Novel Targeting to XCR1+ Dendritic Cells Using Allogeneic T Cells for Polytopical Antibody Responses in the Lymph Nodes.
ABSTRACT: Vaccination strategy that induce efficient antibody responses polytopically in most lymph nodes (LNs) against infections has not been established yet. Because donor-specific blood transfusion induces anti-donor class I MHC antibody production in splenectomized rats, we examined the mechanism and significance of this response. Among the donor blood components, T cells were the most efficient immunogens, inducing recipient T cell and B cell proliferative responses not only in the spleen, but also in the peripheral and gut LNs. Donor T cells soon migrated to the splenic T cell area and the LNs, with a temporary significant increase in recipient NK cells. XCR1+ resident dendritic cells (DCs), but not XCR1- DCs, selectively phagocytosed donor class I MHC+ fragments after 1 day. After 1.5 days, both DC subsets formed clusters with recipient CD4+ T cells, which proliferated within these clusters. Inhibition of donor T cell migration or depletion of NK cells by pretreatment with pertussis toxin or anti-asialoGM1 antibody, respectively, significantly suppressed DC phagocytosis and subsequent immune responses. Three allogeneic strains with different NK activities had the same response but with different intensity. Donor T cell proliferation was not required, indicating that the graft vs. host reaction is dispensable. Intravenous transfer of antigen-labeled and mitotic inhibitor-treated allogeneic, but not syngeneic, T cells induced a polytopical antibody response to labeled antigens in the LNs of splenectomized rats. These results demonstrate a novel mechanism of alloresponses polytopically in the secondary lymphoid organs (SLOs) induced by allogeneic T cells. Donor T cells behave as self-migratory antigen ferries to be delivered to resident XCR1+ DCs with negligible commitment of migratory DCs. Allogeneic T cells may be clinically applicable as vaccine vectors for polytopical prophylactic antibody production even in asplenic or hyposplenic individuals.
Project description:Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2-/- ?c-/- mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein ? (SIRP?) is a key modulator of the recipient's innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRP? to recipient CD47 and was modulated by the strength of the SIRP?-CD47 interaction. Therefore, sensing SIRP? polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells.
Project description:Host dendritic cells (DCs) play a critical role in initiating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL), and separation of GVL from GVHD remains a major challenge in the treatment of hematologic malignancies by allogeneic hematopoietic cell transplantation (HCT). Here, we show that preconditioning with anti-CD3 monoclonal antibody before conditioning with total body irradiation (TBI) prevents GVHD but retains GVL in a HCT model of major histocompatibility complex (MHC)-mismatched C57BL/6 donor to BALB/c host. Prevention of GVHD is associated with inhibition of donor T-cell expression of homing and chemokine receptors, and inhibition of GVHD target tissue expression of chemokines. Furthermore, inhibition of donor T-cell expression of gut homing alpha4beta7 and chemokine receptor (CCR)9 by anti-CD3 preconditioning results from a reduction of CD103(+) DCs in draining mesenteric lymph nodes (LNs), which is associated with down-regulation of DC expression of CCR7, a receptor required for tissue DC migration to draining LNs. These results indicate that anti-CD3 preconditioning reduces not only tissue release of chemokines but also prevents tissue DC migration to draining LNs and subsequently reduces the capacity of DCs of draining LNs to imprint donor T-cell tissue tropism. Therefore, modulation of host DCs by anti-CD3 preconditioning before HCT represents a new approach for separating GVL from GVHD.
Project description:Naive CD8(+) T cell priming during tumor development or many primary infections requires cross-presentation by XCR1(+) dendritic cells (DCs). Memory CD8(+) T lymphocytes (mCTLs) harbor a lower activation threshold as compared with naive cells. However, whether their recall responses depend on XCR1(+) DCs is unknown. By using a new mouse model allowing fluorescent tracking and conditional depletion of XCR1(+) DCs, we demonstrate a differential requirement of these cells for mCTL recall during secondary infections by different pathogens. XCR1(+) DCs were instrumental to promote this function upon secondary challenges with Listeria monocytogenes, vesicular stomatitis virus, or Vaccinia virus, but dispensable in the case of mouse cytomegalovirus. We deciphered how XCR1(+) DCs promote mCTL recall upon secondary infections with Listeria. By visualizing for the first time the in vivo choreography of XCR1(+) DCs, NK cells and mCTLs during secondary immune responses, and by neutralizing in vivo candidate molecules, we demonstrate that, very early after infection, mCTLs are activated, and attracted in a CXCR3-dependent manner, by NK cell-boosted, IL-12-, and CXCL9-producing XCR1(+) DCs. Hence, depending on the infectious agent, strong recall of mCTLs during secondary challenges can require cytokine- and chemokine-dependent cross-talk with XCR1(+) DCs and NK cells.
