Identification of αβ and γδ T Cell Receptor-Positive Cells

Two lineages of T lymphocytes bearing the CD3 antigen can be defined on the basis of the nature of the heterodimeric receptor chain (alpha beta or gamma delta T cell receptor (TCR) expressed. Precise identification of alpha beta and gamma delta TCR+ cells is essential when studying the tissue distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR-delta chain respectively, only reacted with a subpopulation of gamma delta TCR+ cells, whereas another TCR-delta chain recognizing MoAb anti-TCR-delta 1 reacted with all gamma delta TCR+ cells. All MoAb reported to belong to the CD3 group reacted with both alpha beta TCR+ and gamma delta TCR+ cells as expected. Our results indicate that all gamma delta TCR+ cells can be identified with the MoAb anti-TCR-delta 1. Because no MoAb recognizing the TCR-alpha or TCR-beta chains at the cell surface of intact cells are yet available, we suggest that alpha beta TCR+ cells could be identified as CD3+ anti-TCR-delta 1-cells.     

Bio-histochemical aspects of integrins (α2β1, α6β1) in invasive mammary carcinomas: An immunohistochemical study

Immunohistochemical expression of integrins was examined in 39 human invasive mammary carcinomas, of which 34.2% and 43.6% expressed integrins α2β1 and α6β1, respectively. Immuno-electron microscopy clearly demonstrated that the integrins were in the cell membrane of the carcinoma cells. Similar expression of integrin α2β1 or α6β1 in both the intraductal component and invasive portion of the same tumor was seen in 76.9% and 85.7% of cases, respectively. This suggested that invasive carcinoma cells retained their integrin expression after invasion through the basement membrane. Reciprocal expression of integrins α2β1 and α6β1 was seen in 20 cases. Expression of α2β1 was seen significantly less frequently in scirrhous carcinoma than in the more differentiated papillotubular or solid tubular carcinoma (Chi-squared test, P < 0.05). Intraductal components of carcinoma were present more frequently in cases expressing integrin α2β1 than in those that were negative. This suggests the potential usefulness of integrins as clinical parameters in the surgical treatment of mammary carcinomas, since recent trials of conservative treatment for mammary carcinoma have focused on the intraductal spread of the tumor cells.     

Fc Receptors on Human T Lymphocytes: Loss of Fcμ and Expression of Fcγ Receptors by T Cells Stimulated in Mixed Lymphocyte Reaction

The consequences of allogeneic stimulation on the expression of Fc mu or Fc gamma receptors were analysed in T-cell populations responding in mixed lymphocyte reaction (MLR). Unfractionated T-cell populations were cultured with a pool or irradiated allogeneic T-depleted cells. The responder E-rosetting cells progressively lost Fc mu and acquired Fc gamma receptors. The change of the original Fc receptor phenotype is not the consequence of a preferential proliferative response of TG versus TM cells but is likely due to a de novo expression of Fc gamma receptors by T cells lacking detectable Fc receptors (T-null) and also to the loss of Fc mu and expression of Fc gamma receptors by TM cells. These data suggest that, after MLR, responder T cells can modify their Fc receptor phenotype.     

Anti-GD 3 Antibodies are Potent Activators of Human γ/δ and α/β Positive T Cells

The ganglioside GD3 has a variety of biological functions. These include stimulatory effects is on proliferation, natural killer activity and cytokine production by freshly isolated peripheral T cells. In this study we have characterized anti-GD3 antibody (MoAb Z21) mediated effects on T cell clones. Our data indicate that α/β TCR CD4⁺ and CD8⁺ as well as γ/δ TCR positive T cells can be stimulated resulting in proliferation and cytokine production. This effect could be blocked by cyclosporin A and did not involve the LFA-3 or CD4 molecule. Apart from IFN-γ and IL-2 production by T helper I and T helper 0 cells we have observed production of IL-4 and IL-10 by T helper 2 cells indicating that the GD3 molecule is not a marker for a certain functional T cell subset. In contrast to anti-CD3 mediated activation, the responsiveness of T cells to stimulation via GD3 was dependent on the cell surface expression of the molecule and could be enhanced by costimulation via CD2, CD3, CD26 or CD28. In addition, anti-GD3 antibodies delivered a potent costimulatory signal for antigen-induced proliferation of CD4⁺ T lymphocytes. In summary, our experiments illuminate the mechanisms of anti-GD3 antibody induced T cell activation.     

