Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues

Abstract

Lymphocytes travel throughout the body to carry out immune surveillance and participate in inflammatory reactions. Their path takes them from blood through tissues into lymph and back to blood. Molecules that control lymphocyte recruitment into extralymphoid tissues are well characterized, but exit is assumed to be random. Here, we showed that lymphocyte emigration from the skin was regulated and was sensitive to pertussis toxin. CD4(+) lymphocytes emigrated more efficiently than CD8(+) or B lymphocytes. T lymphocytes in the afferent lymph expressed functional chemokine receptor CCR7, and CCR7 was required for T lymphocyte exit from the skin. The regulated expression of CCR7 by tissue T lymphocytes may control their exit, acting with recruitment mechanisms to regulate lymphocyte transit and accumulation during immune surveillance and inflammation.

Figure 1. Tissue exit is non-random

Splenocytes were labeled with CFSE or PKH26 and injected into the footpads of mice. 12 h after transfer, the draining popliteal lymph nodes were analyzed for migrated CFSE⁺ or PKH26⁺ lymphocytes. (a) Flow cytometric analysis of the total injected and migrated populations. (b) Flow cytometric analysis of gated CD4⁺ T cells in the injected and migrated populations. (c, left panel) Migration of total cells and CD8⁺, CD4⁺, and B (CD19⁺) lymphocytes is presented as the percent of injected cells of a given subset recovered from the isolated lymph nodes of individual mice. (c, right panel) The migration of CD8⁺ T cells and CD19⁺ B cells, normalized to that of CD4⁺ T cells in each analyzed mouse. One representative experiment (a, b, and c left panel) of 4 independent experiments using 4–10 mice each, or data points representing individual mice and the median of all experiments (c, right panel) are shown.

Figure 2. Tissue exit involves G_αi protein-coupled receptor signaling

Splenocytes were treated with PTX or mock-treated and labeled with CFSE and PKH26, respectively. Untreated PKH26⁺ cells were injected into one footpad and an equal number of CFSE⁺ PTX-treated cells into the contralateral footpad. (a) Flow cytometric analysis of injected and recovered cells. (b) Cell counts for recovered CFSE⁺ and PKH26⁺ cells from the draining popliteal lymph nodes of individual mice. One representative staining (a) or experiment (b) of 4 independent experiments using 5–10 mice each are shown.

Figure 3. T cells require expression of CCR7 to exit the skin and reach the draining lymph node

PKH26⁺ wild-type spleen cells were mixed with equal numbers of either CFSE⁺ Ccr7^−/− cells or CFSE⁺ wild-type cells, and injected into the footpads of mice. 12 h after transfer, the draining popliteal lymph nodes were analyzed for migrated CFSE⁺ and PKH26⁺ cells. (a) Cell counts for total recovered CFSE⁺ and PKH26⁺ cells from the draining popliteal lymph nodes of individual mice injected with wild-type PKH26⁺ and wild-type CFSE⁺ cells (left panel), or wild-type PKH26⁺ mixed with CCR7-deficient CFSE⁺ cells (right panel). (b) The effect of CCR7 deficiency on the migration of total lymphocytes, total CD4⁺ T cells, naïve (CD45RB^hi) and memory (CD45RB^lo) CD4⁺ T cells, CD8⁺ T cells, and B cells. In each case, the ratio of migrated CFSE⁺ to PKH26⁺ cells (admixed wild-type internal standard cells) of the indicated subset was determined by flow cytometry. Results are normalized to the mean ratio of CFSE⁺ wild-type to PKH26⁺ wild-type cells for each subset (set as 100%). Each data point represents an individually analyzed mouse from a group of 4–5 mice; the mean migration of each group is indicated. One representative experiment of a minimum of three performed is shown.

Figure 4. Transduction of CCR7 rescues Ccr7^−/− CD4⁺ T cell exit from the skin and migration to the draining lymph node

CD4⁺ T cells from spleens of wild-type or Ccr7^−/− (KO) mice were transduced with a retroviral vector encoding GFP from an IRES (control vector) or CCR7 and IRES-GFP (CCR7). A mixture of GFP⁻ (non-transduced) and GFP⁺ (transduced) cells was analyzed. (a) Binding of CCL19-Ig by wild-type (left panel) and CCR7-deficient T cells (middle panel) transduced with control vector or Ccr7^−/− cells transduced with CCR7-encoding vector (right panel) was assessed by flow cytometry. (b) Chemotaxis of wild-type (left panel) and Ccr7^−/− cells transduced with control vector (middle panel) or Ccr7^−/− cells transduced with CCR7-encoding vector (right panel) toward CCL21 was tested in a Transwell chemotaxis assay. Results are expressed as the percentage of GFP⁻ and GFP⁺ Thy1.2⁺ CD4⁺ T cells that migrated to the lower chamber, and data points represent the mean ± SD of triplicate wells at each concentration. (c) The percent migration to the popliteal lymph node of injected GFP⁻ and GFP⁺ wild-type (left panel), Ccr7^−/− cells transduced with control vector (middle panel), or Ccr7^−/− cells transduced with CCR7-encoding vector (right panel) was determined 12 h after injection into the footpad in individual mice. Note, a different y-axis scale is shown in the left panel. Data are representative of three independent experiments (a–c) analyzing 4–5 individual mice in each group (c).

Figure 5. Ovine T cells in the afferent lymph express functional CCR7

Peripheral blood and afferent lymph cells were collected after venopuncture or catheterization of skin draining afferent or pseudoafferent lymph vessels of sheep, respectively. (a) The binding of CCL19-Ig by CD45R⁺ naïve and CD45R⁻ memory CD4⁺ and CD8⁺ T cells was assessed by flow cytometry. (b) Chemotaxis of CD4⁺ and CD8⁺ T cells toward human CCL19 or murine CCL21 was tested ex vivo in a Transwell chemotaxis assay. Results are expressed as the percentage of T cells of the respective subset that migrated to the lower chamber, and data points represent the mean ± SD of triplicate wells at each concentration. Data are representative of four individually analyzed sheep (a) and one out of two independent experiments with similar results (b).