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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: J Immunol. 2014 Mar 3;192(7):2965–2969. doi: 10.4049/jimmunol.1400019

Control of Mycobacterium tuberculosis infection by a subset of lung parenchyma homing CD4 T cells

Shunsuke Sakai *, Keith D Kauffman *, Jason M Schenkel †, Cortez C McBerry *, Katrin D Mayer-Barber ‡, David Masopust †, Daniel L Barber *
PMCID: PMC4010124  NIHMSID: NIHMS566529  PMID: 24591367

Abstract

Summary

Th1 cells are critical for containment of Mycobacterium tuberculosis (Mtb) infection, but little else is known about the properties of protective CD4 T cell responses. Here we show that pulmonary Th1 response against Mtb is composed of two populations that are either CXCR3hi and localize to lung parenchyma or are CX3CR1hiKLRG1hi and retained within lung blood vasculature. Mtb-specific parenchymal CD4 T cells rapidly migrate back into the lung parenchyma upon adoptive transfer, while the intravascular effectors produce the highest levels of IFNγ in vivo. Importantly, parenchymal T cells displayed greater control of infection compared to the intravascular counterparts upon transfer into susceptible T cell deficient hosts. Thus, we have identified a subset of naturally generated Mtb-specific CD4 T cells with enhanced protective capacity, and show that control of Mtb correlates with the ability of CD4 T cells to efficiently enter the lung parenchyma rather than produce high levels of IFNγ.

Introduction

Mycobacterium tuberculosis (Mtb) is a major contributor to global human morbidity and mortality with 8.6 million new cases of disease and 1.3 million deaths annually (1). The only available vaccine, Bacillus Calmette-Guérin (BCG), protects against disseminated tuberculosis (TB) in children, but it confers little or no protection against pulmonary disease in adults. Indeed, the development of novel vaccines for Mtb has proven very difficult (2).

CD4 T cell deficient HIV-infected individuals, mice and non-human primates depleted of CD4 T cells (3-5), humans with inborn errors in genes involving IFNγ signaling, and mice deficient in IFNγ signaling or T-bet are all extremely susceptible to Mtb infection (6-8), indicating that Th1 polarized effector responses play a central role in host resistance to TB. There is also evidence, however, that CD4 T cells can mediate control of Mtb in an IFNγ□ independent manner(9, 10) and IFNγ responses do not predict host resistance to Mtb infection (11). In fact, there are no validated correlates of protection against TB, and there is a great need for a better understanding of the properties of a protective host response against Mtb infection.

Recently, it has become clear that following the resolution of acute viral infection peripheral non-lymphoid tissues harbor a subset of non-recirculating, tissue-resident CD4 and CD8 T cells that are distinct from memory T cells in secondary lymphoid tissue (12-14). However, it is not clear if recirculating and tissue-localizing subsets of effector CD4 T cells exist in the context of chronic Mtb infection. Here, we show that two types of Th1 cells with different phenotypic, migratory and host protective capacities populate the lung parenchyma and vasculature. These results identify a subpopulation of the Mtb-specific CD4 T cell response with enhanced lung homing ability as a key cell type in control of pulmonary Mtb infection.

Materials and Methods

Mice and Mtb infections

C57BL/6, B6.SJL (CD45.1) and TCRα−/− mice were obtained from Taconic Farms (Germantown, NY). All animals were housed at the AAALAC-approved facility at the NIAID, NIH according to the National Research Council Guide for the Care and Use of Laboratory Animals. Mice were used according to an animal study proposal approved by the NIAID Animal Care and Use Committee. Mice were aerosol exposed to □100 CFU of the H37Rv strain of Mtb (Glas-Col, LLC., Terre Haute, IN).

Intravascular staining and flow cytometry

Mice were injected intravenously with 2.5 μg of fluorochrome-labeled anti-CD45.2 or anti-CD45 Ab, and after 3 min, peripheral blood (PBL), bronchoalveolar lavage fluid (BAL) and lungs were harvested. For direct ex vivo intracellular cytokine staining (ICS), lungs were processed in the presence of brefeldin A (eBioscience, San Diego, CA). For T cell stimulations, cells were incubated with 5 μg/ml ESAT-61–20 peptide. I-AbESAT-64–17 and I-AbEsxG46–61 MHC tetramers were produced by the NIAID Tetramer Core Facility (Emory University, Atlanta, GA).