Effects of Heat Shock on Cytolysis Mediated by NK Cells, LAK Cells, Activated Monocytes and TNFs α and β

We have recently shown that a heat treatment of a murine target cell line, WEHI 164, induces resistance to lysis mediated by tumour necrosis factors alpha (TNF-α) and beta (TNF-β). In the present study the effect of the heat shock of target cells on cytotoxicity mediated by natural killer cells (NK cells), lymphocyte-activated killer cells (LAK cells), activated monocytes, TNF-α, and TNF-β was investigated, First, WEHI 164 cell line and six human cell lines (ME 180, K 562, U 937, HeLa, MCF 7, and SK-OV 3) were screened for their sensitivity to different forms of lysis, and then sensitive cell lines were heat-treated. Pretreatment of target cells at 42° C for 45-60 min also rendered human target cell lines more resistant to lysis by rTNFs, and the acquired resistance was accompanied by an increased resistance to activated monocytes, but not to NK cells or LAK cells. Thus, the heat-induced resistance mechanisms capable of protecting target cells from lysis by rTNFs and by activated monocytes do not elicit resistance to lysis by NK cells and LAK cells, supporting the hypothesis that mediators other than TNFs are involved in NK cell-and LAK cellmediated killing.     

An architectural perspective on signaling by the pre-, αβ and γδ T cell receptors

The T cell antigen receptor (TCR) is a multimeric complex composed of an antigen‐binding clonotypic heterodimer and a signal transducing complex consisting of the CD3 dimers (CD3γε and CD3δε) and a TCR‐ζ homodimer. In all jawed vertebrates there are two T cell lineages, αβ and γδ, distinguished by the clonotypic subunits contained within their TCRs (TCR‐α and ‐β or TCR‐γ and ‐δ, respectively). A third receptor complex, the preTCR, is only expressed on immature T cells. The preTCR, which contains the invariant pre‐Tα (pTα) chain in lieu of TCR‐α, plays a critical role in the early development of αβ lineage cells. The subunit composition of the signal transducing complexes of the pre‐, αβ‐ and γδTCRs was previously thought to be identical. However, recent data demonstrate that there are significant differences in the signal transducing complexes of these three TCRs. For example, αβTCRs contain both CD3γε and CD3δε dimers, whereas γδTCRs contain only CD3γε dimers. Moreover, preTCR function appears to be unaffected in the absence of CD3δ, suggesting that CD3δε dimers are dispensable for pre‐TCR assembly. In this review, we summarize current data relating to the subunit composition of the pre‐, αβ‐ and γδTCRs and discuss how these structural differences may impact receptor signaling and αβ/γδ lineage determination.     

Dermatitis herpetiformis and celiac disease are both primarily associated with the HLA-DQ (α1*0501, (β1*02) or the HLA-DQ (α1*03, (β1*0302) heterodimers

HLA-DRB1, -DQA1, and -DQB1 genomic typing of 50 patients with dermatitis herpetiformis and of 290 healthy blood donors was performed. Genes encoding the DQ (α1*0501, (β1*02) heterodimer were carried by 43 (86%) of the patients and 72 (25%) of the controls. Of die remaining seven patients six (12% of all the patients) carried genes encoding the DQ (α1*03, β1*0302) heterodimer. These HLA associations are very similar to those observed in patients with celiac disease. We thus conclude that dermatitis herpetiformis and celiac disease are associated to the very same HLA-DQαβ heterodimers.     

