A critical role for CD40-CD40 ligand interactions in amplification of the mucosal CD8 T cell response

Abstract

The role of CD40 ligand (CD40L) in CD8 T cell activation was assessed by tracking antigen-specific T cells in vivo using both adoptive transfer of T cell receptor transgenic T cells and major histocompatibility complex (MHC) class I tetramers. Soluble antigen immunization induced entry of CD8 cells into the intestinal mucosa and cytotoxic T lymphocyte (CTL) differentiation, whereas CD8 cells in secondary lymphoid tissue proliferated but were not cytolytic. Immunization concurrent with CD40L blockade or in the absence of CD40 demonstrated that accumulation of CD8 T cells in the mucosa was CD40L dependent. Furthermore, activation was mediated through CD40L expressed by the CD8 cells, since inhibition by anti-CD40L monoclonal antibodies occurred after adoptive transfer to CD40L-deficient mice. However, mucosal CD8 T cells in normal and CD40(-/-) mice were equivalent killers, indicating that CD40L was not required for CTL differentiation. Appearance of virus-specific mucosal, but not splenic, CD8 cells also relied heavily on CD40-CD40L interactions. The mucosal CTL response of transferred CD8 T cells was MHC class II and interleukin 12 independent. The results established a novel pathway of direct CD40L-mediated CD8 T cell activation.

Figure 1

CD40L blockade inhibits mucosal CD8 T cell expansion. B6-Ly5.1 OT-I–RAG^−/− cells (2.5 × 10⁶) were transferred to B6-Ly5.2 mice, and 2 d later mice were immunized with 5 mg OVA by intraperitoneal injection, or were not immunized (NO ANTIGEN). 1 d before immunization and continuing each day, mice were treated with 200 μg of MR1 (αCD40L) or control hamster Ig. 3 d after immunization, lymphocytes from the indicated tissues were analyzed by flow cytometry for the presence of donor cells by virtue of Ly5.1 and CD8 expression.

Figure 2

Mucosa-specific inhibition of CD8 T cell expansion by CD40L blockade. 4 × 10⁶ naive OT-I–RAG^−/− cells (Ly5.1) were labeled with CFSE and transferred to normal mice (Ly5.2). Mice were treated daily with αCD40L or control antibody starting at 1 d before immunization. 2 d after transfer mice were immunized with 5 mg OVA, and 3 d later PLN and MLN cells (A) and LP cells and IEL (B) were isolated and analyzed by flow cytometry. CFSE staining of the gated donor cells is shown (right), with open histograms representing αCD40L-treated mice and filled histograms representing control antibody-treated mice. The asterisked histogram indicates CFSE-labeled OT-I cells from the PLNs of naive mice.

Figure 2

Mucosa-specific inhibition of CD8 T cell expansion by CD40L blockade. 4 × 10⁶ naive OT-I–RAG^−/− cells (Ly5.1) were labeled with CFSE and transferred to normal mice (Ly5.2). Mice were treated daily with αCD40L or control antibody starting at 1 d before immunization. 2 d after transfer mice were immunized with 5 mg OVA, and 3 d later PLN and MLN cells (A) and LP cells and IEL (B) were isolated and analyzed by flow cytometry. CFSE staining of the gated donor cells is shown (right), with open histograms representing αCD40L-treated mice and filled histograms representing control antibody-treated mice. The asterisked histogram indicates CFSE-labeled OT-I cells from the PLNs of naive mice.

Figure 3

Direct inhibition of T cell expansion by CD8 T cell CD40L blockade. 2.5 × 10⁶ naive OT-I–RAG^−/− cells (Ly5.2) were transferred to Ly5.1 CD40L^−/− mice. Mice were treated daily with αCD40L or control antibody starting at 1 d before immunization. 2 d after transfer mice were immunized with 5 mg OVA, and 3 d later MLN and LP cells were isolated and analyzed by flow cytometry for the presence of donor cells by analysis of Ly5.2 and CD8 expression. In unimmunized mice, OT-I cells comprised 0.4% of MLN cells and were undetectable in LP cells (data not shown).

Figure 4

Optimal proliferation of activated mucosal CD8 T cells requires CD40. 4 × 10⁶ naive OT-I–RAG^−/− cells (Ly5.2) were transferred to C57BL/6J or B6-CD40^−/− mice, and 2 d later the mice were immunized with 5 mg OVA intraperitoneally. 4 d later, lymphocytes were isolated from the indicated sites and analyzed for the presence of donor CD8 T cells by flow cytometry.

Figure 5

Amplification of the mucosal antiviral CD8 T cell response requires CD40. B6 or CD40^−/− mice were infected by intravenous injection of 1 × 10⁶ PFU of VSV. 6 d later, spleen and LP cells were isolated and stained for three-color flow cytometry with allophycocyanin-labeled H-2K^b–N peptide tetramers (N-tet-APC), anti-CD8–PE, and anti-CD11a–FITC. CD8⁺ cells were positively gated and then analyzed for tetramer and CD11a staining. Negative control staining of cells from infected mice was performed with H-2K^b–OVA peptide tetramers, and no staining was observed (data not shown).

Figure 6

Mucosa-specific induction of CD8 lytic activity does not require CD40. Naive OT-I–RAG^−/− cells (Ly5.2) were transferred to C57BL/6J or B6-CD40^−/− mice, and 2 d later the mice were immunized intraperitoneally with 5 mg OVA. 3 d later, lymphocytes were isolated and tested for cytolytic activity against SIINFEKL-coated (filled symbols) or untreated (open symbols) ⁵¹Cr-labeled EL4 target cells. The effector to target ratios shown indicate actual percentages of OT-I cells based on flow cytometric data. •, ○: B6 IELs; ▪, □: CD40^−/− IELs; ▾, ▿: B6 spleen cells. Spontaneous ⁵¹Cr release was <10%.

Figure 7

Mucosal induction of CD8 T cell lytic activity occurs independently of MHC class II and IL-12. Naive OT-I–RAG^−/− cells (4 × 10⁶; Ly5.2) were transferred to C57BL/6J, or B6–Aβ^b−/−, or B6–IL-12^−/− mice, and 2 d later the mice were immunized intraperitoneally with 5 mg OVA. 3 d later IELs were isolated and tested for cytolytic activity against SIINFEKL-coated ⁵¹Cr-labeled EL4 target cells. The effector to target ratios shown indicate actual percentages of OT-I cells based on flow cytometric data. Host mice were (A) ▪, B6; ▾, MHC class II^−/−; (B) •, B6; ♦, IL-12^−/−. Specific lysis in the absence of peptide was <10%. Spontaneous ⁵¹Cr release was <10%.