Cell sorting and adoptive transfer

Mtb-infected CD45.1 congenic mice were intravenously injected with anti-CD45-PE on day 30 post-infection (pi) and then live CD45+ or CD45− CD4 T cells were sorted to > 98% purity with a FACSAria II (BD Biosciences). For migration experiments, ~5 × 105 cells of each population were transferred into infection matched CD45.2 congenic recipient mice. For the protection experiments, TCRα−/− mice that had been infected with Mtb 7 days before adoptive transfer were used as recipients.

Results and Discussion

Compartmentalization of pulmonary Ag-specific CD4 T cells during Mtb infection

To determine the distribution of CD4 T cells between the airways, tissue parenchyma and blood vasculature within the lungs during Mtb infection, we employed a well-established intravascular (iv) staining technique (15, 16) (Fig. 1A). As expected, CD4 T cells in the PBL all stained with the injected Ab, and cells in the BAL were uniformly negative (Fig. 1B). In contrast, CD4 T cells in the lung were clearly divided into iv stain negative and positive populations, corresponding to cells in the lung parenchyma and vasculature, respectively. The anatomical localization of CD4 T cells was confirmed by immunofluorescence microscopy, which showed that cells staining with the injected anti-CD45 Ab were exclusively located within the CD31+ blood vessels (Fig. 1C).

FIGURE 1. Mtb-specific CD4 T cells are highly enriched in both the lung parenchyma and lung vasculature.

FIGURE 1

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(A) Iv staining of Mtb-infected mice. (B) Representative FACS plots of CD4 T cells on day 30 pi. Data are representative of at least three independent experiments. (C) Immunofluorescence microscopy of day 30 lung sections from anti-CD45 (red) injected mice. CD4 is green and CD31 is blue. Scale bar represents 100μm. (D) Frequency and total number of iv− CD4 T cells after Mtb infection. Data are pooled from at least two independent experiments (n≥3 per experiment). (E) Quantification of I-AbESAT-64–17 and I-AbEsxG46–61-specific cells by MHC class II staining on day 30 pi. Each connecting line shows an individual animal. Data are pooled from two independent experiments (n=5 per experiment).

We next examined the kinetics of CD4 T cell recruitment into the lungs after Mtb infection. Very low numbers of CD4 T cells were recovered from the lungs of naïve mice, and <5% were iv− (Fig. 1D). At the peak of CD4 T cell numbers at day 30, ~45% of lung CD4 T cells were in the parenchyma. The percentage of CD4 T cells that were iv− continued to increase until reaching a plateau of ~65% at day 60, which was stable for at least 6 months pi. Interestingly, the frequency of I-AbESAT-64-17 and I-AbEsxG46-61-specific CD4 T cells was highest in the vasculature (Fig. 1E). Moreover, the frequency of Ag-specific CD4 T cells in the lung vasculature was >5 fold higher than the circulating blood, indicating that iv+ T cells are not simply “blood contamination.” We also found that Mtb-specific CD8 T cells were most enriched in the lung vasculature (Suppl. Fig. 1). Together, these data indicate that significant populations of Mtb-specific CD4 T cells are located in both the lung parenchyma and vasculature throughout the course of Mtb infection.

Function and phenotype of Mtb-specific CD4 T cells in the lung parenchyma and vasculature

We next compared the function of lung iv− and iv+ Mtb-specific CD4 T cells. Similar to the increased frequencies of tetramer+ cells (Fig. 1E), a higher percentage of the iv+ cells produced IFNγ compared to the iv− cells after in vitro stimulation with ESAT-61-20 peptide (Fig. 2A). To more directly compare cytokine production by the iv− and iv+ Ag-specific cells in vivo, lung lymphocytes were isolated in the presence of brefeldin A, and IFNγ production by CD4 T cells were assessed by direct ex vivo ICS. Surprisingly, we observed ~2 fold more IFNγ+ I-AbESAT-64–17-specific CD4 T cells in the lung vasculature compared to the parenchyma by direct ICS (Fig. 2B), and iv+ Mtb-specific CD4 T cells also expressed higher levels of T-bet (Fig. 2C). When accounting for the increased overall number of CD4 T cells in the vasculature and their higher cytokine producing activity, ~75% of the total direct ex vivo IFNγ+ CD4 T cells in the lung were in the vasculature at 4 weeks pi (data not shown). These data indicate that at the peak of T cell clonal expansion the majority of the active Th1 response against Mtb infection (as measured by IFNγ production) occurs in the blood vasculature, not in the parenchyma.

