Our previous studies have demonstrated that Gαi3 antagonizes Gαi2 in CXCR3-mediated signaling by interacting with the receptor without activation and dissociation [18
]. This translated into accelerated migration of T cells stimulated with all three CXCR3 agonists in the absence of Gαi3. In contrast, deletion of Gαi2 ablated T cell migration towards these agonists [18
]. A severer GVHD in immunocompromised mice adoptively transferred with Gαi3−/−
T cells than those mice transferred with WT T cells, but lacking such a disease in mice receiving Gαi2−/−
T cells, argues strongly that Gαi3 inhibits Gαi2-mediated signaling in vivo
and is critical in directing activated Tcells from lymphoid tissues to target organs. Our data showed that activated Gαi3−/−
T cells were capable of migrating to target organs more efficiently than Gαi2−/−
Tcells, which contributes, at least in part, to earlier mortality of the recipients, whereas activated Gαi2−/−
T cells were inadequately trapped in the lymphoid tissues, unable to elicit GVHD. Apart from reciprocal regulation of pathogenic T cell trafficking, lack of either Gαi3 or Gαi2 resulted in more efficient T cell homing to lymphoid tissues than their presence, suggesting similarity of Gαi2 and Gαi3 in directing trafficking of naive T cells. These observations provide compelling evidence that T cell ingress and egress of various tissues at different activation stages are coordinately controlled by a specific array of chemokines and receptors as well as different heterotrimeric G proteins at multiple levels. Gαi2- and Gαi3-KO mice provide a unique system to address the effects of multiple chemokine receptors on spatial and temporal regulation of T cell trafficking in vivo
during the disease development.
A body of evidence has shown increased production of chemokines such as CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, MIP-1α and β, MCP-1 and MCP-2, and so on, at varying stages during GVHD development [7
]. This study details the effects of Gαi2 and Gαi3 on the activities of the corresponding receptors and addresses how they are involved in eliciting GVHD at varying stages. Among them, the chemokine receptor CXCR3 is a key receptor in guiding donor T cells to inflammatory areas and T cells lacking CXCR3 fail to elicit GVHD or reject allograft [28
]. Our current study extends these observations by showing that more efficient migration of activated T cells exacerbates GVHD in the absence of Gαi3, concomitant with enhanced activity of the CXCR3 receptor. On the other hand, abrogation of CXCR3 signaling by Gαi2 deletion hampers T effector cell migration, preventing the disease. The lack of GVHD in mice receiving Gαi2−/−
T cells may be also ascribed partially to aberrant chemotaxis of these T cells to CCL5, an inflammatory chemokine being essential in attracting and sustaining T effector cells to the liver [31
]. We detected little migratory responses over background even in ConA-activated T cells when stimulated with chemokines MIP-1β (CCL4), MIP-1α (CCL3), MCP-1 and MCP-2 at concentrations ranging from 10 to 200 ng/mL (data not shown) [7
]. The role of these chemokines in the reciprocal effects of Gαi2- or Gαi3-deficency on the onset of GVHD is not known at present. In addition, although lack of Gαi2 or Gαi3 has little impact on differentiation of T regulatory cells and their function (our unpublished data) [26
], its influence over infiltration of T regulatory cells versus
effector cells may differ, which can also contribute to the described disparity of the disease induced by Gαi2- and Gαi3-KO T cells, a possibility that requires further investigation.
Our study stresses that migration of not only Tcell effectors but also naive T cells is critically involved in the severity and mortality of GVHD. Increasing numbers of donor Gαi3-deficient T cells and to a lesser extent, Gαi2-deficient T cells were observed in the secondary lymphoid tissues at one day after transfusion, in spite of impeded CXCR4-mediated signaling in these cells. Increased T cell homing to lymphoid tissues probably results from a loss of S1P-mediated inhibition in T cell chemokinesis that facilitates T cell homing elicited by the CCR7 and CXCR4 receptors, with preference of Gαi3−/−
T cells to Gαi2−/−
T cells (). S1P is present at 0.1~1 μM in blood and body fluids and at a decreased level (10~100 nM) in the lymphoid tissues. This elevating S1P concentration gradient directs T cell egress from the lymphoid tissues to blood. Distinguished from egress, the major effect of plasma S1P concentrations on Tcells is to retain them in circulation by dampening their chemotactic responses to various chemokines [27
]. Reduced chemokinesis may be the underlying mechanism whereby a high concentration of S1P dampens chemotaxis induced by CXCR4 and CCR7 ligands. This possibility is supported by the ability of high levels of S1P to inhibit chemotaxis induced by a broad range of chemokines [27
]. Moreover, deletion of either Gαi2 or Gαi3 abolished S1P-mediated suppression in T cell chemokinesis, which was accompanied by increasing Tcell homing mediated by CCR7- and CXCR4 ligands. On the contrary, increased circulating T cells owing to accelerated egress and diminished homing was observed in S1P1
receptor-transgenic mice that over-express the S1P1
receptor in T cells [32
]. These mice are relatively insensitive to T cell-mediated delayed-type hypersensitivity (DTH) responses as compared to that in WT mice.
Despite importance of Gαi2 and Gαi3 in directing of lymphocyte trafficking, gene target deletion of either Gαi2 or Gαi3 gives rise to no gross defect in T cell homing or egress [19
]. Gαi2-deficient mice exhibit impairment in lymph node development in several anatomic locations and in Peyer's patch formation [19
]. The mice also develop inflammatory bowel disease, due at least in part to a lack of Tcell response toTGF-β that results in a Th1-skewed hyperimmune response in the colon [19
]. One unanswered question was that inflammation was not observed in all tissues in the mice, with the notable exception of the colon, although T cells in Gαi2−/−
mice were unresponsive to TGF-β, apparently inconsistent with the systemic inflammation developed in the mice lacking TGF-β or a TGF-β receptor [34
]. Our current study suggests that failure of T cells to reach target organs can make the impaired TGF-β response futile. Indeed, trafficking of pathogenic T cells to the colon seems far less reliant on Gαi2, because of comparable homing of Gαi2-deficeint and WT T cells to the colon.
Ample studies addressing the role of chemokines in GVHD using mice with gene-targeted deletion of individual chemokine receptors or ligands suggest significant redundancy in the chemokine system. In this regard, multiple chemokines can bind to a particular receptor or conversely, multiple receptors can interact with a particular chemokine. The interplay between Gαi2 and Gαi3 proteins introduces another layer of complexity in the control of cell migration velocity, in addition to the directionality that chemokines and their receptors provide [9
]. Gαi2- and Gαi3-KO mouse strains allow us insight into the distinct, overlapping, antagonistic, and additive effects of Gαi2 and Gαi3 on the function of multiple chemokine receptors in vivo
, providing more comprehensive description of cell mobilization during inflammation and immune responses as compared to mice lacking individual chemokines or their receptors.