Defective positioning in granulomas but not lung-homing limits CD4 T-cell interactions with Mycobacterium tuberculosis-infected macrophages in rhesus macaques

Abstract

Protection against Mycobacterium tuberculosis (Mtb) infection requires CD4 T cells to migrate into the lung and interact with infected macrophages. In mice, less-differentiated CXCR3⁺ CD4 T cells migrate into the lung and suppress growth of Mtb, whereas CX3CR1⁺ terminally differentiated Th1 cells accumulate in the blood vasculature and do not control pulmonary infection. Here we examine CD4 T-cell differentiation and lung homing during primary Mtb infection of rhesus macaques. Mtb-specific CD4 T cells simultaneously appeared in the airways and blood ∼21-28 days post exposure, indicating that recently primed effectors are quickly recruited into the lungs after entering circulation. Mtb-specific CD4 T cells in granulomas display a tissue-parenchymal CXCR3⁺CX3CR1^-PD-1^hiCTLA-4⁺ phenotype. However, most granuloma CD4 T cells are found within the outer lymphocyte cuff and few localize to the myeloid cell core containing the bacilli. Using the intravascular stain approach, we find essentially all Mtb-specific CD4 T cells in granulomas have extravasated across the vascular endothelium into the parenchyma. Therefore, it is unlikely to be that lung-homing defects introduced by terminal differentiation limit the migration of CD4 T cells into granulomas following primary Mtb infection of macaques. However, intralesional positioning defects within the granuloma may pose a major barrier to T-cell-mediated immunity during tuberculosis.

Figure 1. Variable disease severity following ultra-low dose Mtb infection of rhesus macaques

Six rhesus macaques were exposed to >1 cfu Mtb Erdman strain via bronchoscope. (A) Only one of the six animals became infected (ZK02) as confirmed by ¹⁸FDG PET/CT scan. (B) The five remaining animals were confirmed negative as described in the text and then re-exposed to 6–10 cfu. ¹⁸FDG PET/CT scans were taken pre-infection and ~2 months post-infection, just prior to necropsy. The day post-infection of the scan is indicated in white letters on each scan. Note that the catheter is visible as a bright spot in the pre-infection scan of animal ZL43. (C) SUVR values were calculated for lung and pulmonary lymph nodes of infected animals. (D) Weight loss was monitored following infection. (E) Correlation between total SUVR in lung and pulmonary lymph nodes and percent weight loss at time of ⁸FDG PET/CT scan. (F) Correlation between SUVR and bacterial loads in individual instillation site lesions and pulmonary lymph nodes.

Figure 2. Ag-specific CD4 T cells simultaneously appear in the peripheral blood and airways following Mtb infection

Weekly blood and BALf samples were collected prior to and following Mtb infection. (A) Bacterial loads were quantified in BAL fluid. (B) Example FACS plots showing intracellular cytokine staining for TNF and IFNγ following a 6-hour in vitro stimulation with ESAT-6/CFP-10 peptide pools. (C) Kinetics of Ag-specific CD4 T cells in blood and BALf following Mtb infection. Note that ZK02 was infected with >1 cfu while the other animals were exposed to 6–10 cfu as described in Figure 1. Numbers represent the frequency of TNF⁺ CD4 T cells following stimulation with ESAT-6/CFP-10 peptide pools.

Figure 3. Ag-specific CD4 T cells in lymphoid tissues and pulmonary lesions of Mtb infected rhesus macaques

Tissues were obtained from animals approximately two months post-infection. (A–B) Example FACS plots (A) and summary quantification (B) of intracellular cytokine staining of TNF and IFNγ following a 6-hr in vitro stimulation of cells from instillation site lesions and lymphoid tissue with ESAT-6/CFP-10 peptide pools. (C) Ag-specific CD4 T cell responses in individually resected pulmonary granulomas as quantified by ICC. (D) Bacterial loads in granulomas. (E) Granuloma bacterial burden did not correlate with the percentage of ESAT-6/CFP-10-specific CD4 T cells. P values are from a one way ANOVA test.

