The Rate of CD4 T Cell Entry into the Lungs during Mycobacterium tuberculosis Infection Is Determined by Partial and Opposing Effects of Multiple Chemokine Receptors

The specific chemokine receptors utilized by Th1 cells to migrate into the lung during Mycobacterium tuberculosis infection are unknown. We previously showed in mice that CXCR3⁺ Th1 cells enter the lung parenchyma and suppress M. tuberculosis growth, while CX3CR1⁺ KLRG1⁺ Th1 cells accumulate in the lung vasculature and are nonprotective.

ABSTRACT

The specific chemokine receptors utilized by Th1 cells to migrate into the lung during Mycobacterium tuberculosis infection are unknown. We previously showed in mice that CXCR3⁺ Th1 cells enter the lung parenchyma and suppress M. tuberculosis growth, while CX3CR1⁺ KLRG1⁺ Th1 cells accumulate in the lung vasculature and are nonprotective. Here we quantify the contributions of these chemokine receptors to the migration and entry rate of Th1 cells into M. tuberculosis-infected lungs using competitive adoptive transfer migration assays and mathematical modeling. We found that in 8.6 h half of M. tuberculosis-specific CD4 T cells migrate from the blood to the lung parenchyma. CXCR3 deficiency decreases the average rate of Th1 cell entry into the lung parenchyma by half, while CX3CR1 deficiency doubles it. KLRG1 blockade has no effect on Th1 cell lung migration. CCR2, CXCR5, and, to a lesser degree, CCR5 and CXCR6 also promote the entry of Th1 cells into the lungs of infected mice. Moreover, blockade of G-protein-coupled receptors with pertussis toxin treatment prior to transfer only partially inhibits T cell migration into the lungs. Thus, the fraction of Th1 cell input into the lungs during M. tuberculosis infection that is regulated by chemokine receptors likely reflects the cumulative effects of multiple chemokine receptors that mostly promote but that can also inhibit entry into the parenchyma.

INTRODUCTION

Tuberculosis (TB) is the leading cause of death due to a single infectious agent and is among the top 10 causes of global human mortality (1). A highly effective vaccine for tuberculosis is desperately needed, but progress in TB vaccine development has been slowed by the lack of information on the immunological mechanisms of protection against Mycobacterium tuberculosis infection. In order for CD4 T cells to protect against M. tuberculosis infection, they must make direct contact with major histocompatibility complex class II molecules on bacillus-laden macrophages in the lung (2, 3). Therefore, migration out of the blood across the vascular endothelium into the lung parenchyma, where the infected macrophages are located, is a critical function of helper T cells. The chemokine receptors (CCRs) that govern CD4 T cell entry into the lung parenchyma during M. tuberculosis infection have not been defined. Identification of the receptors that guide circulating M. tuberculosis-specific CD4 T cells from the blood into the lungs could serve as a useful correlate of protective CD4 T cell responses.

We and others have previously shown in mice that only a subset of M. tuberculosis-specific CD4 T cells is able to enter the lungs. The differentiation state of M. tuberculosis-specific effector Th1 cells determines their chemokine receptor expression pattern and ability to migrate into the lungs to protect against M. tuberculosis infection. Less-differentiated Th1 cells express CXCR3, are able to migrate into the lungs, and suppress the growth of M. tuberculosis (4, 5). In contrast, terminally differentiated Th1 cells that express high levels of CX3CR1 and KLRG1 poorly migrate out of the blood vessels and do not control M. tuberculosis infection. Despite the strong association between lung-homing capacity and the expression of CXCR3 versus CX3CR1, it has previously been shown that CXCR3 (6, 7) and CX3CR1 (8) are not required for either CD4 T cell entry into the lungs or host survival of M. tuberculosis infection. In fact, no chemokine receptor-deficient animal examined to date has shown a major loss of pulmonary CD4 T cell responses following M. tuberculosis infection, indicating that CD4 T cell entry into the lungs during tuberculosis is mediated primarily by yet untested homing receptors or by several receptors, each of which is essential for T cell entry on its own and has a minor contribution.

The intravascular staining technique allows for the discrimination of T cells that are localized in the blood vasculature from those that have migrated into the lung parenchyma, allowing one to carefully track the entry of CD4 T cells into the lungs (9). Here, we used in vivo mixed T cell competitive migration assays and the intravascular staining technique to estimate the rate of entry of CXCR3- or CX3CR1-deficient M. tuberculosis-specific effector CD4 T cells into the lung and to quantify the contribution of several other chemokine receptors. We found that CXCR3 deficiency decreases the rate of CD4 T cell entry into the lungs by about 2-fold, while CX3CR1 deficiency enhances the rate of T cell lung entry by about 2-fold. Using in vivo migration experiments, we also detected minor defects in the migration of less-differentiated CD4 T cells into the parenchyma of M. tuberculosis-infected lungs in the absence of CXCR6, CXCR5, CCR2, or CCR5. Our results indicate that the entry of CD4 T cells into the lungs of M. tuberculosis-infected mice is likely determined by the coexpression of multiple chemokine receptors, each with a relatively small contribution, and is opposed by CX3CR1.

RESULTS

CXCR3 has a minor role in the migration of KLRG1⁻, less-differentiated M. tuberculosis-specific effector CD4 T cells into the lungs.

