Michael Hagen

Activation features of intraepithelial γδ T-cells of the murine vagina

Abstract The epithelium of the murine vagina contains a resident population of γδ T-cells that expresses a homogenous V-4.Vδ1 TCR lacking N-region junctional diversity, implying that these T-cells recognize a very limited array of antigenic structures. The vaginal γδ T-cells express a pattern of surface markers characteristic of memory/effector T-cells that have previously been activated. Although vaginal γδ T-cells do not express the major costimulatory molecules CD28 and CD2, they do proliferate in response to a systemically delivered anti γδ TCR stimulus. Vaginal γδ T-cells contain mRNA that encodes the keratinocyte growth factor raising the possibility that these cells play a role in the repair of vaginal epithelium following injury. While the antigen recognized by the vaginal γδ TCR is unknown, a model is proposed which attempts to relate some of the unusual phenotypic features of vaginal γδ T-cells to the physiological injury and shedding of vaginal epithelium that occurs during the estrous cycle.

Potential role of FcγR in early development of murine lymphoid cells: evidence for functional interaction between FcγR on pre-thymocytes and an alternative, non-Ig ligand on thymic stromal cells

During early development of the murine fetal thymus a fraction of Thy+ thymocytes express Fc gamma receptors. Concurrently, a small fraction of Thy- CD44- thymocytes bind recombinant soluble Fc gamma R. These findings suggested the possibility that Fc gamma R+ pre-T cells interact with a non-Ig ligand present on fetal thymic stromal cells. Evidence in support of this concept was the finding that experimental manipulation of this putative receptor-ligand pair by anti-receptor antibody or recombinant soluble Fc gamma R influenced the developmental pattern of alpha/beta-TCR+ T cells. The restricted expression of CD16 on some pre-B cells raises the possibility that an Fc gamma R-dependent step may play a similar role in B-cell development.

Functional significance of CD23- on CD23-transfected Th2 clone

CD23, a low-affinity IgE Fc receptor, is not displayed on most resting T cells but its expression has been shown to be transiently induced in vivo and in vitro on some CD4+ T cells [1-4] and in vivo on CD8+ T cells by IgE-secreting hybridoma tumors [5]. To investigate the functional role of CD23 on T cells, we inserted a CD23 construct into an expression vector driven by a CD2 promoter and transfected it into a murine Th2 clone D10.G4.1 (D10). We stimulated the transfected D10 cells (D10.3M.24) with anti-TCR antibody in the presence or absence of IgE, and measured IL-4, IL-5 and IL-6 production in the culture supernatants. Activation of D10.3M.24 cells by anti-TCR antibody induced greater levels of IL-4, IL-5 and IL-6 production, when the TCR and CD23 were co-crosslinked by TNP anti-TCR and IgE anti-TNP antibodies. IgG anti-TNP antibody did not enhance lymphokine production by D10.3M.24 cells. The enhanced lymphokine production by IgE was blocked by monoclonal anti-CD23 antibody. IgE anti-TNP antibody did not enhance lymphokine production by the wild-type D10 cells induced by TNP anti-TCR antibody. These studies show that when co-crosslinked with the TCR, CD23 can modulate the lymphokine production in activated Th2 cells. Since CD23 binds to IgE and also binds to CD21 [6], a complement receptor commonly expressed on B cells, T-cell CD23 could play an immunoregulatory role during cognate T-B cell interaction and during IgE antibody responses.

A regulatory role for Fcγ receptors (CD16 and CD32) in hematopoiesis

Abstract Progenitor cells of the T- and B-lineages in mice express (CD32) and FcγRIII (CD16) but as the developing lymphocytes begin to express clonal antigen receptors, CD16 and CD32 are downregulated in T-cells, and CD16 is downregulated in B-cells. Considering that counter-receptors for FcγR occur on thymic and bone marrow stromal cells, the possibility exists that FcγR might participate in some aspect of T- and B-lineage development prior to the stage of antigen receptor expression. Previous studies provided evidence that FcγR can influence murine T-lineage development. In the present studies we found that anti-FcγRII/III mAb accelerated B-lineage development in bone marrow cultures from normal mice, but not in cultures from CD16-/- or CD32-/- mice. Similar results were observed when FACS-purified B-progenitor cells were co-cultured with BMS2, a bone marrow stromal cell line. Fresh bone marrow from CD32-/- mice contained about two-fold more B-lineage cells compared to bone marrow from normal or CD16-/- mice. These studies indicate that the FcγR on B-lineage progenitor cells can influence their further development and add to a growing body of evidence that implicates FcγR as regulatory elements in hematopoiesis.

