tuberculosis-infected macrophages (5 × 104) were cocultured with medium alone, CD20+ B cells (5 × 105), CD25CD4+ T cells (5 × 105), IL-22 CD4+ T cells (5 × 105), IL-22+ CD4+ T cells (5 × 105), or IL-22+ CD4+ T cells (5 × 105) plus anti–IL-22 Ab (10 μg/ml), IL-22+ CD4+ T cells (5 × 105) plus IgG (10 μg/ml) for 4 d. The cultured cells were lysed, and CFU counts in lysate were determined, as described in 1Materials and Methods. Enriched IL-22+ CD4+ T cells (5 × 105) significantly reduced M. tuberculosis CFU counts when compared with control cultures (***p < 0.001, **p < 0.01, *p < 0.05, respectively). Blocking of membrane-bound IL-22 by anti–IL-22 Ab, but not isotype IgG control from the beginning throughout the assay led to a reduced capability of IL-22+ CD4 T cells to inhibit intracellular M. tuberculosis (*p < 0.05). Data were mean values with error bars of SEM derived from four rhesus macaques in five independent experiments. The marked reduction in CFU counts was interpreted as “inhibition” in the text, although “killing” was implicative based on the growth kinetics of M. tuberculosis on days 0, 2, and 4 postinfection (data not shown). D, Bar graphic data show that M. tuberculosis growth in monocyte-derived macrophages was inhibited by contact IL-22+ CD4+ T cells (contacting with infected macrophages), but not those noncontact IL-22+ CD4+ T cells or culture medium containing IL-22 secreted by the IL-22+ CD4+ T effector cells. M. tuberculosis-infected macrophages (5 × 104) were cocultured in Transwell system with medium alone, contact IL-22+ CD4+ T cells (5 × 105), and noncontact IL-22+ CD4+ T cells (5 × 105) for 4 d prior to bacteria counts. Noncontact means that enriched IL-22+ CD4+ T cells were placed in the upper chamber of Transwell culture system without physical contact with infected macrophages that were cultured in the lower chamber. Therefore, noncontact IL-22+ CD4+ T cells did not contact macrophages, but secretory IL-22 in the medium could trans-pass through the microholes and reach macrophages in the lower chamber. Contact means that IL-22+ CD4+ T cells and infected macrophages were cocultured in the lower chamber of Transwell culture system. Data in graph were mean values with error bars of SEM derived from four rhesus macaques. **p < 0.01. NS, no significant difference.

FIGURE 5.