We and others have shown that different subsets of CD4 T cells in the lungs of M. tuberculosis-infected mice vary dramatically in their ability to enter the tissue parenchyma and to reduce bacterial loads (4, 10, 11). The protective CD4 T cells are less differentiated, express CXCR3, and can migrate into the lung parenchyma. In contrast, CD4 T cells that do not protect against M. tuberculosis infection are terminally differentiated effector CD4 T cells that express high levels of KLRG1 and CX3CR1 and preferentially reside in the vasculature (Fig. 1A). Therefore, in order to examine the pathways that mediate CD4 T cell entry into the lungs, it may be useful to distinguish between these two major subsets and focus on the cells that are able to enter the lungs. Given the strong association between these two chemokine receptors and lung-homing ability, we first sought to characterize the role of CXCR3 and CX3CR1 in the migration of T cells into the lung during M. tuberculosis infection. On day 28 postinfection, we measured the percentage of KLRG1⁻ I-A^b ESAT-6_4–17 tetramer-positive (tetramer⁺) CD4 T cells that were intravascular stain negative (iv⁻) in wild-type (WT), CXCR3^−/−, and CX3CR1-green fluorescent protein (GFP)-knock-in (KI) reporter mice. We found that ∼95% of KLRG1⁻ antigen (Ag)-specific CD4 T cells in WT and CX3CR1-deficient mice were iv⁻, while ∼80% of these cells in the CXCR3^−/− mice were iv⁻ (Fig. 1B). Therefore, CXCR3 does have a relatively minor role in the localization of KLRG1⁻ M. tuberculosis-specific effector T cells into the lung parenchyma. However, as previously shown by others, we also found no difference in bacterial loads in WT, CXCR3-deficient (6, 7), and CX3CR1-deficient animals (8) on day 28 postinfection (Fig. 1C), indicating that this small defect in T cell migration is not sufficient to impair control of the infection.

FIG 1.

The migration of M. tuberculosis-specific CD4 T cells into the lung parenchyma is partially dependent upon CXCR3. Lungs were harvested from WT and chemokine receptor-KO mice on day 28 after M. tuberculosis infection. (A) Representative fluorescence-activated cell sorting plots of intravascular CD45 (CD45 iv), CXCR3, CX3CR1, and KLRG1 on WT M. tuberculosis-specific lung CD4 T cells. (B) Summary graph of the frequency of I-A^b ESAT-6_4–17⁺ KLRG1⁻ iv⁻ lung M. tuberculosis-specific effector CD4 T cells in WT, CXCR3^−/−, and CX3CR1^−/− mice. Data were pooled from five independent experiments. ****, P ≤ 0.0001. (C) Numbers of CFU in the lungs of WT, CXCR3^−/−, and CX3CR1^−/− mice. Data were pooled from five independent experiments.

Opposing effects of CXCR3 and CX3CR1 on the rate of CD4 T cell entry into the lung parenchyma of M. tuberculosis-infected mice.

To more carefully evaluate the role of CXCR3 and CX3CR1 in T cell trafficking into the lung tissue in M. tuberculosis infection, we next quantified the impact of these chemokine receptors on the rate of CD4 T cell entry into the lungs. To do so, we measured the kinetics of effector T cell input into the lungs in a three-way competitive in vivo migration experiment. We isolated CD4 T cells from the lungs of M. tuberculosis-infected WT (Thy1.2⁺ CD45.1⁺ CD45.2⁺), CXCR3-knockout (KO) (Thy1.2⁺ CD45.1⁻ CD45.2⁺), and CX3CR1 GFP-KI (Thy1.2⁺ CD45.1⁺ CD45.2⁻) reporter mice and adoptively transferred a 1:1:1 mixture of all three CD4 T cell populations into congenically disparate (Thy1.1⁺ CD45.1⁻ CD45.2⁺) infection-matched WT recipients. This allowed us to separately track the migration of all three genotypes of T cells into the lungs of the same mouse. Effector CD4 T cell entry into the lungs of the recipient mice was measured as the percentage of donor CD4 T cells that became intravascular stain negative over the course of 36 h posttransfer (Fig. 2A). Given their major difference in homing potential, we tracked the migration of both less-differentiated effector cells (KLRG1⁻ CX3CR1⁻) and terminal effector cells (KLRG1⁺ CX3CR1⁺) separately for each of the three chemokine receptor genotypes. In the case of the CX3CR1-deficient cells, we were able to track the CX3CR1⁺ terminal effector cells, despite their lack of CX3CR1 protein expression, by their GFP fluorescence reporting CX3CR1 promoter activity (Fig. 2B). As expected, KLRG1⁻ CX3CR1⁻ T cells migrated much faster than the KLRG1⁺ CX3CR1⁺ terminal effector cells, regardless of chemokine receptor expression (Fig. 2C and D).

FIG 2.

CXCR3 and CX3CR1 oppose each other in the migration of CD4 T cells into the lung parenchyma during M. tuberculosis (Mtb) infection. (A) Schematic of experimental setup (iv, intravenous). (B) Representative fluorescence-activated cell sorting plots of the gating strategy used to identify each donor population, CD45.2 CXCR3^−/−, CD45.1/CD45.2 WT, and CD45.1 CX3CR1-GFP-KI T cells, in the lungs of Thy1.1 recipient mice. Donor cells were further gated as KLRG1⁻ CX3CR1⁻ and KLRG1⁺ CX3CR1⁺ populations, and histograms represent CD45 intravascular staining for each population. (C) A kinetic graph summarizing the frequencies of KLRG1⁻ CX3CR1⁻ and KLRG1⁺CX3CR1⁺ CD4 donor T cells migrating into the lungs of infection-matched recipients at 4, 10, 16, 24, and 36 h posttransfer and fits of the mathematical model to these data (see Materials and Methods for more detail). Data were pooled from two independent experiments. The fit is excellent in both cases, as judged by the lack-of-fit test (P = 0.38 and P = 0.90 for early and late effectors, respectively). (D) Estimated average rate of entry (m̄) of KLRG1⁻ CX3CR1⁻ and KLRG1⁺ CX3CR1⁺ CD4 donor T cells migrating into the lungs of infection-matched recipients. Data were pooled from two independent experiments. For early effectors, the estimated migration rates and 95% confidence intervals were 0.087(0.065 − 0.11)/h for m₁, 0.163(0.141 − 0.191)/h for m₂, and 0.064(0.038 − 0.094)/h for m₃. For late effectors, the estimated migration rates and 95% confidence intervals were 0.155(0.086 − 0.323)/h for m₁, 0.144(0.091 − 0.224)/h for m₂, and 0.141(0.068 − 0.314)/h for m₃. The subscript numbers (i) in the parameter estimates denote WT cells (i = 1), CX3CR1-deficient cells (i = 2), and CXCR3-deficient cells (i = 3). (E) Estimated frequency of KLRG1⁻ CX3CR1⁻ and KLRG1⁺ CX3CR1⁺ CD4 donor T cells migrating into the lungs of infection-matched recipients. Data were pooled from two independent experiments.