The HLA-DQ(α1*0102, β1*0602) heterodimer may confer susceptibility to multiple sclerosis in the absence of the HLA-DR(α1*01, β1*1501) heterodimer

The frequencies of DR2, DQ6-related DRB1, DQA1, DQB1 haplotypes were compared in 181 multiple sclerosis patients and 294 controls in Norway. All individuals carried either DR2 or DQ6, i.e., the DQ(α1*0102, β1*0602) heterodimer. The DR(α1*01,β1*1501) and the DQ(α1*O102,β1*O602) heterodimers were carried by 171 of the patients (94%) and 289 (98%) of the controls. Seven of the patients and one of the controls carried the DQ(α1*0102, β1*0603) heterodimer together with the DR(α1*01, β1* 1501) heterodimer. Two patients carried the DQ(α1*0102, β1*O602) heterodimer in the absence of the DR(α1*O1, β1*1501) heterodimer. The DR(α1*01, β1*1501) heterodimer was not observed in the absence of the DQ(α1*0102, β1*O602) heterodimer or the DQ(α1*O102, β1*0603) heterodimer, neither in the patients nor in therols. Our findings indicate that the genes encoding the DQ(α1*0102,β1*0602) heterodimer may confer susceptibility to developing multiple sclerosis in the absence of the DRB 1* 1501 allele.     

Crucial function of the pre-T-cell receptor (TCR) in TCRP selection, TCRβ allelic exclusion and αβ versus γδ lineage commitment

Summary: The analysis of T-cell receptor (TCR) βselection, TCRβ allelic exclusion and TCRβ rearrangement in γδ T cells from normal and pre-TCR-deficient mice has shown that the pre-TCR has a crucial role in T-lyinpbocyte development:

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  The pre-TCR is by far the most effective receptor that generates large numbers of CD4⁺8⁺ T cells with productive TCRβ rearrangements.
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  In the absence of the pre-TCR, TCRβ rearrangement proceeds in developing cells irrespective of whether they already contain a productive TCRβ gene.
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  The pre-TCR directs developing T cells to the αβ lineage because y5 T cells from pTα^-/- mice proceed much further in TCRβ rearrangement than γδ T cells from wild-type mice. It is argued that the pre-TCR commits developing T cells to the αβ lineage by an instructive mechanism, which has largely replaced an evolutionarily more ancient mechanism that involves stochastic αβ lineage commitment.

     

Production of Interferon-α/β by Murine Dendritic Cell Lines Stimulated by Virus and Bacteria

The interferon-α and -β (IFN-α/β) producing ability of the two murine dendritic cell (DC) lines D2SC/1 and FSDC was studied. The D2SC/1 cells produced IFN-α and -β when stimulated by herpes simplex virus (HSV), Sendai virus (SV) or by the bacteria Escherichia coli or Staphylococcus aureus Cowan I. Precultivating (priming) D2SC/1 cells with recombinant IFN-β or a combination of IFN-β and granulocyte–macrophage colony-stimulating factor increased production of IFN-α/β induced by HSV or the bacteria, but not by SV. Also, the kinetics of IFN-α/β responses were different for SV compared to HSV and the bacteria, suggesting different induction mechanisms. The FSDC cells differed from the D2SC/1 cells mainly in that predominantly IFN-β was produced, that little or no IFN-α/β production was induced by the bacteria, and that the IFN-α/β responses were most efficiently primed by IFN-γ. Priming the DC lines with tumour necrosis factor-α, interleukin-10 (IL-10) or IL-4 did not affect the IFN-α/β response induced by HSV. The results show that the two DC lines provide a convenient tool to study the induction and control of the IFN-α/β response, as well as the immunoregulatory role of IFN-α/β produced by DC.     

Mitogenic Properties of Rabbit Anti-Human β2-Microglobulin for Murine B Cells

Rabbit antisera against human β₂-microgluhulin were found to be mitogenic for mouse spleen cells, giving rise to a peak DNA synthetic response on day 2 in cultures containing serum-free medium. The mitogenic effect was shown on cells from spleens of nude 'athymic' mice and on spleen B cells, but no effect was found on cortisone resistant mouse thymocytes or on spleen T cells. Thus, it was concluded that the serum was mitogenic for mouse B cells but not for T cells. This conclusion was confirmed by experiments showing that the antiserum was able to induce polyclonal antibody synthesis in mouse spleen cells in culture. The activity of the scrum was absorbed by pure human β₂-microglobulin as well as by mouse thymocytes and bone marrow cells     

Receptors for the FC Portion of Immunoglobulins (FCR) on Murine Oral Mucosal T Cells