Membrane-bound IL-22+ CD4+ T cells inhibited intracellular M. tuberculosis replication in monocyte-derived macrophages. A, Flow cytometry characterization of IL-22R expression induced by M. tuberculosis infection of macrophages. Monocyte-derived macrophages were stained with IL-22R and CD14, and analyzed by flow cytometry. Left panel, A representative flow cytometry histogram shows that only 1.5% of IL-22R–expressing CD14+ macrophages (gated on CD14) were observed in monocyte-derived macrophages without M. tuberculosis infection. Right panel, A representative flow cytometry histogram shows that after in vitro M. tuberculosis infection, ∼18% of CD14+ macrophages (gated on CD14) expressed IL-22R (upper right quadruple). Similar results were seen in other five independent experiments. B, Representative flow cytometry data show that immunomagnetic bead-based purification could enrich IL-22+ CD4+ T cells to the level of 70–96%. Similar results were observed in six other repeated experiments. C, Bar graphic data show that IL-22+ CD4+ T cells inhibited intracellular M. tuberculosis replication in monocyte-derived macrophages. M. tuberculosis-infected macrophages (5 × 104) were cocultured with medium alone, CD20+ B cells (5 × 105), CD25CD4+ T cells (5 × 105), IL-22 CD4+ T cells (5 × 105), IL-22+ CD4+ T cells (5 × 105), or IL-22+ CD4+ T cells (5 × 105) plus anti–IL-22 Ab (10 μg/ml), IL-22+ CD4+ T cells (5 × 105) plus IgG (10 μg/ml) for 4 d. The cultured cells were lysed, and CFU counts in lysate were determined, as described in 1Materials and Methods. Enriched IL-22+ CD4+ T cells (5 × 105) significantly reduced M. tuberculosis CFU counts when compared with control cultures (***p < 0.001, **p < 0.01, *p < 0.05, respectively). Blocking of membrane-bound IL-22 by anti–IL-22 Ab, but not isotype IgG control from the beginning throughout the assay led to a reduced capability of IL-22+ CD4 T cells to inhibit intracellular M. tuberculosis (*p < 0.05). Data were mean values with error bars of SEM derived from four rhesus macaques in five independent experiments. The marked reduction in CFU counts was interpreted as “inhibition” in the text, although “killing” was implicative based on the growth kinetics of M. tuberculosis on days 0, 2, and 4 postinfection (data not shown). D, Bar graphic data show that M. tuberculosis growth in monocyte-derived macrophages was inhibited by contact IL-22+ CD4+ T cells (contacting with infected macrophages), but not those noncontact IL-22+ CD4+ T cells or culture medium containing IL-22 secreted by the IL-22+ CD4+ T effector cells. M. tuberculosis-infected macrophages (5 × 104) were cocultured in Transwell system with medium alone, contact IL-22+ CD4+ T cells (5 × 105), and noncontact IL-22+ CD4+ T cells (5 × 105) for 4 d prior to bacteria counts. Noncontact means that enriched IL-22+ CD4+ T cells were placed in the upper chamber of Transwell culture system without physical contact with infected macrophages that were cultured in the lower chamber. Therefore, noncontact IL-22+ CD4+ T cells did not contact macrophages, but secretory IL-22 in the medium could trans-pass through the microholes and reach macrophages in the lower chamber. Contact means that IL-22+ CD4+ T cells and infected macrophages were cocultured in the lower chamber of Transwell culture system. Data in graph were mean values with error bars of SEM derived from four rhesus macaques. **p < 0.01. NS, no significant difference.

Close modal

In the current study, we made an unexpected discovery that IL-22–producing CD4+ T cells in M. tuberculosis-infected macaques and humans could retain or held IL-22 in cell membrane after de novo production. This observation was confirmed by flow cytometry-based surface immune staining, confocal microscopy, and, most importantly, NSOM/QD-based nanoscale imaging. The NSOM/QD-based nanoscale imaging on cell surface indeed provides most direct evidence for the membrane-bound IL-22 as NSOM scans and detects molecules or signals exclusively on the cell surface, but not the underneath when cell surface is scanned (32, 39). NSOM/QD imaging of cross-sectional membrane in tissue sections also suggests that membrane IL-22 molecules are distributed in membrane sharing distribution patterns with membrane CD3. IL-22 is therefore one of the very few cytokines with both secretory and membrane-bound forms (24, 25), although conventional protein chemistry and immunology studies suggest that the mature IL-22 protein, like other cytokines, is most likely secreted to the extracellular environment (40). Nevertheless, low-level secretory human or macaque IL-22 proteins appeared to exist as well in TB bronchoalveolar lavage fluid and culture medium after in vitro immune stimulation (21) (Fig. 1G). The precise mechanism whereby IL-22 retains in the membrane is currently not known. Both human and macaque mature IL-22 do not carry classical trans-membrane sequences, and we found no evidence that TB could induce variant IL-22 mRNA encoding additional trans-membrane domains in the C terminal (data not shown). It is important to note that membrane-bound IL-22+ CD4+ T cells can directly be detected without in vitro Ag stimulation in spleen cells and lymphocytes from lung tissues containing TB granuloma, suggesting that membrane-bound IL-22 possesses long t1/2 and sustains even in TB inflammatory environments.

