Inducible costimulator controls migration of T cells to the lungs via down-regulation of CCR7 and CD62L

Abstract

We and others reported that inducible costimulator-deficient (ICOS(-/-)) mice manifest a defect in Th2-mediated airway inflammation, which was attributed to reduced Th2 differentiation in the absence of ICOS signaling. Interestingly, the number of CD4 T cells present in the airways and lungs after sensitization and challenge is significantly reduced in ICOS(-/-) mice. We now show that this reduction is not attributable simply to a reduced proliferation of ICOS(-/-) cells, because significantly more ICOS(-/-) than wild-type activated CD4 T cells are present in the lymph nodes, suggesting that more ICOS(-/-) CD4 T cells than wild-type CD4 T cells migrated into the lymph nodes. Further investigation revealed that activated ICOS(-/-) CD4 T cells express higher concentrations of the lymph node homing receptors, CCR7 and CD62L, than do wild-type CD4 T cells, leading to a preferential return of ICOS(-/-) cells to the nondraining lymph nodes rather than the lungs. Blocking reentry into the lymph nodes after the initiation of Th2-mediated airway inflammation equalized the levels of CD4 and granulocyte infiltration in the lungs of wild-type and ICOS(-/-) mice. Our results demonstrate that in wild-type CD4 T cells, co-stimulation with ICOS promotes the down-regulation of CCR7 and CD62L after activation, leading to a reduced return of activated CD4 T cells to the lymph nodes and a more efficient entry into the lungs.

Figure 1.

Fewer activated Inducible costimulator-deficient (ICOS^−/−) CD4 T cells are found in inflamed airways and lungs than wild-type cells. C57Bl/6 (B6) and ICOS^−/− mice were sensitized and challenged with inactivated Schistosoma mansonii eggs and soluble egg antigen (SEA). On Day 4 after challenge, mice were killed, and airway (bronchoalveolar lavage; BAL) and lung (tissue digest) cells were analyzed by flow cytometry. (A) CCR3⁺SSC^high eosinophils and CD3⁺CD4⁺ T cells from BAL. (B) CD4⁺ T cells and CD44⁺CD62L⁻ activated CD4⁺ T cells from the lungs. This experiment was repeated three times (n ≥ 4 for each group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2.

Activated antigen-specific ICOS^−/− CD4 T cells accumulate more in the lymph nodes, yet proliferated less than wild-type cells. Naive hosts received wild-type or ICOS^−/− CD4⁺ KJ1–26⁺ cells, were subsequently immunized, and 3–5 hosts were killed at each time-point shown for each group (wild-type or ICOS^−/−). (A) Splenocytes and lymph node (axial, brachial, and inguinal; draining and nondraining) cells were counted and then analyzed by flow cytometry for CD4⁺KJ1–26⁺ cells. The number of CD4⁺KJ1–26⁺ cells was calculated by multiplying the percent CD4⁺KJ1–26⁺ cells within the organ by the total number of cells in the organ. (B) At the peak of response on Day 4, proliferation was assessed by flow cytometry as CFSE dilution in CD4⁺KJ1–26⁺ cells. The number of CD4⁺KJ1–26⁺ cells in each generation of division is shown. Lower graphs in B illustrate representative CFSE dilution plots of DO11.10 (gray line) and ICOS^−/− DO11.10 (black dotted line) cells. This experiment was repeated twice (n ≥ 4 for each group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.

ICOS^−/− CD4 T cells preferentially migrate into the lymph nodes. (A) Wild-type (WT) and ICOS^−/− DO11.10 cells were mixed and co-transferred into Balb/c hosts, which were then immunized. In unimmunized mice (leftmost squares) and on Day 4 after immunization, the ratio of ICOS^−/−/wild-type CD4⁺KJ1–26⁺ cells was quantified. The mice were injected intraperitoneally with Mel-14 on Days 0 and 2 (right, upside-down triangles), treated with FTY720 daily by oral gavage (middle, triangles), or left untreated after immunization (second from left, open circles). This experiment was repeated three times for Mel-14–treated mice and untreated mice, and twice for FTY720-treated mice (n ≥ 4 for each group). (B) Cells were harvested from the draining lymph nodes (mediastinal) of wild-type and ICOS^−/− mice (sensitized and challenged as described in Figure 1), and wild-type and ICOS^−/− cells were transferred together into sensitized and challenged recipient wild-type mice. Sixteen hours later, the migration of CD4 T cells back to the draining lymph nodes versus the spleen was assessed. This experiment was replicated twice (n ≥ 3 for each group). ns, no significance. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4.

