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Kindlin-3 is an integrin-binding focal adhesion adaptor absent in patients with leukocyte and platelet adhesion deficiency syndrome and is critical for firm integrin-dependent leukocyte adhesion. The role of this adaptor in leukocyte diapedesis has never been investigated. In the present study, the functions of Kindlin-3 in this process were investigated in effector T lymphocytes trafficking to various lymphoid and nonlymphoid tissues. In vitro, Kindlin-3-deficient T cells displayed severely impaired lymphocyte function antigen-1-dependent lymphocyte adhesion but partially conserved very late antigen-4 adhesiveness. In vivo, the number of adoptively transferred Kindlin-3-deficient T effectors was dramatically elevated in the circulating pool compared with normal effectors, and the Kindlin-3 mutant effectors failed to enter inflamed skin lesions. The frequency of Kindlin-3-deficient T effectors arrested on vessel walls within inflamed skin-draining lymph nodes was also reduced. Strikingly, however, Kindlin-3-deficient effector T cells accumulated inside these vessels at significantly higher numbers than their wild-type lymphocyte counterparts and successfully extravasated into inflamed lymph nodes. Nevertheless, on entering these organs, the interstitial motility of these lymphocytes was impaired. This is the first in vivo demonstration that Kindlin-3-stabilized integrin adhesions, although essential for lymphocyte arrest on blood vessels and interstitial motility, are not obligatory for leukocyte diapedesis.

The bone marrow (BM) hosts memory lymphocytes and supports secondary immune responses against blood-borne antigens, but it is unsettled whether primary responses occur there and which cells present the antigen. We used 2-photon microscopy in the BM of live mice to study these questions. Naïve CD8(+) T cells crawled rapidly at steady state but arrested immediately upon sensing antigenic peptides. Following infusion of soluble protein, various cell types were imaged ingesting the antigen, while antigen-specific T cells decelerated, clustered, upregulated CD69, and were observed dividing in situ to yield effector cells. Unlike in the spleen, T-cell responses persisted when BM-resident dendritic cells (DCs) were ablated but failed when all phagocytic cells were depleted. Potential antigen-presenting cells included monocytes and macrophages but not B cells. Collectively, our results suggest that the BM supports crosspresentation of blood-borne antigens similar to the spleen; uniquely, alongside DCs, other myeloid cells participate in crosspresentation.

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donorderived central memory CD8(+) T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti-third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

The CX3C chemokine family is composed of only one member, CX3CL1, also known as fractalkine, which in mice is the sole ligand of the G protein-coupled, 7-transmembrane receptor CX3CR1. Unlike classic small peptide chemokines, CX3CL1 is synthesized as a membrane-anchored protein that can promote integrin-independent adhesion. Subsequent cleavage by metalloproteases, either constitutive or induced, can generate shed CX3CL1 entities that potentially have chemoattractive activity. To study the CX3C interface in tissues of live animals, we generated transgenic mice (CX3CL1cherry:CX3CR1gfp), which express red and green fluorescent reporter genes under the respective control of the CX3CL1 and CX3CR1 promoters. Furthermore, we performed a structure/function analysis to differentiate the in vivo functions of membrane-tethered versus shed CX3CL1 moieties by comparing their respective ability to correct established defects in macrophage function and leukocyte survival in CX3CL1-deficient mice. Specifically, expression of CX3CL1(105Δ), an obligatory soluble CX3CL1 isoform, reconstituted the formation of transepithelial dendrites by intestinal macrophages but did not rescue circulating Ly6Clo CX3CR1hi blood monocytes in CX3CR1gfp/gfp mice. Instead, monocyte survival required the full-length membrane-anchored CX3CL1, suggesting differential activities of tethered and shed CX3CL1 entities.