Notably, membrane-bound IL-22 cytokine molecules are engaged as ∼100–200 nm nanoclusters or ∼300–600 nm high-density nanodomains on outer membrane of CD4+ T effector cells that mature in vivo. It is also noteworthy that IL-22 nanoclusters or nanodomains coexist with CD4 and CD3 nanoclusters or nanodomains on membrane of IL-22+ T effector cells. T cells bearing membrane nanoclusters/nanodomains of IL-22, CD4, and CD3 appear to be highly activated effector cells during M. tuberculosis infection or in granulomas formation, because we and others show that occurrence of CD3 and CD4 nanoclusters or nanodomains is a structured engagement of T cell activation for efficient TCR interaction with APC and for sustaining T cell activation (38, 39). More importantly, such membrane IL-22 nanoclusters or nanodomains may confer high-affinity interaction with IL-22R on responsive cells. Thus, membrane-bound IL-22+ CD4+ T cells appear to be immunologically selected to exert prolonged and high-affinity effects on IL-22R–expressing cells in infections.

Our results provided the first evidence, to our knowledge, that purified IL-22+ CD4+ T cells could mediate inhibition of intracellular M. tuberculosis replication in monocyte-derived macrophages. Th1, Th2, Th17, and T regulatory cells are four major CD4 Th subsets that can differentiate from naive CD4+ T cells during infections (1). To date, antimicrobial effector function of human Th17, Th1, and Th2 subsets themselves has remained poorly defined, because it is impossible to isolate or purify each of these Th subsets for functional analyses. The membrane-bound IL-22 as surface marker allowed us to isolate IL-22+ Th22 cells and evaluate their antimicrobial function. Using this approach, we demonstrated that IL-22+ T cells themselves could inhibit intracellular M. tuberculosis in macrophages in the culture system. To our knowledge, this is also the first demonstration that a purified Th subset is able to directly mediate the inhibition of intracellular bacterium in vitro, although soluble IL-22 has been shown to limit M. tuberculosis growth (41). The membrane IL-22 bioactivity appeared to be supported by the fact that membrane-bound IL-22 in purified T cells could be detected for 2–3 d in cultures (data not shown), and that anti–IL-22 Ab incubation with purified IL-22+ CD4+ T cells from the beginning throughout the assay could reduce the effector cells’ ability to limit M. tuberculosis growth. The result from Transwell system-based experiments also suggests that membrane-bound IL-22 appears to contribute to the IL-22+ CD4+ T effector-mediated restriction of M. tuberculosis replication in macrophages as M. tuberculosis growth is inhibited by membrane-bound IL-22+ CD4+ T cells contacting with M. tuberculosis-infected macrophages, but not by those noncontacting IL-22+ CD4+ T cells or culture medium containing soluble IL-22 secreted by the effector cells (Fig. 5D). Membrane-bound IL-22 may act on IL-22R expressed in M. tuberculosis-infected macrophages, as our new studies demonstrate that whereas IL-22R is constitutively expressed in epithelial cells, IL-22R expression can also be induced by M. tuberculosis infection of macrophages (Fig. 5A, and D. Huang, G. Zeng, C. Chen, R. Wang, and Z.W. Chen, manuscript in preparation). This is also supported by our previous observation that TB induces up-regulation of IL-22R transcript (IL-22Rβ [IL-10Rβ]) (20). Membrane-bound IL-22 and IL-22R interaction could help to bring together a T effector cell and an infected macrophage as well as to transduce a signal for anti-bacilli effector function. Thus, our results are consistent with the previous findings that IL-22 can exert antimicrobial activity against extracellular or intracellular pathogens (1417, 41). The recent human study suggests that soluble IL-22 can limit M. tuberculosis growth in macrophages through a mechanism of enhancing phagolysosomal fusion (41). However, our data cannot exclude the possibility that other cytokines or interacting molecules of the IL-22+ CD4+ T cells also contribute to or synergize the in vitro anti-M. tuberculosis effector function (42). Our previous observations support the hypothesis that IL-22+ T cells may function as either proinflammatory or antimicrobial elements in M. tuberculosis infection, depending upon the magnitude of bacterial burdens that elicit optimal or overreacting IL-22+ T effector cells (23). Now the new finding from the current study raises the possibility to define the in vivo function of membrane-bound IL-22+ T cells or IL-22 cytokine during M. tuberculosis infection of nonhuman primates.