To quantify the kinetics of migration of different T cell subsets from the blood to the lung parenchyma, we developed several mathematical models (see Materials and Methods for more details); one of these models assumes that there is a variability in the ability of different cells to migrate to the lung parenchyma. In the model, the rate at which T cells initially appear in the lung parenchyma is given by the average migration rate (m̄), and the overall proportion of cells that are found in the parenchyma (asymptote level) defines the fraction of T cells that are capable of migrating to the lung (p). By fitting the model to the time course data on the percentage of M. tuberculosis-specific CD4 T cells in the lung parenchyma (Fig. 2C), we could thus estimate the average migration rate for T cells lacking specific chemokine receptors and compare that to the rate for WT T cells (Fig. 2D) as well as determine the fraction of T cells capable of migrating into the lung (Fig. 2E). For both less-differentiated and terminal effector cells, our analysis suggested that CXCR3^−/− CD4 T cells enter the lung parenchyma at about half the rate of the WT cells, whereas the CX3CR1^−/− CD4 T cells do so at almost double the rate of the WT cells (Fig. 2C and D). WT cells enter the lung at an average rate (m̄) of 0.081/h, which translates into a half-life (t_1/2) of 8.6 h (in this time, half of WT T cells found in the lung vasculature migrate into the lung parenchyma). Interestingly, CX3CR1-deficient CD4 T cells that were CX3CR1-GFP reporter negative migrated into the lungs faster. This may indicate that CX3CR1 is expressed at low levels even on some less-differentiated T cells and impairs their migration. Alternatively, this may also suggest that other genes which are coregulated by CX3CR1 may impact cell migration. Regardless, the role of CXCR3 in promoting T cell entry and CX3CR1 in inhibiting T cell entry is consistent with their expression on parenchymal and intravascular cells, respectively.

We next calculated the percentage of cells capable of entering the lung tissue. As expected, a much larger fraction of the overall population of KLRG1⁻ CX3CR1⁻ effector cells than KLRG1⁺ CX3CR1⁺ terminal effector cells migrated into the lungs (Fig. 2E). Among the less-differentiated KLRG1⁻ CX3CR1⁻ T cells, we found a minor decrease in the fraction of CXCR3^−/− T cells that entered the lungs and no difference in the fractions of CX3CR1^−/− cells and the WT donor cells that entered the lungs (Fig. 2E). Additional analyses showed that there is a reduced efficiency of CXCR3^−/− T cell entry into the lung parenchyma because fitting of the model to data on the migration of CXCR3^−/− T cells and the assumption that all cells are capable of migrating (p = 1) resulted in fits of lower quality (F_1,74 = 9.1, P = 0.003). As for the CX3CR1-expressing terminal effector CD4 T cells, we estimated that CX3CR1 deficiency approximately doubles the fraction of T cells that can enter the lungs, but the average migration rate of these cells was still very low compared to that of less-differentiated effector cells, mostly because few cells were capable of migrating into the lung. Taken together, these data show that although it is not required for entry, CXCR3 does in fact promote CD4 T cell entry into the lungs and CX3CR1 can oppose CD4 T cell entry into the lungs.

KLRG1 blockade does not enhance the entry of CD4 T cells into the lung parenchyma during M. tuberculosis infection.

Our data indicate that the poor migratory ability of terminal effector cells may in part be explained by the high-level expression of CX3CR1. However, CX3CR1-deficient terminal effector cells still displayed relatively poor migration compared to less-differentiated KLRG1⁻ cells, so we next considered the hypothesis that KLRG1 itself may play a role in limiting the lung homing of terminal effector cells during M. tuberculosis infection. KLRG1 interacts with several cadherin molecules, and it has been suggested that KLRG1 may inhibit the transendothelial diapedesis of lymphocytes (12, 13). In fact, KLRG1-deficient mice have been shown to control M. tuberculosis infection better and survive longer than WT mice (14). We asked if KLRG1 alone or in combination with CX3CR1 contributes to their poor migratory capacity. To measure the impact of KLRG1 on effector T cell migration into the lungs during M. tuberculosis infection, we utilized an adoptive transfer approach similar to that described above, where congenically marked WT, CXCR3-deficient, and CX3CR1-deficient CX3CR1-GFP reporter lung effector CD4 T cells were transferred into infection-matched recipient mice. However, here we tracked the migration of the cells with and without preincubation with an anti-KLRG1 blocking monoclonal antibody (MAb) that prevented its binding to cadherins. The blocking antibody is a human IgG1 isotype and does not have depleting activity. As before, we tracked KLRG1⁺ CXCR3⁺ and KLRG1⁻ CX3CR1⁻ cells separately. The blocking anti-KLRG1 MAb also prevented flow cytometry staining for KLRG1, so in order to identify KLRG1-expressing donor cells in the recipient mice, we detected the surface-bound anti-KLRG1 blocking antibody with a secondary antibody against human IgG1 (Fig. 3A). As in the assay whose results are presented in Fig. 2, CX3CR1 expression on CX3CR1-KO cells was detected via the GFP reporter. As expected, CX3CR1⁺ KLRG1⁺ terminal effector cells migrated poorly compared to less-differentiated CXCR3⁻ KLRG1⁻ cells, and CXCR3-deficient cells displayed a slight reduction and CX3CR1-deficient cells displayed a slight increase in migration into the lungs at 16 h posttransfer (Fig. 3B to D). However, KLRG1 blockade had no impact on the migration of either subset of effector T cells, regardless of genotype, indicating that this inhibitory receptor has little role in regulating T cell entry into the lungs in M. tuberculosis infection.

FIG 3.