Activated ICOS^−/− CD4 T cells express higher concentrations of the lymph node homing molecules CCR7 and CD62L than do wild-type CD4 T cells. DO11.10 and ICOS^−/− DO11.10 cells were assessed on Day 4 after adoptive transfer, as described in Figure 2. (A) CD4⁺KJ1–26⁺ cells were evaluated for the expression of CD62L, shown as a representative plot (left, wild-type, gray shaded histogram; ICOS^−/−, black line), the total percent of CD62L^high cells (middle), and the percent of cells in each cell division expressing CD62L (right). (B) The expression of CCR7 was analyzed in CD4⁺KJ1–26⁺ cells. A representative plot is shown (left, wild-type, gray shaded histogram; ICOS^−/−: black line). The median fluoresence intensity (MFI) of CCR7 of all CD4⁺KJ1–26⁺ cells (middle) and the MFI of CCR7 in each cell division (right) were measured. Experimental results in A and B were replicated in two independent experiments (n ≥ 4 for each experiment). (C) OTII and ICOS^−/− OTII cells were labeled with CFSE and adoptively transferred to ICOSL^−/− or WT congenic hosts, which were immunized with inactivated S. mansonii eggs and ovalbumin_323–339 peptide (OVAp). On Day 4, the CD62L expression of T cell receptor-transgenic cells at each cell division was quantified. Representative plots show the CD62L^high and CD62L^low fractions of each generation for each condition. The percent CD62L^high within a generation is calculated as CD62L^high/total in generation (i.e., CD62L^high/[CD62L^high + CD62L^low]). For A and B, significance was determined by unpaired Student's t tests. For C, significance was calculated by 2-way ANOVA. For all results, *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5.

Activated ICOS^−/− CD4 T cells preferentially migrate to nondraining lymph nodes (LNs). (A) B6 and ICOS^−/− mice were sensitized and challenged with inactivated S. mansonii eggs and SEA, as in Figure 1. On Day 4 after challenge, mice were killed, and their lung (tissue digest), draining lymph node (mediastinal), and nondraining lymph node (inguinal, axial, and brachial) cells were analyzed by flow cytometry for recently activated CD4⁺ T cells (CD69⁺). This experiment was replicated twice (n ≥ 4 for each group). (B) Naive hosts received 1 × 10⁴ DO11.10 or ICOS^−/− DO11.10 cells, and were then immunized with S. mansonii eggs and OVAp. On Day 4 after immunization, mice were killed, and their spleen, draining lymph nodes (mediastinal and mesenteric), and nondraining lymph nodes (axial and cervical) were analyzed by flow cytometry for the presence of CD4⁺KJ1–26⁺ antigen-specific cells. This experiment was replicated twice (n ≥ 4 for each group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6.

Blocking entry into the lymph nodes permits ICOS^−/− CD4 T cells to enter lungs and airways and mediate inflammation, similar to wild-type cells. B6 and ICOS^−/− mice were sensitized and challenged with inactivated S. mansonii eggs and SEA, as in Figure 1. On Day 1 after challenge, half of the mice were treated with Mel-14 to block entry into the lymph nodes. On Day 4 after challenge, the mice were killed, and brochoalveolar lavage and lung digests were performed to isolate infiltrating lymphocytes. Lung (A) and BAL (B) cells were analyzed by flow cytometry for Gr-1(Ly6G)⁺ SSC^high granulocytes and activated CD4⁺ T cells. (C) Lung cells were restimulated with SEA, and the number of cells producing IL-5 was quantified by Enzyme-linked immunosorbent spot (ELISPOT) assay. This experiment was repeated twice (n ≥ 3 for each group). *P < 0.05, **P < 0.01, and ***P < 0.001.