Marian Szczepanik

Critical role for the chemokine receptor CXCR6 in NK cell–mediated antigen-specific memory of haptens and viruses

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell–expressed CXCR6.

Cutaneous Immunization Rapidly Activates Liver Invariant Vα14 NKT Cells Stimulating B-1 B Cells to Initiate T Cell Recruitment for Elicitation of Contact Sensitivity

T cell recruitment to elicit contact sensitivity (CS) requires a CS-initiating process mediated by B-1 cells that produce IgM, which activates complement to promote T cell passage into the tissues. We now show that Vα14i NKT cells induce B-1 cell activation likely by releasing IL-4 early postimmunization. The CS initiation process is absent in Jα18−/− and CD1d−/− NKT cell–deficient mice and is reconstituted by populations enriched for Vα14i NKT cells. Transfers are not effective if cells are derived from IL-4−/− mice. Staining with specific tetramers directly showed that hepatic Vα14i NKT cells increase by 30 min and nearly double by 2 h postimmunization. Transfer of immune B-1 cells also reconstitutes CS responses in NKT cell–deficient mice. The B-1 cells act downstream of the Vα14i NKT cells to restore CS initiation. In addition, IL-4 given systemically to Jα18−/− or CD1d−/− NKT cell–deficient mice reconstitutes elicitation of CS. Further, splenocytes from immune Jα18−/− mice produce less antigen (Ag)-specific IgM antibodies compared with sensitized WT mice. Together these findings indicate that very early after skin immunization Vα14i NKT cells are stimulated to produce IL-4, which activates B-1 cells to produce Ag-specific IgM, subsequently needed to recruit effector T cells for elicitation of CS responses.

B cell-dependent T cell responses: IgM antibodies are required to elicit contact sensitivity.

Contact sensitivity (CS) is a classic example of in vivo T cell immunity in which skin sensitization with reactive hapten leads to immunized T cells, which are then recruited locally to mediate antigen-specific inflammation after subsequent skin challenge. We have previously shown that T cell recruitment in CS is triggered by local activation of complement, which generates C5a that triggers C5a receptors most likely on mast cells. Here, we show that B-1 cell–derived antihapten IgM antibodies generated within 1 day (d) of immunization combine with local challenge antigen to activate complement to recruit the T cells. These findings overturn three widely accepted immune response paradigms by showing that (a) specific IgM antibodies are required to initiate CS, which is a classical model of T cell immunity thought exclusively due to T cells, (b) CS priming induces production of specific IgM antibodies within 1 d, although primary antibody responses typically begin by day 4, and (c) B-1 cells produce the 1-d IgM response to CS priming, although these cells generally are thought to be nonresponsive to antigenic stimulation. Coupled with previous evidence, our findings indicate that the elicitation of CS is initiated by rapidly formed IgM antibodies. The IgM and challenge antigen likely form local complexes that activate complement, generating C5a, leading to local vascular activation to recruit the antigen-primed effector T cells that mediate the CS response.

Comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in NOD-scid/γc−/−, Balb/c-Rag1−/−γc−/−, and C.B-17-scid/bg immunodeficient mice

Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics, and function of developing human immune cells following transfer of CD34(+) hematopoietic stem cell (HSC) preparations originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/gammac(-/-), Balb/c-Rag1(-/-)gammac(-/-), and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/gammac(-/-) and Balb/c-Rag1(-/-)gammac(-/-) harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/gammac(-/-) mice harbored higher levels of human T cells. NOD-scid/gammac(-/-) mice engrafted with HFL CD34(+) HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens and delayed-type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/gammac(-/-) mice that is well suited for future studies toward the development of a fully competent humanized mouse model.

An Hour after Immunization Peritoneal B-1 Cells Are Activated to Migrate to Lymphoid Organs Where within 1 Day They Produce IgM Antibodies That Initiate Elicitation of Contact Sensitivity

Elicitation of contact sensitivity (CS), a classic example of T cell-mediated immunity, requires Ag-specific IgM Abs to trigger an initiation process. This early process leads to local recruitment of CS-effector T cells after secondary Ag challenge. These Abs are produced by the B-1 subset of B cells within 1 day after primary skin immunization. In this study we report the surprising observation that B-1 cells in the peritoneal cavity are activated as early as 1 h after naive mice are painted with a contact-sensitizing Ag on the skin of the trunk and feet to begin the initiation of CS. B-1 cells in the spleen and draining lymph nodes produce the initiating Abs by 1 day after immunization, when we found increased numbers of Ag-specific IgM Ab-producing cells in these tissues by ELISPOT assay. Importantly, we show that contact-activated peritoneal B-1 cells migrate to these lymphoid tissues and then differentiate into Ag-specific IgM Ab-producing cells, resulting in specific CS-initiating IgM Abs in the serum by 1 day. Furthermore, pertussis toxin, which is known to inhibit signaling via G protein-coupled chemokines, inhibited the migration of contact-activated peritoneal B-1 cells to the lymphoid tissues, probably due to BLR-1 (Burkitt lymphoma receptor-1). These findings indicate that within 1 h after contact skin immunization, B-1 cells in the peritoneal cavity are activated to migrate to the lymphoid tissues by chemokine-dependent mechanisms to produce serum Ag-specific IgM Abs within 1 day after immunization, leading to local recruitment of CS-effector T cells.

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity.

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5+, CD19+, CD22+, B220+, Thy1+, and Mac1+, suggesting that they are B-1 B cells. DTH initiation is absent in μMT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.