The incidence and distribution of immunoglobulin receptors on oral mucosal T cells were investigated. Enrichment of either Thy-1.2, L3T4 or Lyt-2.2-positive cells was achieved by the use of a panning procedure following cell extraction by an optimized digestion method that preserved all three T-cell markers. The percentage of cells possessing Fc receptors was determined by using immunoglobulin-coated (IgM, IgG or IgA) fluorescent microspheres in a multipoint rosetting assay. We report that approximately 60% of Thy-1.2, L3T4 and Lyt-2.2 positive cells bear FcyR whereas less than 5% of T cells of any subset bear FcaR or FcjiR. In frozen tissue sections, FcyR+ cells were mainly scattered throughout the basal and suprabasal epithelium and were observed at a lower frequency in the minor salivary gland network. From their distribution, it is anticipated that FcyR+ cells may be involved in the surveillance of the epithelium while the minor FcocR+ L3T4+ T lymphocyte population may promote the expression of slgA by resident slgM-bearing B cells, and their differentiation into IgA plasma cells.     

Receptors for the FC Portion of Immunoglobulins (FCR) on Murine Oral Mucosal T Cells

The incidence and distribution of immunoglobulin receptors on oral mucosal T cells were investigated. Enrichment of either Thy-1.2, L3T4 or Lyt-2.2-positive cells was achieved by the use of a panning procedure following cell extraction by an optimized digestion method that preserved all three T-cell markers. The percentage of cells possessing Fc receptors was determined by using immunoglobulin-coated (IgM, IgG or IgA) fluorescent microspheres in a multipoint rosetting assay. We report that approximately 60% of Thy-1.2, L3T4 and Lyt-2.2 positive cells bear FcyR whereas less than 5% of T cells of any subset bear FcaR or FcjiR. In frozen tissue sections, FcyR+ cells were mainly scattered throughout the basal and suprabasal epithelium and were observed at a lower frequency in the minor salivary gland network. From their distribution, it is anticipated that FcyR+ cells may be involved in the surveillance of the epithelium while the minor FcocR+ L3T4+ T lymphocyte population may promote the expression of slgA by resident slgM-bearing B cells, and their differentiation into IgA plasma cells.     

Lysosomal enzyme levels in human amniotic fluid cells in tissue culture: II. α-galactosidase, β-galactosidase and α-arabinosidase

Changes in the activities of α-galactosidase, β-galactosidase and a-arabinosidase in amniotic fluid cells with time in culture were studied. Marked fluctuations in all three enzymes occurred with passage. In certain cell strains, β-galactosidase showed a marked rise in activity correlated with passage. The activity of all three enzymes, in amniotic fluid cells at the third passage, was correlated with the total time taken to reach con-fluency. There was no consistent pattern of enzyme activity associated with the time after subculture. Enzyme levels in cell strains derived from serial samples of amniotic fluid from several women showed large differences in activity unrelated to gestational age.     

Aggregated Immunoglobulin Protects Immune T Cells from Suppression: Dependence on Isotype, Fc Portion, and Macrophage FcγR

We determined the regulatory properties of heat-aggregated immunoglobulins (HA-Ig) that possess many activities of immune complexes (IC), such as binding and activation of cells via immunoglobulin Fc γ receptors (FcγR). HA-Ig protected contact sensitivity (CS) effector T cells from antigen-specific immunosuppression, while monomeric IgG were inactive. This anti-suppressive activity of HA-Ig was antigen non-specific, and depended on the species from which Ig was derived, i.e. mouse and rat HA-Ig were protective in mice, and of other species were inactive. The protecting activity of HA-Ig was confined to IgG2a and IgG3, and to a lesser degree to IgG1 isotypes, and resided in the Fc domain. Removal of phagocytic cells from the CS-immune target cells, or blocking with anti-FcγR mAb, abolished HA-Ig protection of CS-effector T cells from suppression. We suggest that HA-Ig multimers acted via Fc domains, in one of two ways: by binding to FcγR of macrophages to produce positive-acting cytokines, or by blocking FcγR on macrophages, to compete with suppressive factors that can also bind to FcγR. If HA-Ig protection of T cells is generalized, it is likely that IC in vivo may non-specifically overcome suppression of responses to antigen that normally are under the control of T suppressive cells, and thus may contribute to the development of autoimmunity.