FIGURE 2. Lung parenchymal and intravascular Mtb-specific CD4 T cells are functionally and phenotypically distinct subsets.

FIGURE 2

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(A) IFNγ staining after in vitro stimulation with ESAT-61–20 peptide on day 30 pi. Plots are gated on lung CD4 T cells, and data are pooled from three independent experiments (n=4 per experiment). (B) Direct ex vivo ICS for IFNγ–producing I-AbESAT-64–17-specific CD4 T cells in the lung at 30 days pi. Each connecting line shows an individual animal (n=5). Data are representative of three independent experiments. (C) T-bet and (D) PD-1, CD69 and KLRG1 expression in total naïve (CD44loFoxp3−) and iv+ or iv− I-AbESAT-64–17 tetramer+ lung CD4 T cells on day 30 pi. Numbers represent the MFI or percent+ for the iv− (top) and iv+ (bottom). (E) CXCR3 or CX3CR1 staining gated on total lung CD4 T cells on day 30 pi (left panels), and expression of the chemokine receptors and KLRG1 gated on iv− and iv+ I-AbESAT-64–17 tetramer+ cells (middle and right panels). Cells in panels C–E were pooled from n≥4 mice per experiment for FACS analysis. Data are representative of three independent experiments.

We next compared the phenotype of I-AbESAT-64-17-specific CD4 T cells in both lung compartments. We found that iv− Mtb-specific T cells exhibited much higher levels of activation markers including CD69 and PD-1 (Fig. 2D and Suppl Figure 2), while iv+ cells expressed a high level of KLRG1, which has been associated with terminal effector T cells (17). Among chemokine receptors analyzed (Suppl. Fig. 2), strikingly, the majority of iv− CD4 T cells showed a high level of CXCR3 expression, whereas most of the iv+ cells expressed CX3CR1 (Fig. 2E). I-AbESAT-64-17-specific CD4 T cells in the iv− and iv+ compartments displayed the same preferential expression of CXCR3 and CX3CR1, respectively (Fig. 2E). Collectively, these data demonstrate a phenotypic dichotomy in Mtb-specific CD4 T cells whereby CXCR3 marks highly activated, parenchyma-localized cells, and CX3CR1 and KLRG1 identify cells capable of the highest IFNγ production that are enriched within the lung blood vessels.

Migratory potential of the lung parenchymal and intravascular CD4 T cells

We next compared the ability of the parenchymal and intravascular subsets to migrate into Mtb-infected lungs by transferring FACS purified iv− or iv+ CD4 T cells into infection matched congenic recipient mice. We found that ~60% of the I-AbESAT-64-17 -specific CD4 T cells derived from the iv− donors efficiently migrated back into the lung parenchyma, while only ~5% of the iv+ donors migrated into the lung tissue in the same time period (Fig. 3A). A similar difference was observed between iv− and iv+ total effector (CD44hiFoxp3−) donor CD4 T cells (Fig. 3B-C). It has been shown that during naïve T cells can migrate into Mtb-infected lungs (18), thus we also analyzed the ability of naïve (CD44loFoxp3−) CD4 T cells to migrate into the lungs of Mtb-infected recipients. Naïve T cells derived from either the parenchyma or vasculature equally migrated into the lung parenchyma after adoptive transfer (Fig. 3B-C). Interestingly, naïve T cells migrated more efficiently into the lung than the iv+ effector T cells, and were even comparable to the parenchymal donor effector T cells. Together, these data indicate that the intravascular effector CD4 T cells are unexpectedly poor at migrating into the lung parenchyma.

FIGURE 3. KLRG1− parenchymal effector CD4 T cells migrate most efficiently into the lung while intravascular KLRG1+ cells are the least efficient.