Project description:Natural killer (NK) cells have long been considered as potential agents for adoptive cell therapy for solid cancer patients. Until today most studies utilized autologous NK cells and yielded disappointing results. Here we analyze various modular strategies to employ allogeneic NK cells for adoptive cell transfer, including donor-recipient HLA-C mismatching, selective activation and induction of melanoma-recognizing lysis receptors, and co-administration of antibodies to elicit antibody-dependent cell cytotoxicity (ADCC). We show that NK cell activation and induction of the relevant lysis receptors, as well as co-administration of antibodies yield substantial anti-cancer effects, which are functionally superior to HLA-C mismatching. Combination of the various strategies yielded improved effects. In addition, we developed various clinically-compatible ex vivo expansion protocols that were optimized according to fold expansion, purity and expression of lysis receptors. The main advantages of employing allogeneic NK cells are accessibility, the ability to use a single donor for many patients, combination with various strategies associated with the mechanism of action, e.g. antibodies and specific activation, as well as donor selection according to HLA or CD16 genotypes. This study rationalizes a clinical trial that combines adoptive transfer of highly potent allogeneic NK cells and antibody therapy.
Project description:Direct presentation of foreign MHC molecules expressed by donor-derived dendritic cells (DCs) has generally been considered the dominant pathway of allorecognition in acute transplant rejection. However, recent studies implicate preferential activation of the indirect pathway by host DCs. The respective importance of each pathway and the mechanisms that determine their relative contributions remain to be clearly established. In this study, using two-photon microscopy, we visualized host NK cell interactions with syngeneic and allogeneic DCs within intact lymph nodes of mice. Upon contact with allogeneic DCs, NK cells formed prolonged interactions that led directly to target cell lysis. This rapid elimination limited the ability of allogeneic DCs to stimulate primary and recall T cell responses. To discriminate whether donor or host DCs are principally involved in presenting Ag derived from allografts, we used CD11c-diphtheria toxoid receptor mice to conditionally ablate CD11c(+) DCs and to show that direct presentation by donor DCs is alone insufficient to elicit acute allograft rejection. We thus propose that rapid elimination of allogeneic DCs limits direct Ag presentation and thereby favors the indirect pathway of alloreactivity.
Project description:A functionally responsive natural killer (NK)-cell repertoire requires the acquisition of inhibitory NKG2A and killer immunoglobulin-like receptors (KIR) through pathways that remain undefined. Functional donor NK cells expressing KIRs for non-self class I MHC ligands contribute to a positive outcome after allogeneic hematopoietic stem cell transplantation (alloHSCT) by targeting HLA-matched recipient leukemic cells. Insofar as circulating donor conventional dendritic cells (DC) reconstitute with comparable kinetics with donor NK cells after alloHSCT, we used hyporesponsive KIRnegNKG2Aneg precursor cells to evaluate how specific DC subtypes generate a functionally active NK-cell repertoire. Both monocyte-derived DCs (moDC) and Langerhans-type DCs (LC) induce KIRnegNKG2Aneg precursor cells to express the inhibitory receptors NKG2A and KIR, without requiring cell proliferation. Poly(I:C)-matured moDCs significantly augmented the expression of NKG2A, but not KIR, in an IL12p70-dependent manner. Although all DC-stimulated KIRnegNKG2Aneg cells were able to acquire cytolytic activity against class I MHC-negative targets, the ability to secrete IFN? was restricted to cells that were stimulated by IL12p70-producing, poly(I:C)-matured moDCs. This critical ability of poly(I:C)-matured moDCs to provide IL12p70 to developing KIRnegNKG2Aneg precursors results in a dom4inant, multifunctional, NKG2Apos NK-cell population that is capable of both cytolysis and IFN? production. Poly(I:C)-matured moDCs are, therefore, the most effective conventional DC subtype for generating a functionally competent NK-cell repertoire by an IL12p70-dependent mechanism.