KLRG1 expression has no effect on the migration of effector CD4 T cells into the lung parenchyma during M. tuberculosis infection. Infection-matched Thy1.1 recipient mice received a mixture of Thy1.2 CD45.1/CD45.2 WT, CD45.2 CXCR3^−/−, and CD45.1 CX3CR1^−/− CD4 T cells with or without anti-KLRG1 human IgG1 (huIgG1) treatment, and the lungs were harvested at 16 h after cell transfer. (A) Representative fluorescence-activated cell sorting plots of the gating strategy used to identify each donor population, CD45.2 CXCR3^−/−, CD45.1/CD45.2 WT, and CD45.1 CX3CR1-GFP-KI T cells, in the lungs of Thy1.1 recipient mice. Donor cells were further gated as KLRG1⁻ CX3CR1⁻ and KLRG1⁺ CX3CR1⁺ populations. (B) Representative histograms of CD45 intravascular staining of the donor KLRG1⁺ CX3CR1⁺ CD4 T cells. (C and D) Summary graphs of the frequency of parenchymal (CD45⁻) WT, CXCR3^−/−, or CX3CR1^−/− KLRG1⁻ CX3CR1⁻ (C) and KLRG1⁺ CX3CR1⁺ (D) effector CD4⁺ donor T cells. Data were pooled from two independent experiments. ns, not significant (P > 0.1); untx, untreated.

Analysis of M. tuberculosis-specific CD4 T cell migration in intact chemokine receptor-deficient mice.

The relatively minor contribution of CXCR3 to CD4 T cell entry into the lungs led us to examine the role of other chemokine receptors in M. tuberculosis infection. CXCR6 is highly expressed by tissue-resident memory CD4 T cells and by M. tuberculosis-specific vaccine-elicited CD4 T cells that migrate into the lungs (15–17). We examined CXCR6 expression and its role in CD4 T cell migration using CXCR6-GFP-knock-in reporter mice. CXCR6 was expressed by all intravascular stain-negative I-A^b ESAT-6_4–17-specific CD4 T cells at high levels and was also expressed by a significant fraction of CX3CR1⁺ cells in the vasculature, albeit at a lower mean fluorescence intensity relative to that for iv⁻ cells (Fig. 4A). KLRG1⁻ I-A^b ESAT-6_4–17-specific CD4 T cells displayed a slight defect in localization into the parenchyma (93% for WT cells versus 89% for CXCR6-deficient cells), indicating that CXCR6 is largely dispensable for T cell migration into the lung parenchyma during M. tuberculosis infection. Mice deficient in CCR2 are susceptible to high-dose intravenous infection with M. tuberculosis H37Rv (18, 19) or low-level infection with the hypervirulent strain HN878 (20), but in both of these settings, this seems to be due to defective myeloid cell migration. CCR2-KO mice do not display the enhanced susceptibility to low-dose M. tuberculosis H37Rv infection (21). We found that CCR2 was expressed by a fraction of both the iv⁻ and intravascular stain-positive (iv⁺) CD4 T cells (Fig. 2A) but that KLRG1⁻ CD4 T cells in CCR2^−/− mice displayed no defect in localization compared to WT mice after low-dose aerosol H37Rv infection (Fig. 4B). Therefore, consistent with previous studies, we found that CCR2 is not required for CD4 T cell entry into the lungs. CCR5-deficient mice have been shown to display normal control of pulmonary M. tuberculosis infection (21, 22), and we found that KLRG1⁻ Ag-specific CD4 T cells displayed no defect in their localization to the lung parenchyma relative to the WT controls, although CCR5 was found to be widely expressed by M. tuberculosis-specific CD4 T cells (Fig. 4A and B). CXCR5 has been shown to be required for the control of M. tuberculosis infection in mice (23) and to be required for the long-term maintenance of Ag-specific CD4 T cells in M. tuberculosis-infected mice (10). However, we found that KLRG1⁻ CD4 T cells localized to the lung parenchyma similarly in WT and CXCR5^−/− mice (Fig. 4B). Lastly, we also found that CD4 T cells in CCR6-, CCR1-, and EBi2-deficient mice displayed no defect in migration into the lungs (Fig. 4B). Overall, these data show that while M. tuberculosis-specific CD4 T cells express a large number of different chemokine receptors that may be expected to be important in their recruitment to the lung parenchyma, only ablation of CXCR3 had an appreciable impact on the entry rate of effector CD4 T cells into the lung parenchyma in this analysis of infected gene-deficient mice.

FIG 4.

Pulmonary M. tuberculosis-specific effector CD4 T cells express multiple chemokine receptors. (A) Representative fluorescence-activated cell sorting plots of intravascular stain, CD69, CXCR3, and CX3CR1 versus CXCR6, CCR2, and CCR5 on I-A^b ESAT-6_4–17⁺ CD4 T cells. Lungs were harvested from WT and chemokine receptor-KO mice on day 28 after M. tuberculosis infection. (B) Summary graph of the frequency of I-A^b ESAT-6_4–17⁺ KLRG1⁻ (white dots) or CX3CR1⁻ (gray dots) iv⁻ lung M. tuberculosis-specific effector CD4 T cells in WT and various chemokine receptor-KO mice. Data were pooled from 2 independent experiments per chemokine receptor-KO mouse.

Competitive in vivo migration experiments reveal minor roles for multiple chemokine receptors in CD4 T cell migration into the lungs during M. tuberculosis infection.

Analysis of intact M. tuberculosis-infected mice revealed only slight defects in the localization of less-differentiated effector cells into the lung parenchyma in CXCR3-deficient and, to a lesser extent, CXCR6-deficient mice. However, the fraction of lung T cells that are localized to the parenchyma is a function of many factors (e.g., T cell input and exit rates and tissue dwell times) that may mask the impact of chemokine receptor deficiency on T cell entry into the lungs when bulk populations are examined. Therefore, we sought to more carefully measure the role of these chemokine receptors in the entry of effector T cells into the lung using a series of competitive mixed-adoptive-transfer in vivo migration experiments, where migration into the lung parenchyma was measured after intravenous injection of a bolus of donor T cells. Donor CD4 T cells were obtained from the lungs of M. tuberculosis-infected WT or chemokine receptor-deficient (KO) mice and mixed together prior to injection into infection-matched WT recipient mice. Both WT and KO donor cell populations were CD45.2⁺, and recipient mice were CD45.1⁺. WT donor cells were marked with Thy1.1, and the different KO CD4 T cells were marked with Thy1.2, allowing us to directly compare the migration of different KO T cells to a cotransferred population of control WT T cells (Fig. 5A).

