Depletion of eosinophils in mice through the use of antibodies specific for C-C chemokine receptor 3 (CCR3).

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor, C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells, dendritic cells, or cells from the thymus, lymph node, or spleen of normal mice. Unlike human Th2 cells, mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge, the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection, isolation, and in vivo depletion of eosinophils.

The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb.

A protective immune response to the intracellular parasite Leishmania major requires the development of a Th1 CD4+ T cell phenotype. We demonstrate herein that BALB/c mice, which normally develop a susceptible Th2 response to L. major infection, are protected when co-injected with an agonistic anti-murine CD40 mAb. Anti-CD40 mAb-mediated protection in this system was found to be T cell dependent, since it was not observed in C57BL/6 x 129 mice that were rendered T cell deficient (TCR beta-/- x TCR delta-/-) and L. major susceptible. Anti-CD40 mAb stimulation of L. major-infected BALB/c mice was accompanied by increased IL-12 and IFN-gamma production in draining lymph nodes, analyzed either by direct expression, or in an antigen-specific in vitro recall assay. The protective role of these cytokines was indicated by the finding that anti-CD40 mAb-mediated protection of L. major-infected BALB/c mice could be reversed by co-treating the animals with neutralizing anti-IL-12 and/or anti-IFN-gamma mAb. Collectively, these data suggest that BALB/c mice develop a protective Th1 CD4+ T cell response to L. major infection when co-injected with anti-CD40 mAb. While the CD40-CD40L interaction has been previously shown to be vital in the control of murine Leishmaniasis, the current study establishes in vivo that anti-CD40 mAb treatment alone is sufficient to protect BALB/c mice from L. major infection and raises the possibility of utilizing this approach for vaccination strategies.

CD40 signaling induces interleukin-4-independent IgE switching in vivo.

T cell-deficient T cell receptor (TCR) beta-/- x TCR delta-/- knockout mice lack circulating IgE and fail to produce antigen-specific IgE in response to stimulation with T cell-dependent antigens. We show here that these animals are able to produce significant levels of circulating polyclonal IgE when injected with an agonistic anti-mouse CD40 monoclonal antibody. CD40-mediated induction of circulating polyclonal IgE in T cell-deficient mice was only partially reduced when the animals were co-treated with neutralizing anti-interleukin-4 (IL-4) antibody. The IL-4 independence of this response was further supported by experiments showing that anti-CD40 antibodies induced circulating IgE when injected into IL-4 knockout mice, and sterile RNA epsilon transcript production when cultured with purified B cells from the same mice. These data strongly suggest that CD40 signaling causes IL-4-independent IgE switching in mice.

CD38 expression on mouse T cells: CD38 defines functionally distinct subsets of alpha beta TCR+CD4-CD8- thymocytes.

We have examined CD38 expression on mouse lymphocytes using the rat mAb NIM-R5 and demonstrate that CD38 expression is restricted to approximately 8% of thymocytes. Although CD38 is absent from the majority of CD4+CD8- and CD4-CD8+ T cells, we detected a strong correlation between CD38 expression and alpha beta+CD4-CD8- T cells in the thymus, with nearly 80% of alpha beta TCR+CD4-CD8- thymocytes being CD38+. Using heat stable antigen (HSA) and CD38, we divided alpha beta+CD4-CD8- thymocytes into four subsets: HSA+CD38-, HSA-CD38hi, HSA-CD38low and HSA-CD38-. Two established characteristics of alpha beta TCR+CD4-CD8- cells, bias towards V beta 8.2 TCR expression and high levels of IL-4 production, were used to establish a possible relationship between the above thymocyte subsets. Our present data show that the HSA+CD38- subset is not biased towards V beta 8.2 TCR expression whereas the HSA-CD38- subset does show this bias (approximately 47%). Neither of these subsets make IL-4 upon CD3 mediated stimulation. In contrast, the CD38+ subsets are heavily biased toward V beta 8.2 expression and produce large amounts of IL-4 upon stimulation, particularly the CD38low cells. Taken together, these data suggest that these four subsets represent various stages of a possible differentiation pathway for alpha beta TCR+CD4-CD8- cells, with the HSA+CD38- subset being the most immature while the HSA-CD38low subset is the most functionally mature. These characteristics support the view that alpha beta TCR+CD4-CD8- T cells represent an independent lineage with a distinct, but as yet obscure, role in immunity.

Differential production of interferon-gamma and interleukin-4 in response to Th1- and Th2-stimulating pathogens by gamma delta T cells in vivo.

Exposure to various pathogens can stimulate at least two patterns of cytokine production by CD4-positive T cells. Responses that result in secretion of interferon-gamma (IFN-gamma), lymphotoxin and interleukin-2 (IL-2) are classified as T-helper-1 (Th1); CD4+ T-cell production of IL-4, IL-5, IL-9, IL-10 and IL-13 is called a T-helper-2 response (Th2). Differentiation of CD4+ T cells into either Th1 or Th2 cells is influenced by the cytokine milieu in which the initial antigen priming occurs. Here we use flow cytometry to identify the presence of intracellular cytokines (cytoflow) and analyse T-cell production of IFN-gamma and IL-4 from mice infected with Listeria monocytogenes or Nippostrongylus brasiliensis. We show that T cells bearing gamma delta receptors discriminate early in infection between these two pathogens by producing cytokines associated with the appropriate T-helper response. Our results demonstrate that gamma delta T cells are involved in establishing primary immune responses.