Experimental studies had previously shown that the CD31 inhibitory pathway mediated by the ITIMs is engaged during T-cell responses elicited by TCR stimulation (7
). We have recently shown that the lack of T-cell CD31-signaling favors auto-immune responses in vitro
and in vivo
) supporting a T-cell regulatory role for this transmembrane ITIM-bearing receptor (1
). In this study we show that TCR stimulation drives the cleavage and shedding of the extracellular T-cell CD31 comprising Ig-like domains 1 to 5. We also show that the immunosuppressive CD31 peptide aa 551–574 (26
) is highly homophilic and probably acts by homo-oligomerizing with the truncated CD31 remaining after its cleavage/shedding.
Although CD31 is known to interact with heterophilic ligands on blood and vascular cells (29
), the original feature of CD31 signaling resides in its trans- and cis-homophilic nature. According to the model proposed by Newton et al. (33
), a low-affinity trans-homophilic engagement of the distal domains is necessary to drive the cis-homodimerization of the membrane-proximal portion of the molecule. This, in turn, would be the critical step for triggering the phosphorylation of the ITIMs, and the recruitment and activation of SH2-containing phosphatases. This hypothesis is supported by the fact that cross-linking of the CD31 molecule is necessary to induce the phosphorylation of ITIMs (34
) and inhibition of antigen-driven T-cell activation (7
CD31 expression is constitutive but peripheral blood T-cells of the memory/activated phenotype lack this molecule at their surface (11
). We demonstrate here that the assumption that CD31 molecules are absent on activated T-lymphocytes is based on incomplete information. Indeed, the observed loss is due to a cleavage and shedding of the extracellular CD31 comprising domains 1–5. Since the membrane-proximal 6th
domain remains anchored to all peripheral blood T-lymphocytes, we propose to use the term CD31shed
rather that CD31 “negative” cells for the designation of those lymphocytes that are not detected by monoclonal antibodies directed towards the distal CD31 Ig-like domains. CD31 shedding occurs rapidly upon T-cell activation and leads to an accumulation of the truncated molecule in the supernatant. The same truncated form of CD31 is also specifically detected in human plasma and could serve as a biomarker of pathologic T-cell activation.
We propose that the broad overlap of values between patients and controls in the previous studies concerning the predictive value of soluble CD31 levels (36
) is due to the fact that i) circulating CD31 represents a mixture of the transmembraneless and the truncated form and ii) it was not possible, until now, to discriminate between the two soluble forms of CD31.
In a therapeutic perspective, it is therefore necessary to find alternative strategies to rescue CD31-mediated T-cell regulation in situations in which pathogenic T-cells have already undergone activation and hence CD31 shedding.
Previously, it had been documented that the presence of antibodies targeting the 23 juxta-membrane amino acid sequence of CD31 (LYP21) inhibited the mixed lymphocyte reaction (MLR) in a specific and dose-dependent manner (26
). Intriguingly, a synthetic 23-mer peptide corresponding to the epitope of the LYP21 antibody (aa 551–574) equally inhibited mixed lymphocyte reactions, which showed dispersed small aggregates of cells, rather than the single large aggregate observed in control mixed lymphocyte reactions (26
). Thus it appears that the CD31 peptide is able to reproduce the physiologic active detachment signal which is mutually driven in live leukocytes by CD31-CD31 interactions (6
). However, stimulated T-cells typically lose CD31 at their surface (13
) and Zehnder et al. demonstrated that the peptide was effective also on CD31 negative enriched T cells (26
). As a consequence, the mechanism involved in the immunosuppressive effect exerted by the CD31 peptide 551–574 was left unresolved.
The present study demonstrates that the extracellular portion of CD31 recognized by MBC 78.2/PECAM-1.2 antibody [domain 6 (23
)], which is located N-terminal of the aa 551–574 (26
), remains expressed on CD31shed
T cells. We have also documented that the CD31 551–574 immunosuppressive peptide is highly homophilic. We therefore hypothesized that it can homo-oligomerize with the truncated CD31 on CD31shed
cells and thus acts by mimicking the second step of the CD31 homophilic engagement described by Newton et al (33
): the cis-homo-dimerization of the molecule. Indeed, the analysis of the CD31 domain 6 distribution showed that the peptide clusters localize in between CD31 domain 6 clusters. We thus postulate that the peptide rescues the CD31-ITIM inhibitory signaling in CD31shed
T-cells by “bridging” the distant truncated CD31 molecules over the cell surface. This hypothesis is supported by the fact that either antibody-mediated CD31 domain 6 crosslinking or treatment with the CD31 551–574 peptide durably enhanced the phosphorylation of CD31 ITIMs and of SHP2 of T-cells and inhibited their TCR-induced activation and proliferation in vitro
. Our data cannot rule out that the peptide clusters may exert other and as yet unknown effects in T-cells. Of note, we also show that the CD31 peptide inhibits antigen-induced T-cell responses in vivo
(in the delayed type hypersensitivity model) pointing to a potential immunosuppressive therapeutic effect for this peptide in chronic inflammatory diseases.
In conclusion, we demonstrate that, upon cell activation, the loss of T-cell CD31 is due to its cleavage and shedding of the truncated portion of its ectodomain into biological fluids. CD31 shedding results in the loss of its inhibitory function, as the necessary trans-homophilic engagement of the molecule cannot be established by CD31shed
cells because they lack the distal Ig-like domain 1. However, cis-homo-oligomerization, which is induced by the trans-homophilic engagement of the molecule, is the key event in CD31 inhibitory signal transduction. It appears therefore possible to use a homophilic peptide or an appropriate peptidomimetic compound to mimic the cis-homo-oligomerization step and rescue the lost physiological immunoregulatory function of CD31 on activated T-cells. Such a therapeutic approach may be useful for treating debilitating chronic immuno-inflammatory diseases in which CD31-signaling is suspected to play an important protective role, such as in rheumatoid arthritis (9
), multiple sclerosis (8
), inflammatory liver disease (41
) and atherothrombosis (24