Selective generation of gut tropic T cells in gut-associated lymphoid tissue (GALT): requirement for GALT dendritic cells and adjuvant

Abstract

In the current study, we address the underlying mechanism for the selective generation of gut-homing T cells in the gut-associated lymphoid tissues (GALT). We demonstrate that DCs in the GALT are unique in their capacity to establish T cell gut tropism but in vivo only confer this property to T cells in the presence of DC maturational stimuli, including toll-like receptor-dependent and -independent adjuvants. Thus, DCs from mesenteric LNs (MLNs), but not from spleen, supported expression of the chemokine receptor CCR9 and integrin alpha4beta7 by activated CD8+ T cells. While DCs were also required for an efficient down-regulation of CD62L, this function was not restricted to MLN DCs. In an adoptive CD8+ T cell transfer model, antigen-specific T cells entering the small intestinal epithelium were homogeneously CCR9+alpha4beta7+CD62Llow, and this phenotype was only generated in GALT and in the presence of adjuvant. Consistent with the CCR9+ phenotype of the gut-homing T cells, CCR9 was found to play a critical role in the localization of T cells to the small intestinal epithelium. Together, these results demonstrate that GALT DCs and T cell expression of CCR9 play critical and integrated roles during T cell homing to the gut.

Figure 1.

DCs from MLN, but not from spleen, are necessary and sufficient to induce a CCR9⁺α₄β₇ ⁺CD62L^low phenotype on Ag-specific CD8⁺ T cells. CFSE-labeled OT-1 cells were stimulated with SIINFEKL peptide-pulsed CD11c⁺ DCs and CD11c⁻ non-DCs or anti-CD3/CD28 mAbs. After 4 d of culture, the fraction of responding OT-1 cells expressing CCR9, CD62L, α₄β₇, and CXCR3 was determined by flow cytometry. (A) Representative results obtained for CCR9 and CD62L. (B) Percentages ± SD of responding OT-1 cells expressing indicated phenotype after stimulation with DCs (DC, n = 6), DC-depleted APCs (−DC, n = 3), or anti-CD3/CD28 mAbs (n = 3–5). Gray and white bars represent APCs from MLN and spleen, respectively. (C) Percentages ± SD of OT-1 cells expressing indicated phenotype as a function of cell division (n = 3). (D) Responsiveness of MLN DC (gray bars) and spleen DC (white bars) stimulated OT-1 cells to CCL25 and CXCL10. One representative experiment is shown.

Figure 2.

MLN CD8α¹ and CD8α⁻ DC subsets regulate OT-1 cell expression of CCR9, α4β7, and CD62L in a similar manner. CFSE-labeled OT-1 cells (10⁵) were stimulated for 4 d with Ag-pulsed CD8α⁺ and CD8α⁻ DC subsets from MLN (5 × 10⁴ of each) or by anti-CD3/CD28 mAbs, and CCR9, α₄β₇, and CD62L were analyzed by flow cytometry. The percentage of responding OT-1 cells expressing a CCR9⁺, α₄β₇ ⁺, or a CD62L^low phenotype is indicated for each analysis.

Figure 3.

In vivo gut-homing OT-1 cells express a CCR9⁺α₄β₇ ⁺CD62L^low phenotype which is induced efficiently only in GALT and after adjuvant-triggered inflammation. OT-1 cells were injected i.v. into C57BL/6J-Ly5.1 mice, and 2 d later mice received OVA i.p., either alone or in combination with adjuvant. (A) Flow cytometry analysis of OT-1 cells in indicated organs 3 d after immunization with OVA and pI:C. (B) Phenotype of OT-1 cells in the MLN and IEL compartment 3 d after immunization with OVA alone or OVA with adjuvant. (C) CFSE-labeled OT-1 cells were transferred into C57BL/6J-Ly5.1 mice, and donor cells in MLN and spleen were analyzed by flow cytometry 2 d after immunization with OVA or OVA plus pI:C. Results are representative of two to three performed experiments.

Figure 4.

OT-1 lymphocyte localization to the small intestinal epithelium after immunization with OVA and adjuvant is CCR9 dependent. CCR9^−/− (Ly5.1⁻Ly5.2⁺) and WT (Ly5.1⁺Ly5.2⁺) OT-1 cells were coinjected (3–5 × 10⁶ total cells) i.v. into C57BL/6J-Ly5.1 (Ly5.1⁺Ly5.2⁻) mice. Mice received OVA and LPS i.p. 2 d after cell transfer, and the percentage of CCR9^−/− and WT OT-1 cells among CD8β¹T cells was determined in each organ by flow cytometry analysis 3 d later. (A and B) Representative flow cytometry analysis from one experiment of three performed. (C). The CCR9^−/− to WT OT-1 cell ratio in the MLN and IEL. Organ ratio was determined by dividing the percentage of CCR9^−/− (Ly5.2⁺) OT-1 cells with the percentage of WT (Ly5.1⁺Ly5.2⁺) OT-1 cells in each organ. Results are mean (SEM) of four mice in each group and from one representative experiment of three performed. *P = 0.0286. Dotted line represents the CCR9^−/− to WT OT-1 cell input ratio.