CEACAM1-S: The Virtues of Alternative Splicing in Gut Immunity

Abstract

Immunoglobulin A (IgA) is the main intestinal antibody. In this issue of Immunity, Chen et al. (2012) show that intestinal T cells enhance protective IgA responses by expressing a short isoform of the CEACAM1 protein.

The intestine is home to trillions of commensal microbes that confer many metabolic capabilities to the host and fill a niche that would otherwise be accessible to pathogens (Sansonetti, 2004). In spite of expressing a wealth of microbial sensors, intestinal tissues peacefully coexist with commensals without inducing inflammation. This homeostatic balance involves a continuous dialog between microbes and intestinal immune cells, which leads to the generation of a dynamic state of hyporesponsiveness against commensals and active readiness against pathogens. A key mediator of intestinal homeostasis is immunoglobulin A (IgA), an antibody isotype produced by B cells through class switching (Cerutti, 2008). IgA confers a noninflammatory tone to the intestine by shaping the composition of commensals (Wei et al., 2011). IgA production and release onto the mucosal surface require a close interplay of B cells with T follicular helper (Tfh) cells, T regulatory (Treg) cells, dendritic cells, follicular dendritic cells, and epithelial cells (Cerutti, 2008; Suzuki et al., 2010; Tsuji et al., 2009). In spite of recent advances, the mechanisms regulating intestinal IgA responses remain unclear.

In this issue of Immunity,Chen et al. (2012) show that intestinal T cells control commensal bacteria and protect against pathogens by inducing IgA through a pathway involving preferential expression of a short isoform of carcinoembryonic antigen cell adhesion molecule like 1 (CEACAM1), a transmembrane protein mediating intercellular interactions and intracellular signaling (Gray-Owen and Blumberg, 2006). CEACAM1 is expressed on the surface of various hematopoietic and nonhematopoietic cells, except natural killer and T cells. However, T cells upregulate CEACAM1 expression in response to cytokines or engagement of T cell antigen receptor (TCR) by antigen. CEACAM1 exists in multiple isoforms, each composed of an extracellular domain with an amino-terminal Ig variable-like motif mediating homophilic cell-to-cell interaction and a carboxy-terminal cytoplasmic domain mediating signal transduction. Alternative splicing generates CEACAM1 molecules that contain either a long (L) or a short (S) cytoplasmic domain. The L domain includes two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that negatively regulate signaling from various activating receptors, including the TCR. The S domain lacks ITIM motifs, but includes a calmodulin-binding sequence that transduces stimulating signals, at least in vitro.

In addition to mediating homophilic cell-to-cell interactions, the extracellular domain of CEACAM1 recognizes bacteria by binding conserved microbial signatures (Gray-Owen and Blumberg, 2006). This property promptedChen et al. (2012) to explore the function of CEACAM-1 in the intestine (Figure 1), an environment heavily colonized by bacteria. Compared to peripheral T cells, intestinal T cells expressed more CEACAM1 and displayed a CEACAM1-S/CEACAM1-L ratio unexpectedly skewed toward CEACAM1-S. Adoptive transfer experiments indicated that preferential CEACAM1-S expression was not irreversibly imprinted in intestinal T cells but rather required reversible induction of the CEACAM1 messenger RNA (mRNA) splicing machinery by cues originating from the local micro-environment. Induction of CEACAM1-S followed a microbe-independent pathway, because CEACAM1-S expression remained elevated in intestinal T cells from mice that either lacked the intestinal microbiota or had defective signaling via microbial sensors such as Toll-like receptor (TLR) molecules and nucleotide oligomerization domain (NOD) receptors.

Figure 1. Alternative Splicing of CEACAM1 mRNA Generates a CEACAM1-S Isoform That Is Required for Gut Humoral Immunity.

Microbe-independent signals generated by the intestinal microenvironment induce alternative splicing of CEACAM1-encoding mRNA to generate a CEACAM1-S protein with a short (S) cytoplasmic domain in T cells. Signals from CEACAM1-S stimulate the activation of T effector memoy (Tem) cells, T central memory (Tcm) cells, Tfh cells, and Treg cells, which in turn promote induction of the noninflammatory antibody isotype IgA. This induction involves the differentiation of B cells expressing IgM (purple surface antibody) into plasma cells expressing IgA (intracellular red antibody). Subsequent translocation of secreted IgA across epithelial cells generates secretory IgA antibodies that enter the intestinal lumen to control the composition of the commensal microbiota and prevent tissue invasion by pathogens.

Chen et al. (2012) elegantly demonstrated the mucosal functions of CEACAM1-S by overexpressing CEACAM1-S or CEACAM1-L in T cells from either wild-type (WT) or CEACAM1-deficient mice. In spite of showing elevated T cell apoptosis, WT mice expressing a CEACAM1-S transgene (4STg) had fewer naive T cells but more central memory T cells and effector memory T cells. These mice also showed an increased production of cytokines typically associated with T helper type-1 (Th1), Th2, Th17, and Treg cells, including interleukin-2 (IL-2), IL-4, IL-10, IL-17, interferon-γ (IFN-γ), and transforming growth factorβ1 (TGF-β1). Furthermore, T cells from 4STg mice showed increased proliferation, cytokine production, and activation-induced cell death in response to TCR stimulation. A similar phenotype was observed in Ceacam1^−/− mice expressing a CEACAM1-S transgene (Ceacam1^−/−-4STg), whereas gene-targeted mice expressing a CEACAM1-L transgene (Ceacam1^−/−-4LTg) showed blunted signaling via the TCR. These findings indicate that CEACAM1-S functions as an autonomous signal transducing receptor that regulates the function of T cells independently of its ability to oppose the inhibitory function of CEACAM1-L.