FIG 5.

Migration of effector CD4 T cells into the lung parenchyma during M. tuberculosis infection is determined by the minor effects of multiple chemokine receptors. WT and chemokine receptor-deficient CD4 T cells were mixed and adoptively transferred into infection-matched congenic recipients, and the lungs were harvested at 16 h after cell transfer. (A) Representative fluorescence-activated cell sorting plots of the gating strategy used to identify donor effector WT or chemokine receptor-KO CD4 T cells, CX3CR1 expression, and CD45 intravascular staining. (B and C) Summary graphs of the ratio of KO to WT donor iv⁻ frequencies for KLRG1⁻ or CX3CR1⁻ (B) and KLRG1⁺ or CX3CR1⁺ (C) effector donor CD4 T cells. White dots indicate that KLRG1 expression is used to determine terminal effectors. Gray dots indicate that CX3CR1 expression is used to determine terminal effectors. Data were pooled from five independent experiments. Two-tailed t tests compared KO to WT cell frequencies. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. d29, day 29.

We distinguished the early effectors from the terminally differentiated cells with either KLRG1 or CX3CR1 staining, depending on the particular experiment, but in any case, KLRG1 or CX3CR1 expression marked the terminal effector cells, while the less-differentiated cells were negative for those markers (Fig. 5A to C). By normalizing the percentage of cells that were intravascular stain negative for the KO donor cells compared to that for the WT internal control donor cells, we could express the data as the percentage of cells that migrated compared to that for the WT cells. We found that CXCR3-deficient less-differentiated effector T cells displayed an ∼50% reduction in the average rate of migration into the lungs (Fig. 5B; see also Fig. S3 in the supplemental material). This is consistent with the data in Fig. 1 showing that even in intact CXCR3^−/− mice, a defect in the localization of T cells into the lung parenchyma could be detected. Using this adoptive transfer approach, we also found that CCR2-, CCR5-, CXCR5-, and CXCR6-deficient less-differentiated T cells displayed slight reductions in their ability to enter the lung (Fig. 5B). In contrast, terminal effector cells showed only minor migration defects in the absence of CXCR3, CCR5, and CXCR6 (Fig. 5C). Additionally, we found no role for CCR6, CCR1, or Ebi2 (GPR183) in the migration of either donor effector cell subset into the lungs of the recipient mice (Fig. 5B and C). Interestingly, this analysis showed that deletion of CX3CR1 did not have a significant impact on T cell migration into the lung parenchyma, which may contradict our analysis of kinetic data suggesting a 50% increase in the average migration rate (compared to that for WT cells). However, in our kinetic data the difference in the percentage of lung WT and CX3CR1-deficient T cells at 16 h after T cell transfer (and compared to that at 10 h) was not large. This indicates that measuring T cell accumulation at later time points after T cell transfer may not allow detection of migration differences if cells migrate rapidly.

While we were unable to detect roles for CCR2, CCR5, and CXCR5 in the migration of host-protective KLRG1⁻ effector CD4 T cells into the lung parenchyma by simply examining intact gene-KO mice, the adoptive transfer approach used here was able to reveal roles for each of these molecules. These data indicate that multiple chemokine receptors are utilized by CD4 T cells to migrate from the blood into the lung parenchyma. However, defects in any of these individual chemokine receptors have relatively small effects on the migration of the population.

Pertussis toxin treatment only partially inhibits effector CD4 T cell migration into the lungs of M. tuberculosis-infected mice.

Given the overall relatively minor role for chemokine receptors in the migration of effector CD4 T cells into the lungs of M. tuberculosis-infected mice, we next examined the role of G-protein-coupled receptors in T cell lung entry. Day 26 lung cells from CD45.1 mice were treated with pertussis toxin and mixed with cells from day 26 CD45.2 mice. The donor cell mixture was then injected into infection-matched CD45.1/CD45.2 recipient mice, and migration of the donor T cells was measured at 16 h posttransfer (Fig. 6A). As before, we tracked KLRG1⁺ CX3CR1⁺ terminal effector cells and KLRG1⁻ CX3CR1⁻ cells separately. Interestingly, pertussis toxin treatment only partially inhibited the migration of both effector cell subsets into the lung (Fig. 6B and C). Calculations based on the heterogeneous migration mathematical model (using equation 2 [see Materials and Methods]) suggested that pertussis toxin treatment decreased the rate of T cell migration into the lungs by 68%. These results indicate that effector CD4 T cell entry into the lungs during M. tuberculosis infection may be only partially dependent on chemokine receptor signaling, consistent with our overall conclusion that most chemokine receptors have relatively minor contributions to the accumulation of CD4 T cells in the lung parenchyma.

FIG 6.

Effector CD4 T cell migration into the lungs during M. tuberculosis infection is only partially inhibited by pertussis toxin treatment. (A) Lung cells isolated from day 26 M. tuberculosis-infected CD45.1 mice were treated with pertussis toxin and mixed with untreated control lung cells from CD45.2 infection-matched mice. (B and C) The mixture of donor cells was injected into CD45.1/CD45.2 infection-matched recipient mice, and 16 h later the migration of treated and untreated I-A^b ESAT-6_4–17-specific KLRG1⁺ CX3CR1⁺ (B) and KLRG1⁻ CX3CR1⁻ (C) donor CD4 T cells was measured using the intravascular stain. Data are for 5 mice per group. Data are representative of those from four similar experiments. Two-tailed t tests were used to compare untreated (UNTX) to pertussis toxin-treated (PTX) T cell frequencies. ***, P ≤ 0.001; ****, P ≤ 0.0001. Using equation 2, we estimated that pertussis toxin-treated T cells had 68% reduced rates of migration to the lung parenchyma compared to untreated, control T cells.