We thank Dr. M.L. Chen for confocal microscopic technical assistance, Jewell Graves, Brenda Paige, and Juan Chen for flow cytometry assistance, Dr. Lisa Halliday for animal work assistance, and P.Y. Teng for NSOM data analysis.

This work was supported by National Institutes of Health R01 Grants HL64560 and RR13601 (to Z.W.C.). Near-field scanning optical microscopy imaging was supported in part by Sino National Natural Science Foundation Grant NNSFC 30828028.

Abbreviations used in this article:

MOI

multiplicity of infection

NSOM

near-field scanning optical microscopy

PB

Pacific blue

PPD

purified protein derivative

QD

quantum dot

TB

tuberculosis.

1
Zhou
L.
,
Chong
M. M.
,
Littman
D. R.
.
2009
.
Plasticity of CD4+ T cell lineage differentiation.
Immunity
30
:
646
655
.
2
Zhu
J.
,
Paul
W. E.
.
2008
.
CD4 T cells: fates, functions, and faults.
Blood
112
:
1557
1569
.
3
Luci
C.
,
Reynders
A.
,
Ivanov
I. I.
,
Cognet
C.
,
Chiche
L.
,
Chasson
L.
,
Hardwigsen
J.
,
Anguiano
E.
,
Banchereau
J.
,
Chaussabel
D.
, et al
.
2009
.
Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin.
Nat. Immunol.
10
:
75
82
.
4
Zenewicz
L. A.
,
Yancopoulos
G. D.
,
Valenzuela
D. M.
,
Murphy
A. J.
,
Stevens
S.
,
Flavell
R. A.
.
2008
.
Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease.
Immunity
29
:
947
957
.
5
Cella
M.
,
Fuchs
A.
,
Vermi
W.
,
Facchetti
F.
,
Otero
K.
,
Lennerz
J. K.
,
Doherty
J. M.
,
Mills
J. C.
,
Colonna
M.
.
2009
.
A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity.
Nature
457
:
722
725
.
6
Bettelli
E.
,
Korn
T.
,
Oukka
M.
,
Kuchroo
V. K.
.
2008
.
Induction and effector functions of T(H)17 cells.
Nature
453
:
1051
1057
.
7
Andoh
A.
,
Zhang
Z.
,
Inatomi
O.
,
Fujino
S.
,
Deguchi
Y.
,
Araki
Y.
,
Tsujikawa
T.
,
Kitoh
K.
,
Kim-Mitsuyama
S.
,
Takayanagi
A.
, et al
.
2005
.
Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts.
Gastroenterology
129
:
969
984
.
8
Ikeuchi
H.
,
Kuroiwa
T.
,
Hiramatsu
N.
,
Kaneko
Y.
,
Hiromura
K.
,
Ueki
K.
,
Nojima
Y.
.
2005
.
Expression of interleukin-22 in rheumatoid arthritis: potential role as a proinflammatory cytokine.
Arthritis Rheum.
52
:
1037
1046
.
9
Zheng
Y.
,
Danilenko
D. M.
,
Valdez
P.
,
Kasman
I.
,
Eastham-Anderson
J.
,
Wu
J.
,
Ouyang
W.
.
2007
.
Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis.
Nature
445
:
648
651
.
10
Ma
H. L.
,
Liang
S.
,
Li
J.
,
Napierata
L.
,
Brown
T.
,
Benoit
S.
,
Senices
M.
,
Gill
D.
,
Dunussi-Joannopoulos
K.
,
Collins
M.
, et al
.
2008
.
IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation.
J. Clin. Invest.
118
:
597