FIGURE 3

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FACS purified iv− and iv+ CD4 T cells from lungs of day 30 infected mice (CD45.1) were transferred into infection-matched congenic recipient mice (CD45.2). After 1.5 days, migration of the donor cells into the lung tissue was monitored with a second iv stain in the recipient mice. (A) Migration of I-AbESAT-64–17 tetramer+ donor CD4 T cells into the parenchyma. Plots are electronically concatenated from each group of mice (n=5). Data are representative of two independent experiments. (B) Gating strategy to identify naïve, KLRG1hi and KLRG1lo effector donor CD4 T cells. Plots are concatenated from each group of mice (n=5). (C) Summary of donor cells migrating into the lung parenchyma of recipients. Data are pooled from two independent experiments (n=5 per experiment).

While the vast majority of the iv+ effector CD4 T cells were KLRG1+, we routinely observed that a small subpopulation of KLRG1− iv+ effector cells expressed CXCR3 at levels similar to the iv− effectors (Fig. 2D-E). Thus, we next asked if the intravascular KLRG1+ and KLRG1− effector CD4 T cells differed in their ability to migrate into the parenchyma by gating on the subsets after recovery from the recipients. We found that the KLRG1− iv+ donor cells migrated efficiently into the lung parenchyma upon adoptive transfer (Fig. 3B-C). In contrast, the KLRG1+ iv+ donor cells were extremely poor at migrating into the tissue. Collectively, these data show that the anatomical localization of pulmonary effector T cells largely reflects the cell’s migratory ability.

Protective capacity of the lung parenchymal and intravascular CD4 T cells

We compared the ability of lung iv− and iv+ CD4 T cells to control bacterial replication upon adoptive transfer into infected recipients. As it is often found that it is difficult to observe protection of Mtb-infected mice by transfer of CD4 T cells into normal recipients, we chose to use T cell deficient mice as hosts. Although the use of lymphopenic mice is a caveat of this experiment, it does allow for us to observe large effects of transferred CD4 T cells on bacterial control. FACS purified iv− or iv+ CD4 T cells isolated from the lungs at 30 days pi were injected into TCRα−/− mice that had been infected with Mtb 7 days previously, and the recipients were euthanized on day 28 pi. A total of 5×105 CD4 T cells were transferred, which equated to 3.2×105 iv− and 3.3×105 iv+ CD44hiFoxp3− effector cells and 2.2×104 iv− and 2.1×104 iv+ I-AbESAT-64-17 specific T cells. We found that donor CD4 T cells initially isolated from either the lung parenchyma or vasculature of the donor mice gave rise to both iv− and iv+ cells in the recipient lungs (Fig. 4A). The total number of donor CD44hiFoxp3− effector and I-AbESAT-64-17 specific T cells CD4 T cells in the parenchyma was ~2 fold higher in the lungs of mice reconstituted with the iv− cells compared to iv+ donors Figure 4A. While the iv− donor cells repopulated the parenchyma more efficiently, the iv+ donors produced higher levels of IFNγ in the direct ICS assay (Fig. 4B). This increased cytokine secretion by the iv+ donor cells was observed when comparing either the expanded iv− or iv+ cells.

FIGURE 4. Parenchyma homing CD4 T cells display greater control of Mtb infection compared to the intravascular cells.

FIGURE 4

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Iv− and iv+ CD4 T cells were FACS purified from day 30 infected mice and adoptively transferred into TCRα−/− mice that had been infected with Mtb 7 days before, and lungs were harvested on day 28 pi. (A) Absolute number of CD44hiFoxp3− effector and I-AbESAT-64–17 tetramer+ donor CD4 T cells in the recipient lungs. (B) Direct ex vivo IFNγ staining of donor CD4 T cells. (C) Bacterial loads in the lungs of recipient mice. All data are representative of two independent experiments (n=5 per experiment).

This experimental design allowed us to compare bacterial loads in the setting of higher parenchymal effector cell numbers versus higher in vivo IFNγ production. Both groups of recipient mice showed reduced bacterial loads in the lungs compared to the unreconstituted mice (Fig. 4C). However, TCRα−/− mice injected with iv+ donor cells showed a ~4 fold reduction in bacterial loads while the recipients of iv− donors showed an ~18 fold decrease in CFU compared to the unreconstituted controls (Fig. 4C). Therefore, CD4 T cell-mediated protection against Mtb infection was associated with a higher number of CD4 T cells in the lung parenchyma rather than a higher frequency of IFNγ+ cells in the vasculature.