DISCUSSION

KLRG1⁻, lung-homing, M. tuberculosis-specific effector CD4 T cells express multiple chemokine receptors. However, individual chemokine receptor-deficient mice display little to no defect in the control of M. tuberculosis infection following low-dose aerosol exposure with typical lab strains like H37Rv. Here we used the intravascular staining technique to show that there are no appreciable defects in CD4 T cell entry into the lungs of several relevant intact chemokine receptor-KO animals, apart from CXCR3-deficient mice. We speculated that by examining the intact gene-KO mice at a single time point, minor effects may be masked. Using competitive adoptive transfer approaches to examine CD4 T cell migration in the setting of various chemokine receptor deficiencies, we show that CXCR3, CXCR5, and, to a lesser degree, CXCR6, CCR2, and CCR5 each has a partial, nonredundant role in CD4 T cell entry into the lung tissue during M. tuberculosis infection. Because we did not test all homing receptors, we cannot rule out the possibility that there may be other receptors that play a much greater role in the lung homing of M. tuberculosis-specific effector CD4 T cells. However, these data indicate that CD4 T cell entry into the lungs during M. tuberculosis infection is likely mediated by the cumulative effects of multiple different chemokine receptors, each with a relatively small contribution. Even CXCR3, which displayed the largest effect that we observed, lowered the rate of T cell input into the lungs by only ∼2-fold, and nearly similar fractions of WT and CXCR3-deficient cells eventually entered the lung. Interestingly, this impact of CXCR3 deficiency on T cell migration rate into the lungs with no appreciable effect on M. tuberculosis growth in the lungs raises the possibility that the speed at which effector CD4 T cells exit the blood and enter the lungs may not be a critical determinant of their protective capacity in M. tuberculosis infection, so long as they eventually accumulate to sufficient numbers. Perhaps this is due to the very low rate at which M. tuberculosis replicates in the lungs of mice (24). Alternatively, the specific location where T cells enter the lung may also be important; e.g., T cells entering the lung near or within a granuloma are likely to be more effective in M. tuberculosis control than cells entering the lung elsewhere.

Our data also provide insight into factors that inhibit CD4 T cell input into the lung parenchyma during M. tuberculosis infection, as CX3CR1-deficient effector CD4 T cells enter the lung parenchyma at about twice the rate of WT cells. The negative role for CX3CR1 in the entry of CD4 T cells into the lung tissue is consistent with the localization of lung CX3CR1⁺ CD4 T cells almost exclusively in the blood vasculature. It is not clear how CX3CR1 impairs CD4 T cell accumulation in the lung parenchyma, but it may relate to the expression pattern of its ligand. CX3CL1, the ligand for CX3CR1, is expressed on the luminal surface of inflamed vascular endothelium and may contribute to retaining cells within the vessels, thereby slowing their progress through into the lung parenchyma (25, 26). Although CX3CR1 deficiency enhanced the migration of CX3CR1⁺ KLRG1⁺ terminal effector CD4 T cells, it did not restore their migratory capacity to the levels of KLRG1⁻ CXCR3⁺ CD4 T cells, indicating that the defect in the migration of terminal effector Th1 cells is not as simple as CX3CR1-mediated suppression of lung entry. Therefore, the mechanistic basis for the inability of most terminal effector Th1 cells to accumulate in the lung parenchyma is still not clear. We have previously shown that while CX3CR1⁺ CD4 T cells in the lungs of rhesus macaques are exclusively localized to the blood vasculature, primary M. tuberculosis-specific effector CD4 T cells in macaques do not express CX3CR1 (27). Therefore, our results showing that CX3CR1 promotes intravascular localization are likely generalizable to CX3CR1-expressing CD4 T cells in nonhuman primates (NHP), but in the specific setting of primary M. tuberculosis infection, these data are more likely relevant to mice rather than NHP.

We also found that pertussis toxin treatment only partially inhibited CD4 T cell migration into the lungs of M. tuberculosis-infected mice. This may be due to inefficiencies with the toxin treatment, yet the treatment did reduce the relative migration rate more than deletion of any other chemokine receptor did. Interestingly, a recent report failed to find any impact of pertussis toxin treatment on CD4 T cell migration into M. bovis BCG-infected lungs (28). This is consistent with our difficulty in identifying a single chemokine receptor with a major role in the extravasation of effector CD4 T cells into the lungs. However, identification of the molecular mechanisms of pertussis toxin-insensitive migration is needed to firmly establish the importance of chemokine-receptor independent T cell lung homing during M. tuberculosis infection.

We should also point out that we specifically measured the entry of CD4 T cells into the lung parenchyma from the vasculature, and other functions of these chemokine receptors were not addressed here. There may be important roles for the chemokine receptors studied here in the localization of CD4 T cells into different regions of the lung after they extravasate out of the blood vessels. For example, CXCR5 has been shown to play a role in the localization of CD4 T cells into lymphoid follicle-like structures in the lungs of M. tuberculosis-infected mice (23). In the setting of CD8 T cell priming in the spleen following vaccinia virus infection, CXCR3 has been shown to promote the formation of KLRG1⁺ CD8 T cells by facilitating the clustering of clonally expanding T cells with antigen-presenting cells in the marginal zone that produce interleukin-12 and alpha interferon and promote terminal differentiation (29). Moreover, M. tuberculosis-specific CD8 T cells in M. tuberculosis-infected CCR5/CXCR3 double-deficient mice showed increased expression of CD127, indicating that the cells remained less differentiated (30). While we did not observe a lack of M. tuberculosis-specific KLRG1⁺ CD4 T cells in CXCR3-deficient animals (data not shown), we also cannot rule out the possibility that some of these chemokine receptors may impact the quality of the responding T cells. Lastly, we did not examine the role of these receptors in regulating the dwell time within the lung parenchyma or exit from the lungs via the lymphatics. Additional work is needed to better understand the chemokine receptors that determine the input, output, and spatial distribution of T cells in the lungs during M. tuberculosis infection.

