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. 2014 May;7(3):615-24.
doi: 10.1038/mi.2013.80. Epub 2013 Oct 9.

Characterizing CEACAM5 interaction with CD8α and CD1d in intestinal homeostasis

G Roda  1 , X Jianyu  1 , M S Park  1 , L DeMarte  2 , Z Hovhannisyan  1 , R Couri  1 , C P Stanners  2 , G Yeretssian  1 , L Mayer  1
Affiliations

Characterizing CEACAM5 interaction with CD8α and CD1d in intestinal homeostasis

G Roda et al. Mucosal Immunol. 2014 May.

Abstract

Normal intestinal epithelial cells (IECs) could act as non-professional antigen-presenting cells, selectively activating CD8(+)-suppressor T cells. An epithelial cell surface glycoprotein, gp180, recognized by monoclonal antibodies B9 and L12 was determined to be critical in this process. Purification and sequence analysis of mAb B9 reactive material revealed amino-acid sequence homology with CEACAM5. We demonstrate that CEACAM5 has properties attributed to gp180, such as CD8α binding and activation of CD8-associated Lck. CEACAM5 is the only CEACAM member interacting with CD1d through the B3 domain. Its N domain (recognized by B9) is required for CD8α binding. Removal of the N-domain glycosylated residues reduces B9 recognition, CD8α binding affinity, and activation of LcK. Therefore, conformational changes in CEACAM5 glycosylation site are critical for its interaction with CD8α. CEACAM5-activated CD8(+) T cells acquire the ability to suppress the proliferation of CD4(+) T cells in vitro in the presence of interleukin (IL)-15 or IL-7. We provide new insights into the role of CEACAM5 and define its specific immunoregulatory properties among the CEACAMs expressed on IECs. We suggest that unique set of interactions between CEACAM5, CD1d, and CD8 render CD1d more class I-like molecule, facilitating antigen presentation and activation of CD8(+)-suppressor regulatory T cells.

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Conflict of interest statement

DISCLOSURE

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. CEACAM5 share homology with gp180 and is recognized by B9 mAb
(a) Amino-terminal sequence of the specific target protein of the mAb B9, gp180 and CEACAM5. The first 25 amino acids showed 100% sequence homology with CEACAM5. (b) Immunoblotting for CEACAM5 in lysates obtained from HT29, as well as FO-1 and 293T cells transfected with CEACAM5. B9 and Col-1 monoclonal antibodies were used for Western blotting. Non-transfected FO-1 and 293T cells were used as negative control. Data are representative of three independent experiments.
Figure 2
Figure 2. B9 recognizes different CEACAMs expressed on IECs but specifically binds to a unique region in the N-domain of CEACAM5
(a) Cytofluorimetric profiles of CHO cells transfected with wild type CEACAM5, a N42RQII deletion mutant, and point mutants K35A, and N70,81A and double-stained with mAb T84.66 and mAb B9. B9 reactivity was reduced with cells expressing the deletion mutant and the sugarless mutant of CEACAM5. (b) Cyto-fluorimetric profiles of CHO cells expressing CEACAM5 or CEACAM1-4L, CEACAM6 and CEACAM8, labeled with B9 or B18 mAbs. B9 can recognize all four CEACAM members. Data are representative of three independent experiments.
Figure 3
Figure 3. CEACAM5, but not CEACAM1 or CEACAM6 binds to CD1d
293T cells were co-transfected with pcDNA3.1-IRES-CD1d alone or together with CEACAM1, CEACAM5 or CEACAM6. The cell lysate was co-immunoprecipitated with mouse IgG2b anti-CD1d (D5), or with mouse IgG1 anti-gp180 (B9) antibodies. Western blots were performed with Col-1 and D5 mAbs. Data are representative of three independent experiments.
Figure 4
Figure 4. The B3 domain of CEACAM5 is involved in its interaction with CD1d
293T cells were transfected with expression vectors pCDNA3.1-IRES expressing CD1d and wild-type or truncated CEACAM5. The cell lysate of the transfectants was immunoprecipitated with mouse IgG2b anti-CD1d (D5). Western blot was then performed with Col-1 and D5 mAbs. (a) Schematic rapresantation of deletions of the N-domain, A2 domain and C-terminal truncations (b) WT and CEACAM5 with the deletion of N-domain (N) co-immunoprecipitate with CD1d. WT and CEACAM5 with the deletion of A2-domain (A2) co-immunoprecipitate with CD1d. (c) CEACAM5 with the sequential C-terminal truncations of each domain fails to co-immunoprecipitate with CD1d. CEACAM5 with the deletion of fragment 1 of B3-domain co-immunoprecipitates with CD1d. With further deletion of the B3 domain, CEACAM5 fails to co-immunoprecipitate with CD1d. Data are representative of three independent experiments.
Figure 5
Figure 5. CEACAM5 binds CD8α and induces phosphorlylation of CD8-associated LcK
(a) 3G4 and 3G8 cells were incubated with PIPLC supernatants from CEACAM5 transfectants, vector control 293 T cells, or (b) IECs cell line T84. FO-1 cells were used as negative control. Absorbed or unabsorbed supernatants were subjected for immunoblotting using Col-1 or B9 mAbs. 3G8 but not 3G4 were capable of absorbing CEACAM5. Data are representative of three independent experiments. (c) 3G cells were incubated with purified CEACAM5 for different time points and subjected to immunoblotting for anti-phosphoLcK mAb. At 15′ CEACAM5 induce phosphorylation of LcK. Data are representative of three independent experiments. (d) Flow cytometry analysis of p-Lck staining in human peripheral CD8+ T cells and CD4+ T cells stimulated with Sup1, mAb CD8 and CEACAM5 purified peptide for different time points. H2O2 represents the positive control. Data are representative of five independent experiments.
Figure 6
Figure 6. Removal of N-domain sugar bridge reduces CEACAM5-CD8α binding affinity
(a) Schematic representation of CEACAM5 N-domain mutants. (b) Binding of soluble wild type CEACAM5 (WT) to immobilized CD8α peptide and (c) of wild type CEACAM5 to immobilized recombinant CD4 peptide. (d) KD values of binding of wild Type CEACAM5, N42RQII, K35A and N70,81A to immobilized CD8α peptide.
Figure 7
Figure 7. Suppressor activity of CEACAM5-activated CD8+ T cells
Proliferation of CFSE labeled CD4+ T cells in the presence of anti-CD3/CD28 beads, CD8+ T cells, anti-CD3/CD28 stimulated CD8+ T cells, CEACAM5 +/− IL15 stimulated CD8+ T cells, and OKT8 +/− IL15 stimulated CD8+ T cells. Data are representative of four independent experiments.
Figure 8
Figure 8. Interactions between CEACAM5/CD1d and CEACAM5/CD8α
Intestinal epithelial cells express CEACAM5 and CD1d on their surface. CEACAM5 (gp180) interacts with CD1d through the B3 domain. Fragment 1 of the B3 domain is not required for its interaction with CEACAM5. The sugar bridge in the N domain of CEACAM5 is crucial for it’s binding to CD8α and for the activation of CD8-associated LcK. CEACAM5-activated CD8+ T cells acquire suppressive functions and reduce the proliferation of CD4+ T cells.
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