γδ T Cell Regulation of IFN-γ Production by Central Nervous System-Infiltrating Encephalitogenic T Cells: Correlation with Recovery from Experimental Autoimmune Encephalomyelitis

Interferon-γ has been shown to be important for the resolution of inflammation associated with CNS autoimmunity. Because one of the roles of γδ T cells is the regulation of inflammation, we asked whether γδ T cells were able to regulate CNS inflammation using the autoimmune disease mouse model experimental autoimmune encephalomyelitis (EAE). We show that the presence of γδ T cells was needed to promote the production of IFN-γ by both CD4 and CD8 T cells in the CNS before the onset of EAE. This regulation was shown to be independent of the ability of γδ T cells to produce IFN-γ, and was specific to T cells in the CNS, as no alterations in IFN-γ production were detectable in γδ T cell-deficient mice in the spleen and lymph nodes of mice with EAE or following immunization. Analysis of TCRγδ gene usage in the CNS showed that the only TCRδ V gene families present in the CNS before EAE onset are from the DV7s6 and DV105s1 gene families. We also show that the primary IFN-γ-producing cells in the CNS are the encephalitogenic T cells, and that γδ T cell-deficient mice are unable to resolve EAE disease symptoms like control mice, thus exhibiting a long-term chronic disease course similar to that observed in IFN-γ-deficient mice. These data suggest that CNS resident γδ T cells promote the production of IFN-γ by encephalitogenic T cells in the CNS, which is ultimately required for the recovery from EAE.

γδ T Cells Enhance the Expression of Experimental Autoimmune Encephalomyelitis by Promoting Antigen Presentation and IL-12 Production

Using an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP)-reactive lymph node cells (LNC), we have shown that depletion of γδ T cells from LNC resulted in diminished severity of EAE in recipient mice, both clinically and histopathologically. The reduced potency of γδ T cell-depleted LNC to induce EAE correlated with decreased cell proliferation in response to MBP. The γδ T cell effect upon the threshold of MBP-induced LNC proliferation and EAE transfer was restored by reconstitution of γδ T cells derived from either MBP-immunized or naive mice, indicating that this effect was not Ag specific. The enhancing effect of γδ T cells on MBP-induced proliferation and EAE transfer required direct cell-to-cell contact with LNC. The γδ T cell effect upon the LNC response to MBP did not involve a change in expression of the costimulatory molecules CD28, CD40L, and CTLA-4 on TCRαβ+ cells, and CD40, CD80, and CD86 on CD19+ and CD11b+ cells. However, depletion of γδ T cells resulted in significant reduction in IL-12 production by LNC. That γδ T cells enhanced the MBP response and severity of adoptive EAE by stimulating IL-12 production was supported by experiments showing that reconstitution of the γδ T cell population restored IL-12 production, and that γδ T cell depletion-induced effects were reversed by the addition of IL-12. These results suggest a role for γδ T cells in the early effector phase of the immune response in EAE.

Epicutaneous immunization induces αβ T-cell receptor CD4 CD8 double-positive non-specific suppressor T cells that inhibit contact sensitivity via transforming growth factor-β

Since it was previously shown that protein antigens applied epicutaneously in mice induce allergic dermatitis mediated by production of T helper 2 (Th2) cytokines we postulated that this might induce suppression of Th1 immunity. Here we show that epicutaneous immunization of normal mice with a different protein antigen applied on the skin in the form of a patch induces a state of subsequent antigen‐non‐specific unresponsiveness caused by suppressor T cells (Ts) that inhibit sensitization and elicitation of effector T‐cell responses. Suppression is transferable in vivo by αβ‐T‐cell receptor CD4+ CD8+ double positive lymphocytes harvested from lymphoid organs of skin patched animals and are not major histocompatibility complex‐restricted nor antigen specific. Both CD25+ and CD25– CD4+ CD8+ T cells are able to suppress adoptive transfer of Th1 effector cells mediating cutaneous contact sensitivity. In vivo treatment with monoclonal antibodies showed that the cytokines interleukin (IL)‐4, IL‐10 and transforming growth factor‐β (TGF‐β) are involved in the induction of the Ts cells. Additionally, using IL‐10–/– mice we found that IL‐10 is involved in skin induced tolerance. Further in vitro experiments showed that lymph node cells of skin tolerized mice non‐specifically suppress [3H]thymidine incorporation by antigen‐stimulated immune cells and this effect can be abolished by adding anti‐TGF‐β, but not anti‐IL‐4 nor anti‐IL‐10 antibodies. These studies indicate the crucial role of TGF‐β in skin induced tolerance due to non‐antigen‐specific Ts cells and also show that IL‐4, IL‐10 and TGF‐β play an important role in the induction of epicutaneously induced Ts cell suppression.

TLR-Dependent IL-4 Production by Invariant Vα14+Jα18+ NKT Cells to Initiate Contact Sensitivity In Vivo

LPS stimulated B-1 cell polyclonal in vivo IgM responses depend on IL-4 release by invariant Vα14+Jα18+ NKT (iNKT) cells. The IgM Abs can recruit effector T cells to mediate contact sensitivity. LPS activates the B-1 cell response just 1 day later, and depends on CD1d, iNKT cells, IL-4, TLR4, and MyD88. LPS in vivo and in vitro stimulates rapid preferential production of IL-4 in hepatic iNKT cells within 2 h. TLR4 were demonstrated in iNKT cells by flow cytometry and functional studies. Thus, innate microbial stimulation via TLR can activate iNKT cell and B-1 cell collaboration. The result is polyclonal IgM Ab responses capable of recruiting Ag-specific T cells into tissues. This may be involved in the promotion of autoimmunity by infectious agents.