Figure 4. Ag-specific CD4 T cells do not differentiate into CX3CR1⁺CXCR3⁺ terminal effectors in primary Mtb infection of rhesus macaques

(A–B) Example FACS plots (A) and summary quantifications (B) of CXCR3 and CX3CR1 expression on bulk (grey) or ESAT-6/CFP-10-specific (red dots) CD4 T cells from the blood and BAL. Overlaid Ag-specific CD4 T cells are gated on TNF⁺IFNγ⁺ cells following stimulation with peptide pools. (C–D) Example FACS plots (C) and summary quantifications (D) of chemokine receptor expression on ESAT-6/CFP-10-specific CD4 T cells from various tissues on ~day 60 post-infection.

Figure 5. PD-1 and CTLA-4 expression on Mtb-specific CD4 T cells are elevated at sites of bacterial replication

(A–B) Example FACS plots (A) and quantifications (B) of PD-1 expression in ESAT-6/CFP-10-specific CD4 T cells in different tissues of Mtb infected rhesus macaques. In order to better compare across different necropsy days, the adjusted gMFI of PD-1 expression was calculated as the gMFI of PD-1 on Ag-specific CD4 T cells minus the gMFI of PD-1 on PD-1 negative lymph node CD4 T cells from the matched animal. P values for one way ANOVA with Fisher’s LSD test for multiple comparisons shown to the right of the graph with significant differences highlighted in red. (C–D) PD-1 expression was compared on ESAT-6/CFP-10, PPD and TB10.4-specific CD4 T cells. Example FACS plots are shown in (C) and summary graphs in panel (D). Data from various tissues at necropsy are pooled and only samples where data from all three stimulation conditions were available are included for t test comparison. (E–F) Example FACS plots (E) and summary graphs (F) of the percentage of ESAT-6/CFP-10-specific CD4 T cells that expressed intracellular CTLA-4 following stimulation. P values for one way ANOVA with Fisher’s LSD test for multiple comparisons shown to the right of the graph with significant differences highlighted in red. (G) Correlation of the adjusted gMFI of PD-1 and the percent CTLA-4⁺ in ESAT-6/CFP-10-specific CD4 T cells. Data from various tissues at necropsy are pooled. ****P<0.0001, ***P<0.001 and *P<0.05

Figure 6. CD4 T cells poorly localize to the infected macrophage-rich center of granulomas

(A–C) Individually resected pulmonary granulomas from rhesus macaques infected with Mtb-Erdman were thick-sectioned, stained with an antibody cocktail and imaged for the presence and localization of CD4 T cells. (A) Example image showing the location of CD4⁺ and CD8⁺ cells in relation to CD11b expressing cells. (B) Four different regions of the granuloma were identified as described in the text and CD4 T cells in each region highlighted in a different color: lymphocyte cuff (blue), lacunar zone between the lymphocyte cuff and the macrophage core (green), boundary of the macrophage-rich core (red), and the macrophage-rich core (white). (C) Quantification of the percent of CD4⁺ cells that are in each region of the granuloma. Data shown are from three separate granulomas. (D–E) Two rhesus macaques were infected with ~8 cfu of Mtb-H37Rv-RFP and granulomas were resected 6–7 weeks post infection for analysis of CD4 T cell localization as above. (D) Individual staining of laminin, CD11b, CD4, and RFP-Mtb on the left with a merged tile on the right. (E) Quantification of the percent of CD4⁺ cells that are in each region. Three separate granulomas from these two Mtb-H37Rv-RFP infected animals were analyzed.

Figure 7. Ag-specific CD4 T cells efficiently extravasate out of the lung blood vasculature into parenchymal pulmonary lesions

Just prior to euthanasia and necropsy, αCD45-biotin mAb was intravenously injected into the animals to allow for the detection of intravascular and parenchymal lymphocytes by flow cytometry. (A) Staining for the iv injected αCD45 mAb and CD69 are shown on CD4 T cells in various tissues from an uninfected rhesus macaque. (B) CX3CR1 and iv αCD45 stain on CD4 T cells from the BALf and lung tissue of an uninfected rhesus macaque. (C) Example FACS plots of intravascular staining on ESAT-6/CFP-10-specific CD4 T cells from different tissues of an Mtb-Erdman infected rhesus macaque. (D) Summary graphs of the percent of ESAT-6/CFP-10-specific CD4 T cells that are positive for the intravascular CD45 stain from various tissues of Mtb-Erdman infected rhesus macaques. Ag-specific CD4 T cells were identified by gating on TNF⁺IFNγ⁺ cells following restimulation with peptide pools.