There are also some key differences in the expression of chemokine receptors on murine versus NHP/human M. tuberculosis-specific CD4 T cells, most notably, in the cases of CCR6 and CXCR5. CCR6 is not found on M. tuberculosis-specific CD4 T cells in mice. In contrast, in humans and nonhuman primates, peptide-specific CD4 T cells coexpress CXCR3 and CCR6 (27, 31–34). CXCR5 expression seems to display the opposite trend in expression between species. In mice, CXCR5 is expressed on M. tuberculosis-specific CD4 T cells and contributes to their entry into the lungs. In rhesus macaques, however, we have previously shown that CXCR5 is not observed on tumor necrosis factor-producing, Ag-specific CD4 T cells in lymphoid tissue, blood, airways, or granulomas (27). Therefore, these data for mice may underestimate the role of CCR6 and overestimate the role of CXCR5 in effector CD4 T cell migration into the lungs in human M. tuberculosis infection.

We combined the use of experimental data with mathematical modeling to quantify the kinetics of T cell migration from the lung vasculature to the lung parenchyma. Any mathematical model involves sets of assumptions, and the predictions of models are the direct consequences of these assumptions (35). We found that the two alternative mathematical models can accurately describe the experimental data and, importantly, provide nearly identical estimates of the average entry rate of T cells into the lung. This is in part because in both models the initial slope of the change in the percentage of T cells in the parenchyma is determined by the average migration rate (m̄). However, the two models drastically differ in the interpretation of why the percentage of T cells in the parenchyma saturates at a less than 100% level, especially for terminally differentiated effector T cells: model 1 assumes that it is because of the inability of some T cells to migrate to the lung parenchyma, while model 2 assumes that it is due to differential division/death in the lung vasculature and lung parenchyma. Our data currently cannot discriminate between these alternatives. Future experiments which quantify the total numbers of transferred T cells in the lung vasculature and parenchyma would be instrumental at discriminating between the alternative models. It is now clear that calculating the total number of lymphocytes in nonlymphoid tissues, especially mucosal tissues, by flow cytometry is associated with strong biases due to inefficiencies of cell extraction (36). Therefore, accurate quantification of the cell migration kinetics into the lung most likely will benefit from the use of immunohistochemistry.

Our kinetic experiments suggest that it takes, on average, 8.6 h (95% range, 7.1 to 10.4 h) for half of WT M. tuberculosis-specific KLRG1⁻ effector CD4 T cells to enter the lung parenchyma, which is a relatively long time. Indeed, previous studies measuring the migration of lymphocytes into lymph nodes suggested an extremely efficient entry, taking only 10s of minutes to translocate from the vasculature to the node parenchyma (37, 38). However, lymphocytes may be expected to enter secondary lymphoid tissues more rapidly than they enter peripheral organs. Another recent study estimated that activated T cells spend a substantial amount of time migrating via lung capillaries (∼40 min) (39), and it is not clear why it takes hours for M. tuberculosis-specific effector CD4 T cells to cross the vascular endothelium and enter the lung parenchyma.

There is a need for correlates of protection against M. tuberculosis infection that can be used to guide vaccine design by predicting the effectiveness of the vaccine-elicited response. Identification of the key properties of the most host-protective CD4 T cells, such as lung-homing ability, may provide such a predictor. Collectively, our study indicates that M. tuberculosis-specific effector CD4 T cells that coexpress multiple chemokine receptors, including at least CXCR3, CXCR6, CCR2, and CCR5 (and, most likely, CCR6 in humans), but that do not express CX3CR1 may be the best lung-homing CD4 T cells. Therefore, polychemokine receptor expression pattern may be a useful feature to consider when evaluating the protective quality of M. tuberculosis-specific CD4 T cells.

MATERIALS AND METHODS

Mice.

Through an NIAID and Taconic Farms contract, the following mice were obtained: C57BL/6 CD45a-[KO]CX3CR1-[KI]enhanced green fluorescent protein (EGFP), C57BL/6nTac-[KO]CCR1, C57BL/6J-[KO]CCR2, C57BL/6J-[KO] CCR5, C57BL/6J-[KI]CCR6-EGFP, B6.PL-Thy1a/CyJ N10, and C57BL/6J × B6.SJL-CD45a(Ly5a)/Nai F₁. B6.129S2(Cg)-Cxcr5^tm1Lipp/J (CXCR5^−/−) and B6.129P2-Cxcr6^tm1Litt/J (CXCR6^−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). CXCR3^−/− and Ebi2^−/− mice were maintained at NIAID, NIH, Bethesda, MD. All animals were housed at an Association for the Assessment and Accreditation of Laboratory Animal Care-approved facility at the NIAID according to the National Research Council’s Guide for the Care and Use of Laboratory Animals (40). All experiments involving the use of animals were approved by the NIAID Animal Care and Use Committee under animal study proposal LPD-24E.

Aerosol M. tuberculosis infection.

Mice were exposed to ∼100 CFU of H37Rv in an aerosol machine (Glas-Col, LLC, Terre Haute, IN). Serial dilutions of tissue homogenates were plated on 7H11 agar plates to measure the bacterial loads (Difco, Detroit, MI).

Intravascular staining and lung lymphocyte isolation.

Mice were intravenously injected with 2.5 μg of fluorochrome-labeled anti-CD45 antibody 3 min before euthanasia. Lungs were processed and digested in RPMI supplemented with 1 mg/ml collagenase D (Roche-Diagnostics, Indianapolis, IN), 1 mg/ml hyaluronidase, 50 U/ml DNase I, and 1 mM aminoguanidine (all from Sigma-Aldrich, St. Louis, MO). A single-cell suspension, obtained by pushing the samples through a 100 μm cell-strainer, and lymphocytes were isolated by density centrifugation after resuspension in 37% Percoll (GE Healthcare, Pittsburgh, PA), and red blood cells were lysed with ACK lysing buffer (KD Medical, Columbia, MD).