Here we demonstrate that the pulmonary CD4 T cell response against Mtb is comprised of two major subsets that either enter the lung parenchyma or reside within the vasculature. The parenchymal effectors express CXCR3 and are PD-1hi/CD69hi, which likely reflects their access to Ag within the tissue. In contrast, most of the intravascular CD4 T cells express CX3CR1 and have a more terminally differentiated phenotype (KLRG1hi/T-bethi). A recent study noted that in Mtb-infected mice PD-1hi CD4 T cells are highly proliferative while KLRG1hi cells produce more IFNγ (19). Our results indicate that these differences previously reported between PD-1hi and KLRG1hi CD4 T cells reflects the phenotype of T cells within the parenchyma and blood vessels respectively, highlighting the importance of this iv staining technique in the study of cellular immune responses in Mtb-infected lungs. Indeed, many previous studies of cellular immunity to Mtb may warrant reevaluation with this technique.

We also found that parenchymal CD4 T cells rapidly migrate back into the parenchyma, while only a small population of intravascular cells does so in the same amount of time. Although they are the most abundant subset in the entire lung at the peak of clonal expansion, KLRG1hi intravascular T cells are poor at entering the lung parenchyma (even compared to naïve T cells). In contrast, a small subset of intravascular T cells negative for KLRG1 efficiently migrates into the lung. This indicates that parenchymal precursor cells within blood vessels may be a small population of effector T cells with enhanced lung migratory activity that are distinct from the majority of intravascular cells that have very poor ability to enter the lung. However, further investigation is needed to fully characterize the relationship between the two subsets.

While the lung homing subset of CD4 T cells mediates the most efficient control of Mtb infection, the majority of active IFNγ secretion comes from CD4 T cells in the lung vasculature. It is unclear to what extent Ag recognition and innate cytokine signals contribute to the IFNγ production by intravascular CD4 T cells, but it raises the interesting possibility that intravascular T cells could have some unrecognized contribution to bacterial control or regulation of other cell types during Mtb infection. It is also not clear why the parenchymal CD4 T cells make less IFNγ compared to the T cells in the blood vessels. The parenchymal effector cells could be actively inhibited by signals in the tissue such as PD-1, or the parenchymal and intravascular T cells may represent distinct lineages of Th1 cells with different effector programs. These two scenarios are not mutually exclusive. Regardless, despite their reduced IFNγ production in comparison to the intravascular cells, the parenchymal effector CD4 T cells did produce some IFNγ, and perhaps this amount was sufficient to mediate the IFNγ dependent effects on bacterial control. A recent study has shown that the major role of CD4 T cells in control of Mtb infection is dependent on direct contact between the CD4 T cells and infected antigen presenting cells (20). Therefore, it is likely that the enhanced protective capacity of the parenchymal homing subset is principally due to their ability to gain access to the infected cells in the granuloma, and conversely the relative ineffectiveness of the CX3CR1+ subset despite their enhanced IFNγ production is simply due to their inability to enter the lung tissue. Interestingly, it is also possible that the parenchyma homing subset mediates the still unidentified IFNγ independent effector functions that are able to induce control of Mtb infection (9, 10).

Lastly, the findings reported here might have implications for understanding the correlates of protection against Mtb infection. Our observations that the CD4 T cells producing the highest level of IFNγ are not the same as the cells that efficiently enter the lung and control infection may help explain why IFNγ responses notoriously don’t correlate with resistance to Mtb. These data illustrate how a better understanding of the heterogeneity of Mtb-specific CD4 T cells could provide rationale for the development of novel therapies and vaccine regimens for TB by identifying the properties of protective CD4 T cell responses.

Supplementary Material

1

Acknowledgement

We are grateful to Bishop Hague and Kevin Holmes of the NIAID Flow Cytometry Core Facility for FACS sorting.

Funding

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Footnotes

Disclosures

The authors have no financial interests to declare.

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Supplementary Materials

1
NIHMS566529-supplement-1.pdf (1.6MB, pdf)

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