Regis A. Campos

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.

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.

Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-γ via CXCR3 chemokines

We investigated the role of T helper (Th)1‐ and Th2‐type cytokines in delayed‐type hypersensitivity to soluble protein antigens elicited early postimmunization. Mice were sensitized by intradermal injection without adjuvants, or subcutaneously with complete Freunds adjuvant, and subsequently ear challenged intradermally. As soon as day 3, antigen‐specific eosinophil‐rich responses were elicited in wild‐type mice, but not in T‐cell receptor‐α–/– mice without adjuvant. Draining lymph node T cells stimulated with antigen secreted interleukin (IL)‐4, IL‐5 and interferon‐γ (IFN‐γ). IFN‐γ‐dependent specific immunoglobulin G (IgG)2a and IL‐4‐dependent IgG1 were also generated. Delayed‐type hypersensitivity ear swelling and local eosinophil recruitment were decreased in IL‐5–/–, IL‐4–/– and signal transducer and activator of transcription‐6 (STAT‐6)–/– mice, and with anti‐IL‐4 treatment of wild‐type mice, suggesting Th2 mechanisms. Interestingly, responses were also decreased in IFN‐γ–/– mice, and IFN‐γ protein and the IFN‐γ‐inducible CXC chemokine, IP‐10, were present in 24‐hr ear tissue extracts, suggesting Th1 effects. Finally, ear swelling, total histology and eosinophils were decreased in mice deficient in CXCR3, the chemokine receptor for IP‐10. These results suggest that both a Th2‐like (IL‐5, IL‐4 and STAT‐6) and a Th1‐like (IFN‐γ, IP‐10, CXCR3) pathway contribute to eosinophil recruitment in early delayed‐type hypersensitivity.

Interleukin-4-dependent innate collaboration between iNKT cells and B-1 B cells controls adaptative contact sensitivity

We showed that hepatic Vα14+ invariant natural killer T (iNKT) cells, via their rapid interleukin (IL)‐4 production, activate B‐1 cells to initiate contact sensitivity (CS). This innate collaboration was absent in IL‐4–/– and signal transducer and activator of transcription (STAT)‐6–/– mice and was inhibited by anti‐IL‐4 treatment. These mice have defective CS because they fail to locally recruit the sensitized effector T cells of acquired immunity. Their CS is reconstituted by transfer of downstream‐acting 1‐day immune B‐1 cells from wild‐type mice. Responses were not reconstituted with B‐1 cells from IL‐4 receptor‐α–/– or STAT‐6–/– mice, nor by IL‐4 treatment of B cell‐deficient mice at immunization. Finally, IL‐4 was preferentially and transiently produced by hepatic iNKT cells within 7 min after sensitization to mediate collaboration between innate‐like iNKT cells and the B‐1 B cells that participate in the recruitment of effector T cells in vivo.

Subunits of IgM Reconstitute Defective Contact Sensitivity in B-1 Cell-Deficient xid Mice: κ Light Chains Recruit T Cells Independent of Complement

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP κ L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while κ L chains act via another C-independent but mast cell-dependent pathway.

Airway hyper-reactivity mediated by B-1 cell immunoglobulin M antibody generating complement C5a at 1 day post-immunization in a murine hapten model of non-atopic asthma

Contact skin immunization of mice with reactive hapten antigen and subsequent airway challenge with the same hapten induces immediate airflow obstruction and subsequent airway hyper‐reactivity (AHR) to methacholine challenge, which is dependent on B cells but not on T cells. This responsiveness to airway challenge with antigen is elicited as early as 1 day postimmunization and can be adoptively transferred to naïve recipients via 1‐day immune cells. Responses are absent in 1‐day immune B‐cell‐deficient JH−/− mice and B‐1 B‐cell‐deficient xid male mice, as well as in recipients of 1‐day immune cells depleted of cells with the B‐1 cell phenotype (CD19+ B220+ CD5+). As B‐1 cells produce immunoglobulin M (IgM), we sought and found significantly increased numbers of anti‐hapten IgM‐producing cells in the spleen and lymph nodes of 1‐day immune wild‐type mice, but not in xid mice. Then, we passively immunized naive mice with anti‐hapten IgM monoclonal antibody and, following airway hapten challenge of the recipients, we showed both immediate airflow obstruction and AHR. In addition, AHR was absent in complement C5 and C5a receptor‐deficient mice. In summary, this study of the very early elicited phase of a hapten asthma model suggests, for the first time, a role of B‐1 cells in producing IgM to activate complement to rapidly mediate asthma airway reactivity only 1 day after immunization.

Omalizumab in Chronic Spontaneous Urticaria: A Brazilian Real-Life Experience

Background: Current guidelines on chronic spontaneous urticaria (CSU) suggest a treatment based on a 3-step approach that aims at total symptom control, starting with H1-antihistamines. However, a significant number of patients present an antihistamine-resistant urticaria that must be treated with an alternative third-line therapy such as omalizumab. Methods: Patients with a history of CSU who did not respond to treatment with high doses of modern antihistamines were treated with 150 or 300 mg of omalizumab every 4 weeks. The response to treatment was recorded as complete (CR), partial (PR) or no response. A dose adjustment was proposed according to response. Results: We treated 47 CSU patients with omalizumab (40 females), of whom 39.5% had evidence of autoimmunity. The average number of treatments was 11.4 (range 2-87). All patients had been refractory to high-dose modern antihistamines. A CR was seen in 84.6% of patients who started with 300 mg and in 60% of those who started with 150 mg. Only 1 patient had no response to both the 150- and 300-mg doses. In 6 of the PR patients with 150 mg, a higher dose of 300 mg was proposed and 4 had a CR. Four patients discontinued the treatment. No severe adverse events were reported in the patients who finished the study. Discussion: Although good results were seen in both groups, CR rates were higher in those under a high-dose initial treatment. Our data strongly suggest that the therapy should be individualized.

A subset of AID-dependent B-1a cells initiates hypersensitivity and pneumococcal pneumonia resistance

We propose that there is a special B‐1a B cell subset (“sB‐1a” cells) that mediates linked processes very early after immunization to initiate cutaneous contact sensitivity (CS), delayed‐type hypersensitivity (DTH), and immune resistance to pneumococcal pneumonia. Our published data indicate that in CS and DTH, these initiating processes are required for elicitation of the delayed onset and late‐occurring classical T cell–mediated responses. sB‐1a cells resemble memory B2 cells, as they are stimulated within 1 h of immunization and depend on T helper cytokines—uniquely IL‐4 from hepatic iNKT cells—for activation and rapid migration from the peritoneal cavity to the spleen to secrete IgM antibody (Ab) and Ab‐derived free light chains (FLCs) by only 1 day after immunization. Unlike conventional B‐1a (cB‐1a) cell–produced IgM natural Ab, IgM Ab produced by sB‐1a cells has high Ag affinity owing to immunoglobulin V‐region mutations induced by activation‐induced cytidine deaminase (AID). The dominant cB‐1a cells are increased in immunized AID‐deficient mice but do not mediate initiation, CS, or pneumonia resistance because natural Ab has relatively low Ag affinity because of unmutated germ‐line V regions. In CS and DTH, sB‐1a IgM Ag affinity is sufficiently high to mediate complement activation for generation of C5a that, together with vasoactive mediators such as TNF‐α released by FLC‐sensitized mast cells, activate local endothelium for extravascular recruitment of effector T cells. We conclude by discussing the possibility of functional sB‐1 cells in humans.