The findings by Chen et al. (2012) indicate that CEACAM1-S promotes intestinal homeostasis and immunity by enhancing the activation of local effector T cells. But how does CEACAM1-S signal to T cells? Compared to intestinal T cells from WT mice, intestinal T cells from 4STg mice showed a transcriptional profile consistent with increased signaling via nuclear factor of activated T cells (NF-AT), a transcriptional activator that undergoes nuclear import after being dephosphorylated by a calmodulin-dependent phosphatase known as calcineurin. NF-AT nuclear translocation was increased in intestinal T cells from both 4STg and CEACAM1^−/−-4STg mice, suggesting that CEACAM1-S amplifies signals from TCR or other NF-AT-inducing receptors. Given that NF-AT proteins form a family with multiple immune and metabolic functions, it would be important to elucidate whether CEACAM1-S induces specific NF-AT activation patterns in intestinal T cells and whether such patterns are regulated by local signals, including microbial signals. Additional studies should establish whether the induction of the splicing machinery governing CEACAM1-S expression requires specific members of the NF-AT family.

Remarkably,Chen et al. (2012) found that CEACAM1-S played an important role in steady-state intestinal IgA responses to commensals. Compared to WT and CEACAM1-deficient mice, 4STg and Ceacam1^−/−-4STg mice showed larger intestinal Peyer’s patches that contained an increased proportion of IgA-secreting B220⁻IgA⁺ plasmablasts but an equivalent proportion of IgA class-switched B220⁺IgA⁺ B cells. Tg mice also had more IgA-secreting plasmablasts in the lamina propria and increased secretory IgA in the intestinal lumen. The IgA-enhancing activity of CEACAM1-S was associated with an increased expression of programmed cell death-1 (PD-1), an NF-AT-inducible inhibitory receptor typically expressed by Tfh cells (Crotty, 2011). These effector T cells are specifically required for the activation, expansion, survival, and selection of germinal center B cells expressing class-switched antibodies with high affinity for antigen, including IgA (Crotty, 2011). Tfh cells were increased in Peyer’s patches from 4STg mice but decreased in Peyer’s patches from Ceacam1^−/− mice, suggesting that CEACAM1-S augments intestinal IgA responses by facilitating the cognate interaction of follicular B cells with T_FH cells. Homophilic engagement of CEACAM1-S on T_FH cells by CEACAM1-S on B cells may increase the expression of CD40 ligand (CD40L), an NF-AT-inducible T cell molecule that cooperates with the cytokine TGF-β1 to induce IgA class switching and production in B cells (Cerutti, 2008). In this regard, CEACAM1-S-expressing T cells upregulated the expression of both CD40L and latency-associated peptide (LAP), a cell-bound precursor of the cytokine TGF-β1 (Suzuki et al., 2010).

LAP was expressed by an unusual subset of CD4⁺ Treg cells that lacked the CD25 subunit of the interleukin-2 (IL-2) receptor and yet suppressed colitis via TGF-β as conventional CD25-expressing Treg cells do. Remarkably, CD4⁺CD25⁻LAP⁺ Treg cells preferentially expressed CEACAM1-S and required CEACAM1-S for their induction by an anti-CD3 antibody administered through the oral route. In 4STg and CEACAM1^−/−-4STg mice, CD4⁺CD25⁻LAP⁺ Treg cells were not only expanded, but also exerted an enhanced suppressive activity on the inflammatory activity of IFN-γ-producing Th1 cells. In addition to suppressing inflammation, Treg cells induce noninflammatory IgA production by triggering IgA class switching in B cells via CD40L, TGF-β1, and IL-21 (Tsuji et al., 2009). However, it remains unclear whether also CEACAM1-S-expressing CD4⁺CD25⁻LAP⁺ Treg cells have this effect, because WT and S4Tg mice have comparable numbers of B220⁺IgA⁺ B cells in Peyer’s patches. Furthermore, the presence of CD4⁺CD25⁻LAP⁺ Treg cells in Peyer’s patches remains untested.

CEACAM1-S may optimize the quality of intestinal IgA responses through its ability to enhance the expression of the inhibitory receptor PD-1 on Tfh cells. As shown by recent studies (Kawamoto et al., 2012), increased PD-1 signaling may restrain the clonal expansion and activation of T_FH cells, thereby causing a more stringent selection of high-affinity IgA-expressing B cells in Peyer’s patches. It remains to be tested whether CEACAM1-S also modulates the expression of interleukin-21 (IL-21), a T_FH cell-associated cytokine that cooperates with CD40L and TGF-β1 to induce IgA class switching and production (Cerutti, 2008). CEACAM1-S-mediated regulation of key T_FH cell-associated IgA-inducing molecules could explain the alterations of the gut microbiota thatChen et al. (2012) observed in 4STg and CEACAM1-deficient mice. In addition to facilitating homeostatic IgA responses to noninvasive commensals, CEACAM1-S enhanced IgA responses to invasive pathogens such as Listeria monocytogenes. Indeed, compared to WT and Ceacam1^−/− mice, 4STg mice produced more abundant secretory IgA to Listeria monocytogenes, which correlated with a decreased invasion of intestinal and systemic tissues by this pathogen.

More than 75% of human genes undergo alternative splicing, with a bias toward genes of the immune system, including T cells (Lynch, 2004). This process generates multiple mRNAs that lead to the expression of multiple isoforms of the same protein. The findings byChen et al. (2012) show that intestinal T cells with effector and regulatory functions take advantage of alternative splicing as a means to generate homeostasis and immunity via CEACAM1-S. Future studies should identify the intestinal signals required for CEACAM-S expression by T cells, precisely define the intestinal ligands of CEACAM1-S, and elucidate the mechanisms whereby CEACAM1-S regulates intestinal T cell activation and IgA production.