607
.
11
Wilson
M. S.
,
Feng
C. G.
,
Barber
D. L.
,
Yarovinsky
F.
,
Cheever
A. W.
,
Sher
A.
,
Grigg
M.
,
Collins
M.
,
Fouser
L.
,
Wynn
T. A.
.
2010
.
Redundant and pathogenic roles for IL-22 in mycobacterial, protozoan, and helminth infections.
J. Immunol.
184
:
4378
4390
.
12
Zenewicz
L. A.
,
Yancopoulos
G. D.
,
Valenzuela
D. M.
,
Murphy
A. J.
,
Karow
M.
,
Flavell
R. A.
.
2007
.
Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation.
Immunity
27
:
647
659
.
13
Sugimoto
K.
,
Ogawa
A.
,
Mizoguchi
E.
,
Shimomura
Y.
,
Andoh
A.
,
Bhan
A. K.
,
Blumberg
R. S.
,
Xavier
R. J.
,
Mizoguchi
A.
.
2008
.
IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis.
J. Clin. Invest.
118
:
534
544
.
14
Wolk
K.
,
Kunz
S.
,
Witte
E.
,
Friedrich
M.
,
Asadullah
K.
,
Sabat
R.
.
2004
.
IL-22 increases the innate immunity of tissues.
Immunity
21
:
241
254
.
15
Liang
S. C.
,
Tan
X. Y.
,
Luxenberg
D. P.
,
Karim
R.
,
Dunussi-Joannopoulos
K.
,
Collins
M.
,
Fouser
L. A.
.
2006
.
Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides.
J. Exp. Med.
203
:
2271
2279
.
16
Zheng
Y.
,
Valdez
P. A.
,
Danilenko
D. M.
,
Hu
Y.
,
Sa
S. M.
,
Gong
Q.
,
Abbas
A. R.
,
Modrusan
Z.
,
Ghilardi
N.
,
de Sauvage
F. J.
,
Ouyang
W.
.
2008
.
Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens.
Nat. Med.
14
:
282
289
.
17
Aujla
S. J.
,
Chan
Y. R.
,
Zheng
M.
,
Fei
M.
,
Askew
D. J.
,
Pociask
D. A.
,
Reinhart
T. A.
,
McAllister
F.
,
Edeal
J.
,
Gaus
K.
, et al
.
2008
.
IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia.
Nat. Med.
14
:
275
281
.
18
Wirth
T.
,
Hildebrand
F.
,
Allix-Béguec
C.
,
Wölbeling
F.
,
Kubica
T.
,
Kremer
K.
,
van Soolingen
D.
,
Rüsch-Gerdes
S.
,
Locht
C.
,
Brisse
S.
, et al
.
2008
.
Origin, spread and demography of the Mycobacterium tuberculosis complex.
PLoS Pathog.
4
:
e1000160
.
19
Kaufmann
S. H.
2001
.
How can immunology contribute to the control of tuberculosis?
Nat. Rev. Immunol.
1
:
20
30
.
20
Qiu
L.
,
Huang
D.
,
Chen
C. Y.
,
Wang
R.
,
Shen
L.
,
Shen
Y.
,
Hunt
R.
,
Estep
J.
,
Haynes
B. F.
,
Jacobs
W. R.
 Jr.
, et al
.
2008
.
Severe tuberculosis induces unbalanced up-regulation of gene networks and overexpression of IL-22, MIP-1alpha, CCL27, IP-10, CCR4, CCR5, CXCR3, PD1, PDL2, IL-3, IFN-beta, TIM1, and TLR2 but low antigen-specific cellular responses.
J. Infect. Dis.
198
:
1514
1519
.
21
Scriba
T. J.
,
Kalsdorf
B.
,
Abrahams
D. A.
,
Isaacs
F.
,
Hofmeister
J.
,
Black
G.
,
Hassan
H. Y.
,
Wilkinson
R. J.
,
Walzl
G.
,
Gelderbloem
S. J.
, et al
.
2008
.
Distinct, specific IL-17- and IL-22-producing CD4+ T cell subsets contribute to the human anti-mycobacterial immune response.