Flow cytometry.

The cells were phenotyped by flow cytometry using the following reagents (antibody clone names in parentheses): anti-CD4 (RM4-5), CD44 (IM7), CD45 (30-F11), CD69 (H1.2F3), CXCR3 (CXCR3-173), CX3CR1 (SA011F11), Foxp3 (FJK-16s), KLRG1 (2F1/KLRG1), CD45.1 (A20), CD45.2 (104), Thy1.1 (OX-7), Thy1.2 (53-2.1), and T-bet (4B10) antibodies and fixable viability dye eFluor 780. All antibodies were purchased from BioLegend (San Diego, CA), eBioscience (San Diego, CA), BD Biosciences (San Jose, CA), and Cell Signaling Technologies (Danvers, MA). For tetramer staining, cells were incubated in the dark for 1 h at 37°C with I-A^b ESAT-6_4–17 tetramer from the NIAID tetramer facility (Atlanta, GA). The cells treated with human anti-KLRG1 IgG1 (kindly provided by Abcuro, Newton MA) were stained with a biotin-SP AffiniPure F(ab′)₂ fragment donkey anti-human IgG, Fcγ fragment-specific antibody purchased from Jackson ImmunoResearch Laboratory (West Grove, PA) and secondarily stained with fluorochrome-labeled streptavidin. Samples were acquired on an LSR II Fortessa flow cytometer and analyzed using FlowJo software (BD Biosciences, San Jose, CA).

CD4 T cell isolation and adoptive transfer experiments.

On day 28 after aerosol infection, CD4 T cells were isolated from the lungs of WT and KO mice after administering anti-CD45 by intravenous injection. The lungs were processed into single-cell suspensions, homogenate was plated for determination of the number of CFU, and 1 × 10⁶ cells were stained for day 28 intact phenotype analysis. WT and chemokine receptor-KO samples were pooled, and CD4⁺ cells were enriched using mouse CD4⁺ T cell microbeads (Miltenyi Biotec, Auburn, CA). CD4 T cells from each KO sample (1 × 10⁶ to 3 × 10⁶ cells) were mixed with an equivalent number of WT CD4 T cells and adoptively transferred into infection-matched, congenically disparate WT recipients. Recipient mice were harvested at 16 h posttransfer, and whole lungs were processed into single-cell suspensions and stained for flow cytometric analysis.

Kinetics and blockade experiments.

On day 28 after M. tuberculosis infection, the lungs of Thy1.2 donor mice (CD45.2 CXCR3^−/−, CD45.1 CX3CR1^−/−, and CD45.2 CD45.1 WT mice) were harvested and processed as described above. Following the same procedure used in the adoptive transfer experiments, CD4 T cells were enriched, and 1 × 10⁶ cells of each donor cell type were transferred into Thy1.1 recipient mice by intravenous injection. Lungs from the recipient mice were harvested at 4, 10, 16, 24, and 36 h after adoptive transfer and were processed as described above. For the KLRG1⁻ blockade, recipient mice received 200 μg per mouse of anti-KLRG1 in combination with the donor cells. For experiments blocking G-protein-coupled receptors, isolated lung lymphocytes were treated with 25 ng/ml of pertussis toxin for 1 h at 37°C at 5 × 10⁶ cells/ml in complete medium and washed extensively before they were mixed with an equal number of untreated cells and transferred into infection-matched recipient mice.

Statistical analysis.

Most of the statistical analysis was performed in Prism software (GraphPad Software, La Jolla, CA). A two-tailed t test, paired t test, one-way analysis of variance with Tukey’s multiple comparisons, and Kaplan-Meier tests were used in these analyses. To estimate the rate at which T cells migrate from the blood to the lung parenchyma, we developed a series of alternative mathematical models based on two alternative core mechanisms (35) (see the information in the supplemental material for more detail). In the first set of models, we assumed that T cells have a variable ability to migrate to the lung parenchyma, and in the simplest model, we assumed that the population of T cells consists of 2 subpopulations with proportions p and 1 − p and that only cells in the first subpopulation are able to migrate to the lung parenchyma at rate m. Then, the fraction of T cells in the lung parenchyma f(t) in this model is given by

$$f(t)=p(1-e^{-mt})$$ (1)

where t is time and the average migration rate of the total T cell population is m̄ = pm. In the supplemental material, we also discuss the results of a model in which the T cell population consists of multiple subpopulations with different rates of entry into the lung. In the alternative model, all T cells in the population are capable of migrating, but cells may divide and/or die in the blood or the lung parenchyma, and these processes contribute to the accumulation of intravascularly transferred T cells in the lung. The analytical solution of this alternative model for the faction of T cells in the lung parenchyma is given in the supplemental material. Because the two models gave nearly identical estimates on the rate of T cell migration into the lung, here we present the results only for the first (heterogeneous migration) model; the results from both models are presented in the supplemental material. In the heterogeneous migration model, we characterize T cells based on the fraction (p) of T cells in the blood that are capable of migrating to the lung parenchyma at rate m and the average migration rate of the whole-cell population (m̄). We fitted the model solution (equation 1) to the data on the percentage of T cells that are in the lung parenchyma at different times since T cell transfer by minimizing the sum-of-squared residuals. The confidence intervals for the parameters were estimated using a standard bootstrap approach by resampling the residuals (deviations between the data and the best-fit model) with replacement 1,000 times (41).

In additional cotransfer experiments, we measured the accumulation of WT and chemokine receptor-deficient M. tuberculosis-specific CD4 T cells in the lung parenchyma following the cotransfer of these cell populations into M. tuberculosis-infected mice. We show in the supplemental material that the mathematical model in equation 1 can be used to estimate the relative entry of receptor-deficient T cells into the lung parenchyma (m̄) using the formula

$$\alpha =\frac{\ln (1-f_{\text{KO}})}{\ln (1-f_{\text{WT}})}$$ (2)

where f_KO and f_WT are the frequency of receptor-deficient and WT T cells in the lung parenchyma, respectively, at the same time point.