Preferential migration of effector CD8+ T cells into the interstitium of the normal lung

Abstract

The respiratory tract is a primary site of infection and exposure to environmental antigens and an important site of memory T cell localization. We analyzed the migration and retention of naive and activated CD8+ T cells within the noninflamed lungs and quantitated the partitioning of adoptively transferred T cells between the pulmonary vascular and interstitial compartments. Activated but not naive T cells were retained within the lungs for a prolonged period. Effector CD8+ T cells preferentially egressed from the pulmonary vascular compartment into the noninflamed pulmonary interstitium. T cell retention within the lung vasculature was leukocyte function antigen-1 dependent, while the egress of effector T cells from the vascular to the interstitium functions through a pertussis toxin-sensitive (PTX-sensitive) mechanism driven in part by constitutive CC chemokine ligand 5 expression in the lungs. These results document a novel mechanism of adhesion receptor- and pulmonary chemokine-dependent regulation of the migration of activated CD8+ T cells into an important nonlymphoid peripheral site (i.e., the normal/noninflamed lung).

Figure 1

Activated CD8⁺ T cells but not naive T cells are retained within lungs after adoptive transfer in an antigen-independent manner. (A) Dual modality image (merged image combining γ-ray and x-ray images) following adoptive transfer of ¹¹¹In-labeled naive, early-activated, or effector CD8⁺ T cells into WT (non-Tg) recipient mice (top view, prone mouse). In the merged RGB images shown, the red channel indicates γ-counts. (B) Distribution of ¹¹¹In-labeled CD8⁺ T cells at the indicated time points after transfer. Data is represented as percentage of radioactivity in ROI defined for the lungs, with the fraction of transferred cells/radioactivity localized to the lungs (∼80%) at 1 minute defined as 100%. Results are representative of 6 independent experiments. (C) Dual modality image and (D) quantitation following adoptive transfer of ¹¹¹In-labeled effector CTLs into HA-transgenic (HA-Tg) and WT mice.

Figure 2

Effector CD8⁺ T cells migrate into the interstitium of noninflamed lungs. (A) Kinetic analysis of intravascular versus extravascular compartmentalization of effector CD8⁺ T cells in lungs. We injected 4 × 10⁶ CFSE-labeled CD8⁺ T cells into recipient mice. Anti–CD8α-APC mAbs were injected at 5 minutes or 1, 2, 3, or 6 hours after transfer; then lungs were harvested 10 minutes later. The percentage of CFSE-labeled cells is shown; the percentage of CFSE-labeled interstitial T cells from total percentage of emigrated CFSE-labeled T cells is shown in parentheses. Ab first indicates that anti–CD8α-APC mAbs were injected 2 minutes prior to T cell transfer. (B) Data from A is expressed as the percentage of transferred CD8⁺ T cells in the intravascular or extravascular compartment.

Figure 3

Effector CD8⁺ T cells but not early-activated CD8⁺ T cells transmigrate to the lung interstitium. (A) Effector CTLs and early-activated (48 hours) CD8⁺ T cells (labeled with CFSE and CMTMR, respectively) were injected into WT recipient mice. Anti–CD8α-APC mAbs were injected at 6 hours after transfer; lungs were harvested 10 minutes later. The percentages of CFSE and CMTMR-labeled cells from individual recipients are shown in dot plots. (B and C) Analysis of T cell partitioning between pulmonary vascular and interstitial compartments. The numbers in parentheses reflect the percentages of CFSE-labeled (effector CD8⁺ T cells) or CMTMR-labeled (early-activated CD8⁺ T cells) cells from individual recipients that emigrated into the interstitium from the total number of CFSE- or CMTMR-labeled cells remaining within the lungs. Results represent the mean ± SEM of 4 recipients in a total of 4 independent experiments.