Project description:Modification of allogeneic dendritic cells (DCs) through drug treatment results in DCs with in vitro hallmarks of tolerogenicity. Despite these observations, using murine MHC-mismatched skin and heart transplant models, donor-derived drug-modified DCs not only failed to induce tolerance but also accelerated graft rejection. The latter was inhibited by injecting the recipient with anti-CD8 Ab, which removed both CD8(+) T cells and CD8(+) DCs. The discrepancy between in vitro and in vivo data could be explained, partly, by the presentation of drug-modified donor DC MHC alloantigens by recipient APCs and activation of recipient T cells with indirect allospecificity, leading to the induction of alloantibodies. Furthermore, allogeneic MHC molecules expressed by drug-treated DCs were rapidly processed and presented in peptide form by recipient APCs in vivo within hours of DC injection. Using TCR-transgenic T cells, Ag presentation of injected OVA-pulsed DCs was detectable for ? 3 d, whereas indirect presentation of MHC alloantigen by recipient APCs led to activation of T cells within 14 h and was partially inhibited by reducing the numbers of CD8(+) DCs in vivo. In support of this observation when mice lacking CD8(+) DCs were pretreated with drug-modified DCs prior to transplantation, skin graft rejection kinetics were similar to those in non-DC-treated controls. Of interest, when the same mice were treated with anti-CD40L blockade plus drug-modified DCs, skin graft survival was prolonged, suggesting endogenous DCs were responsible for T cell priming. Altogether, these findings highlight the risks and limitations of negative vaccination using alloantigen-bearing "tolerogenic" DCs.
Project description:The chemokine (C motif) receptor 1 (XCR1) and its ligandXCL1 have been intensively studied in the mouse and human immune systems. Here, we determined the molecular characteristics of cattle XCR1 and XCL1 and their distribution among peripheral blood cells. Cattle XCR1 mRNA expression was mainly restricted to CD26+CADM1+CD205+MHCII+CD11b- cells in blood that were otherwise lineage marker negative (lin-); these represented a subset of classic dendritic cells (DCs), not plasmacytoid DCs. Some of these DCs expressed CD11a, CD44, CD80 and CD86, but they did not express CD4, CD8, CD163 or CD172a. Cattle XCL1 was expressed in quiescent NK cells and in activated CD8+ T cells. Cattle XCR1+ DCs migrated chemotactically in response to mouse, but not to human, XCL1. The distribution characters of cattle XCR1 and XCL1 suggested a vital role in regulation of acquired immune responses and indicated a potential for a DC targeted veterinary vaccine in cattle using XCL1 fused antigens.
Project description:Based on clinical observations, we have previously shown in a murine model that recipient leukocyte infusion (RLI) induces a host-versus-graft reaction in mixed bone marrow chimeras and that rejection of donor cells leads to a specific antitumor response against recipient malignancies. This response is dependent on T cells and IFN-gamma. We investigated the role of NKT cells (NKTs) in this phenomenon. Depletion of recipient NK1.1(+) cells led to loss of an anti-tumor effect induced by RLI in mixed bone marrow chimeras. In recipients specifically lacking host invariant NKT cells (iNKTs), RLI did not induce an antitumor effect, indicating a critical role for recipient iNKTs. Conversely, specific activation of iNKTs enhanced the anti-tumor effect induced by RLI. Following RLI, recipient iNKTs, NK cells, dendritic cells (DCs), and CD8 T cells were activated. CD8 T cells were the major producers of IFN-gamma. Lack of recipient iNKTs resulted in failure of activation of NK cells and DCs by RLI. Our studies demonstrate a central role for iNKTs in promoting RLI-induced anti-tumor effects and suggest that this pathway involved promotion of the activation of recipient NK cells and DCs.
Project description:Natural killer (NK) cells express inhibitory receptors with varied binding affinities to specific major histocompatibility complex class I (MHC-I) haplotypes. NK cells can be classified as licensed or unlicensed based on their ability or inability to bind MHC-I, respectively. The role of donor vs host MHC on their development after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not known. Following reciprocal MHC-disparate allogeneic transplants and during de novo NK-cell recovery, depletion of the licensed and not unlicensed population of NK cells as determined by the licensing patterns of donor MHC-I haplotypes, resulted in significantly increased susceptibility to murine cytomegalovirus (MCMV) infection. A corresponding expansion of the licensed Ly49H(+) NK cells occurred with greater interferon ? production by these cells than unlicensed NK cells in the context of donor MHC-I. Thus, NK licensing behavior to MCMV corresponds to the donor, and not recipient, MHC haplotype after allo-HSCT in mice.