J. Immunol.
180
:
1962
1970
.
22
Zeng
G.
,
Huang
D.
,
Chen
C. Y.
,
Shen
L.
,
Wang
R. R.
,
Chen
Z. W.
.
2009
.
In situ visualization of IL-22 cytokine response in tuberculosis, reply to Vani et al.
J. Infect. Dis.
200
:
482
484
.
23
Yao
S.
,
Zeng
G.
,
Huang
D.
,
Chen
C. Y.
,
Halliday
L.
,
Wang
R. R.
,
Chen
Z. W.
.
2010
.
Differentiation, distribution, and immune regulation of IL-22-producing Th17 cells during M. tuberculosis infection of macaques.
PLoS Pathog.
6
:
e1000789
.
24
Bruns
H.
,
Meinken
C.
,
Schauenberg
P.
,
Härter
G.
,
Kern
P.
,
Modlin
R. L.
,
Antoni
C.
,
Stenger
S.
.
2009
.
Anti-TNF immunotherapy reduces CD8+ T cell-mediated antimicrobial activity against Mycobacterium tuberculosis in humans.
J. Clin. Invest.
119
:
1167
1177
.
25
Ostroukhova
M.
,
Qi
Z.
,
Oriss
T. B.
,
Dixon-McCarthy
B.
,
Ray
P.
,
Ray
A.
.
2006
.
Treg-mediated immunosuppression involves activation of the Notch-HES1 axis by membrane-bound TGF-beta.
J. Clin. Invest.
116
:
996
1004
.
26
Chen
C. Y.
,
Huang
D.
,
Wang
R. C.
,
Shen
L.
,
Zeng
G.
,
Yao
S.
,
Shen
Y.
,
Halliday
L.
,
Fortman
J.
,
McAllister
M.
, et al
.
2009
.
A critical role for CD8 T cells in a nonhuman primate model of tuberculosis.
PLoS Pathog.
5
:
e1000392
.
27
Shen
Y.
,
Zhou
D.
,
Qiu
L.
,
Lai
X.
,
Simon
M.
,
Shen
L.
,
Kou
Z.
,
Wang
Q.
,
Jiang
L.
,
Estep
J.
, et al
.
2002
.
Adaptive immune response of Vgamma2Vdelta2+ T cells during mycobacterial infections.
Science
295
:
2255
2258
.
28
Wei
H.
,
Huang
D.
,
Lai
X.
,
Chen
M.
,
Zhong
W.
,
Wang
R.
,
Chen
Z. W.
.
2008
.
Definition of APC presentation of phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate to Vgamma2Vdelta 2 TCR.
J. Immunol.
181
:
4798
4806
.
29
Wei
H.
,
Wang
R.
,
Yuan
Z.
,
Chen
C. Y.
,
Huang
D.
,
Halliday
L.
,
Zhong
W.
,
Zeng
G.
,
Shen
Y.
,
Shen
L.
, et al
.
2009
.
DR*W201/P65 tetramer visualization of epitope-specific CD4 T-cell during M. tuberculosis infection and its resting memory pool after BCG vaccination.
PLoS One
4
:
e6905
.
30
Zeng
G.
,
Chen
J.
,
Zhong
L.
,
Wang
R.
,
Jiang
L.
,
Cai
J.
,
Yan
L.
,
Huang
D.
,
Chen
C. Y.
,
Chen
Z. W.
.
2009
.
NSOM- and AFM-based nanotechnology elucidates nano-structural and atomic-force features of a Y. pestis V immunogen-containing particle vaccine capable of eliciting robust response.
Proteomics
9
:
1538
1547
.
31
Chen
Y.
,
Qin
J.
,
Chen
Z. W.
.
2008
.
Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations.
J. Lipid Res.
49
:
2268
2275
.
32
Chen
Y.
,
Shao
L.
,
Ali
Z.
,
Cai
J.
,
Chen
Z. W.
.
2008
.
NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal Vgamma2Vdelta2 T-cell expansion.
Blood
111
:
4220
4232
.
33
Yong Chen
J. Q.
,
Cai
J.
,
Chen
Z. W.
.
2009
.
Cold induces micro/nano-scale reorganization of lipid raft markers at mounds of cell-membrane fluctuations
.
PLoS One
4
:
e5386
.
34
Huang
D.
,
Shen
Y.
,
Qiu
L.
,
Chen
C. Y.
,
Shen
L.
,
Estep
J.
,
Hunt
R.
,
Vasconcelos
D.
,
Du
G.
,
Aye
P.
, et al
.
2008
.
Immune distribution and localization of phosphoantigen-specific Vgamma2Vdelta2 T cells in lymphoid and nonlymphoid tissues in Mycobacterium tuberculosis infection.
Infect. Immun.
76
:
426
436
.
35
Gong
G.
,
Shao
L.
,
Wang
Y.
,
Chen
C. Y.
,
Huang
D.
,
Yao
S.
,
Zhan
X.
,
Sicard
H.
,
Wang
R.
,
Chen
Z. W.
.
2009
.
Phosphoantigen-activated V gamma 2V delta 2 T cells antagonize IL-2-induced CD4+CD25+Foxp3+ T regulatory cells in mycobacterial infection.
Blood
113
:
837
845
.
36
Dumoutier
L.
,
Lejeune
D.
,
Colau
D.
,
Renauld
J. C.
.
2001
.
Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cell-derived inducible factor/IL-22.
J. Immunol.
166
:
7090
7095
.
37
Yao
S.
,
Huang
D.
,
Chen
C. Y.
,
Halliday
L.
,
Zeng
G.
,
Wang
R. C.
,
Chen
Z. W.
.
2010
.
Differentiation, distribution and gammadelta T cell-driven regulation of IL-22-producing T cells in tuberculosis.
PLoS Pathog.
6
:
e1000789
.
38
Fooksman
D. R.
,
Vardhana
S.
,
Vasiliver-Shamis
G.
,
Liese
J.
,
Blair
D.
,
Waite
J.
,
Sacristan
C.
,
Victora
G.
,
Zanin-Zhorov
A.
,
Dustin
M. L.
.
2009
.
Functional anatomy of T cell activation and synapse formation.
Annu. Rev. Immunol.
182
:
5179
5182
.
39
Zhong
L.
,
Zeng
G.
,
Lu
X.
,
Wang
R. C.
,
Gong
G.
,
Yan
L.
,
Huang
D.
,
Chen
Z. W.
.
2009
.
NSOM/QD-based direct visualization of CD3-induced and CD28-enhanced nanospatial coclustering of TCR and coreceptor in nanodomains in T cell activation.
PLoS One
4
:
e5945
.
40
Dumoutier
L.
,
Louahed
J.
,
Renauld
J. C.
.
2000
.
Cloning and characterization of IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9.
J. Immunol.
164
:
1814
1819
.
41
Dhiman
R.
,
Indramohan
M.
,
Barnes
P. F.
,
Nayak
R. C.
,
Paidipally
P.
,
Rao
L. V.
,
Vankayalapati
R.
.
2009
.
IL-22 produced by human NK cells inhibits growth of Mycobacterium tuberculosis by enhancing phagolysosomal fusion.
J. Immunol.
183
:
6639
6645
.
42
Canaday
D. H.
,
Wilkinson
R. J.
,
Li
Q.
,
Harding
C. V.
,
Silver
R. F.
,
Boom
W. H.
.
2001
.
CD4(+) and CD8(+) T cells kill intracellular Mycobacterium tuberculosis by a perforin and Fas/Fas ligand-independent mechanism.
J. Immunol.
167
:
2734
2742
.

The authors have no financial conflicts of interest.