Figure 4

Regulation of the retention time within the lungs but not transmigration into the interstitium by LFA-1. (A) Pretreated with anti–LFA-1 mAbs, CFSE-labeled effector CD8⁺ T cells were mixed with CMTMR-labeled–nontreated effector CD8⁺ T cells and injected into WT recipient mice. Anti–CD8α-APC mAbs were injected at 6 hours after transfer; lungs were harvested 10 minutes later. The percentages of labeled CD8⁺ T cells from individual mice are shown in dot plots, the percentages of interstitial cells in parentheses. Results are representative of 6 recipients from a total of 6 independent experiments. (B) CMTMR-labeled LFA-1^–/– activated CD8⁺ T cells were mixed with equal numbers of CFSE-labeled activated (LFA-1^+/+) CD8⁺ T cells and injected into WT recipient mice. The percentages of labeled CD8⁺ T cells from LFA-1^–/– and LFA-1^+/+ donors localized to the lungs over time are shown. CD8⁺ T cells were activated by plate-bound anti-CD3/CD28 Abs. Results are representative of 4 recipient mice in a total of 4 independent experiments.

Figure 5

Migration of effector CD8⁺ T cells into the lung interstitium is PTX-sensitive. PTX-pretreated effector CD8⁺ T cells or nontreated effector CD8⁺ T cells were labeled with CFSE and CMTMR, mixed equally, and injected into WT recipient mice. Anti–CD8α-APC mAbs were injected at indicated time points after transfer, and lungs were harvested 10 minutes later. The percentages of total labeled, PTX-treated or control T cells retained in the lungs over time from individual recipient are indicated. The percentages of cells localized to the interstitium (CFSE⁺/CD8α-APC^–, CMTMR⁺/CD8α-APC^–) are shown in parentheses. Results represent the mean ± SEM from 3 to 9 recipient mice for each time point. In control experiments, cells were treated with mutant PTX (PT9K 129G, provided by E. Hewlett, University of Virginia, Charlottesville, Virginia, USA).

Figure 6

RANTES/CCR5-dependent transmigration of activated effector CD8⁺ T cells to alveolar interstitium. (A) Naive, early-activated, and effector CD8⁺ T cells were analyzed for expression of chemokine receptors. Results show the percentages (± SEM) of chemokine receptor expression for naive (white bars), early-activated (gray bars), and effector (black bars) CD8⁺ T cells from total expression of β-actin as a control. (B) WT recipients were injected with 1 of the following: polyclonal goat anti-RANTES Ab, polyclonal goat Ig, or PBS. After 1 hour, equal numbers of PTX-treated and nontreated effector CD8⁺ T cells labeled with CMTMR and CFSE were injected into recipient mice. After 4 hours, anti–CD8α-APC mAbs were injected, and 10 minutes later, lungs were harvested. Counterplots represent cells gated on CFSE⁺ and CMTMR⁺ T cells. The percentages of emigrated T cells in vascular (CFSE⁺/CD8-APC⁺, CMTMR⁺/CD8-APC⁺) and interstitial (CFSE⁺/CD8-APC^–, CMTMR⁺/CD8-APC^–) compartments from individual mice are shown. The percentage of emigrated interstitial labeled cells is shown in parentheses. The results are representative of 6 independent experiments. (C) Percentage of interstitial PTX-treated and control CD8⁺ T cells in the indicated recipients. *P < 0.05, **P < 0.001 between the percentages of interstitial nontreated T cells in nontreated mice and all other groups. (D) Effector CD8⁺ T cells from CCR5^–/– mice and CCR5^+/+ littermates were labeled with CFSE and CMTMR, respectively, mixed in ratio 1:1, and injected i.v. into recipient mice. After 7 hours, anti–CD8α-APC mAbs were injected, and 10 minutes later, lungs were harvested. Flow cytometry plots represent cells gated on CD45⁺ dye-labeled (CD45⁺/CFSE⁺ or CD45⁺/CMTMR⁺) cells. The percentages of lung-resident transferred CD8⁺ T cells in vascular (CFSE⁺/CD8-APC⁺, CMTMR⁺/CD8-APC⁺) and interstitial (CFSE⁺/CD8-APC^–, CMTMR⁺/CD8-APC^–) compartments are shown.