Functional consequences of the interaction between T-cell antigen receptors and FcγRs on T cells

Two series of experiments are presented indicating that Fc gamma II receptors can interfere with the antigen receptor-induced signaling on T cells. It has been previously described that a 13 amino acid motif on the cytoplasmic portion of Fc gamma II beta 1 can abrogate the antigen receptor-initiated signals mediated through consensus motifs present on the cytoplasmic portion of Ig alpha and Ig beta chains. Similar activating motifs are crucial to T-cell receptor (TCR) signaling. A splenic gamma/delta T-cell hybridoma that expressed the Fc gamma RII beta 1 receptor helped to establish that this receptor can also interfere with TCR-induced activation. The cytoplasmic portion of human Fc gamma RIIa has an activation motif similar to the activation motifs present on the TCR. Using a transgenic mouse in which the T cells express the human Fc gamma RIIa transgene, we demonstrated that despite the common activation motif, the TCR and human Fc gamma RIIa-induced signals are different. Additionally, the human Fc gamma RIIa expressing T cells exhibit an enhanced TCR response both in vitro and in vivo.

The FcϵRIICD23 gene is actively transcribed during all stages of murine B-lymphocyte development☆

It is generally accepted that the expression of FcϵRIICD23 on the surface of B-lineage cells is restricted to the stage of the resting, mature (sIgM+sIgD+) B-lymphocyte. However, it is unknown whether activation of the FcϵRIICD23 gene is also restricted to the stage of the mature B-lymphocyte. To address this question we investigated a panel of B-lineage cell lines for the presence of transcripts encoding FcϵRIICD23. We detected transcripts in 16 of 26 B-lineage cell lines representing the entire spectrum of B-cell development. In most cases (13 of 16) active transcription of the murine FcϵRIICD23 gene was not coupled with the expression of cell surface FcϵRIICD23 protein. The stage-restricted pattern of cell surface FcϵRIICD23 expression did not hold for all murine B-cell lines. One post-switch B-cell line (sIgM−sIgG+) expressed FcϵRIICD23 on the cell surface and another could be induced with IL-4 and LPS to express surface FcϵRIICD23. Transcription of the murine CD23 gene in the absence of cell surface expression of FcϵRIICD23 does not appear to simply be an aberrant feature of transformed B-cells since we found transcripts, but not surface expression, in some normal splenic and peritoneal B-lymphocytes. Our findings suggest that the potential for expression of FcϵRIICD23 may occur over a much broader developmental window of the B-lineage than previously suspected. Transcription of the FcϵRIICD23 gene, in the absence of detectable cell surface protein expression in B-lineage cell lines, and in sort-purified B-lymphocyte subpopulations, implies that in addition to regulatory mechanisms already known, murine CD23 is also regulated through post-transcriptional mechanisms that have not yet been characterized.

Developmental regulation of FcϵRII/CD23 expression in B-lineage cells: evidence for transcriptional and post-transcriptional levels of control☆

The present studies show that the expression of cell surface Fc epsilon RII/CD23, detected with the monoclonal anti-Fc epsilon RII/CD23 antibody B3B4 or by the binding of IgE, is not restricted to the stage of resting mature virgin B lymphocytes. Murine CD23 was detected as a cell surface protein on two sIgG+ B-cell lines. Moreover, we detected full-length transcripts for Fc epsilon RII/CD23 in several members of a panel murine B lymphoid lineage cell lines representative of all stages of murine B lymphocyte development. Our findings suggest that regulation of CD23 translation may differ between B-cell lines at various stages of differentiation. The detection of mRNA transcripts for Fc epsilon RII/CD23 was not restricted to transformed cell lines. Fc epsilon RII/CD23 transcripts were amplified by RT-PCR from peritoneal and splenic B lymphocyte subpopulations that were sorted by flow cytometry into populations that did not express surface Fc epsilon RII/CD23. Our findings suggest that Fc epsilon RII/CD23 transcription and translation are not necessarily restricted to the narrow developmental window of murine B lymphocyte differentiation as previously thought. Our findings imply that Fc epsilon RII/CD23 may be expressed at the protein level at various stages of murine B lymphocyte differentiation. Investigations into the expression patterns and potential mechanisms of regulation of Fc epsilon RII/CD23 could provide insight into the basis for the wide range of immunological functions that have been proposed for Fc epsilon RII/CD23.

The Regulation and Functional Significance of Fc Receptors on Murine Lymphoid Cells: The Special Case of T Cells

Fc receptors (FcR) specific for each of the five major classes of murine Ig heavy chains occur on the surface membranes of all the major lineages of murine lymphoid cells. The number of different classes of FcR expressed, their concentration and level of functional activity, and the developmental window in ontogeny during which the FcR are expressed vary considerably amongst the subsets of lymphoid cells. The regulation of FcR expression on lymphoid cells is a multi-factorial process that involves preprogrammed genetic events and local environmental factors (Lynch and Sandor, 1990).