Humoral immunity is maintained by long-lived plasma cells, constitutively secreting antibodies, and nonsecreting resting memory B cells that are rapidly reactivated upon antigen encounter. The activation requirements for resting memory B cells, particularly the role of T helper cells, are unclear. To analyze the activation of memory B cells, mice were immunized with human cytomegalovirus, a complex human herpesvirus, and tick-born encephalitis virus, and a simple flavivirus. B cell populations devoid of Ig-secreting plasma cells were adoptively transferred into T and B cell–deficient RAG-1−/− mice. Antigenic stimulation 4–6 d after transfer of B cells resulted in rapid IgG production. The response was long lasting and strictly antigen specific, excluding polyclonal B cell activation. CD4+ T cells were not involved since (a) further depletion of CD4+ T cells in the recipient mice did not alter the antibody response and (b) recipient mice contained no detectable CD4+ T cells 90 d posttransfer. Memory B cells could not be activated by a soluble viral protein without T cell help. Transfer of memory B cells into immunocompetent animals indicated that presence of helper T cells did not enhance the memory B cell response. Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is independent of cognate or bystander T cell help.

The human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) induces a malignant lymphocytic disease. The HTLV-1 transactivator protein, Tax, is believed to be crucial for the development of the disease since it is transforming in vitro and induces tumors in transgenic animals. Although the transcriptional modulation of viral and cellular gene expression by Tax has been analyzed thoroughly, it has remained unclear how the Tax functions act on the cell cycle of primary T cells. To investigate the mechanism of Tax-mediated T-cell stimulation, we transduced primary human cord blood T cells with a conditional, tetracycline repressor-based tax expression system. Permanent Tax expression results in an abnormal proliferation of T cells which closely resemble HTLV-1-infected lymphocytes. Suppression of Tax synthesis stopped lymphocyte growth and caused cell cycle arrest in the G 1 phase. Upon reinduction of tax expression, the arrested cells entered the S phase. Thisshowed that Tax has mitogenic activity, which is required for stimulating the G 1 -to S-phase transition of immortalized lymphocytes. In mammalian cells, the G 1 -phase progression is controlled by the serial activation of several cyclin-dependent kinases (Cdks), starting with Cdk4 and Cdk6. In the presence of Tax, both Cdk4 and Cdk6 were activated. The suppression of Tax synthesis, however, resulted in a significant reduction of the Cdk4 and Cdk6 activities but did not influence the expression of Cdk4, Cdk6, or cognate D-type cyclin proteins. These data suggest that Tax induces Cdk4 and Cdk6 activity in primary human T lymphocytes; this Cdk activation is likely to account for the mitogenic Tax effect and for the abnormal T-cell proliferation of HTLV-1-infected lymphocytes.

The differential expression of Ia antigens was studied in freshly isolated rheumatoid nonlymphoid synovial lining cells (SLC)

A transmembrane pump for organic anions was identified in resting murine T helper (Th) 2, but not Th1 lymphocyte cell clones, as revealed by extrusion of a fluorescent dye. Dye extrusion inhibition studies suggested that the pump may be the multidrug-resistance protein (MRP). The different expression of the pump in resting Th1 and Th2 cell clones correlated with their respective levels of MRP mRNA. The pump was inducible in Th1 cells by antigenic stimulation in vitro leading to equal expression in activated Th1 and Th2 cell clones. This suggested that dye extrusion might allow the detection of Th2 (resting or activated) or of activated Th1 cells ex vivo based on a functional parameter. To test this, mice were infected with Leishmania major parasites to activate L. major-specific T cells of either Th1 (C57BL/6 mice) or Th2 (BALB/c mice) phenotype: 2-3% of CD4+ lymph node T cells of both strains of mice extruded the dye, defining a cell subset that did not coincide with subsets defined by other activation markers. Fluorescence-activated cell-sorting revealed that the lymphokine response (Th1 or Th2, respectively) to L. major antigens was restricted to this dye-extruding subset.

Ia antigens (class I1 HLA molecules) have been detected on cells eluted from affected human cartilage in certain disease states, but not on normal cartilage cells. Because the presence of Ia antigens on chondrocytes may play an important role in rheumatic diseases, we investigated the induction of these molecules by yinterferon ( y-IFN), a potent Ia-inducing lymphokine. Human articular chondrocytes were incubated with recombinant y-IFN, and the expression of Ia antigens was studied by cell sorter analysis, using a panel of reagents that detect monomorphic and polymorphic specificities of the DR and DQ Ia antigen families. While the induction of DR antigens, including polymorphic DR specificities, was readily obtained with y-IFN (50-95% positive cells), DQ antigens were negative or were displayed only on a lower percentage of chondrocytes (5-600/0). In addition, incubation with y-IFN led to an increased expression of HLA class I antigens. The expression of various other surface markers either remained unchanged (as in 4F2 and BA-2) or showed

Recently, it has been shown that cloned L1/1 T helper cells of type 2 (TH2-cells), when stimulated with antigen, are able to induce polyclonal B cell proliferation. Here we present evidence that this process is dependent on direct cell-cell interaction between T and B cells, which in the effector phase, i.e., during stimulation of the B cells by activated T cells, can be mediated by a mechanism other than cognate interaction. This conclusion is derived from experiments in which highly purified, small B cells of high density were polyclonally stimulated by L1/1 T cells triggered by an anti-T3 monoclonal antibody in the absence of antigen. The triggering process was independent of the presence of the Fc part of the antibody and occurred in cultures devoid of macrophages. Thus, the well-established cognate recognition does not appear to be the only mechanism of B cell induction by T helper cells.

The presence of activated T lymphocytes bearing interleukin 2 (IL-2) receptors and HLA class II (Ia) antigens accompanied by impaired T cell functions such as a decreased mitogenic responsiveness are characteristic findings, especially in intra-articular sites in chronic inflammatory joint diseases. The objective of the present study was to further characterize these in vivo activated T cells by the investigation of IL-2 production and a possible T cell receptor modulation. IL-2 receptors were found to be expressed primarily in the CD4+ subset. The Ia+ subset expressing both DR and DQ antigens showed a weaker mitogen-induced response as compared to the Ia- fraction. A decreased mitogen-induced IL-2 production and a lower response to anti-CD3 monoclonal antibodies was observed with synovial T lymphocytes as compared to peripheral blood T cells. The density of the CD3 molecule, known to be closely associated with the T cell receptor, was significantly lower in intra-articular sites, while other T cell-specific surface molecules were expressed to a similar extent in both compartments. The decreased synovial T cell mitogenesis was not restored by the addition of lymphokines (IL-1 and IL-2) or blood monocytes, nor by removing CD8+ T cells. These data present further evidence for a significant T cell activation in intra-articular sites in chronic inflammatory joint diseases. The decreased expression of the CD3 glycoprotein suggests a modulation by so far unidentified antigen(s), which could also be responsible for the weak T cell response elicited by polyclonal mitogens.

Large tissue samples from ten patients operated for colorectal cancer were prepared in the operating room in iced phosphate buffered saline, containing ethylene diaminetetraacetic acid and protease inhibitors. After cutting the specimens into small fragments, the tissues were gently pressed through a steel mesh. Membranes were permeabilized in chilled ethanol 70% to allow cytosolic fluoresceine isothiocyanate labeling, performed with anti-cytokeratin (CAM 5.2) antibodies. Samples were quantitatively sorted with a fluorescence activated cell sorter (FACS) and denatured before processing separation by two-dimensional electrophoresis on polyacrylamide gels. This procedure made it possible to sample about 4 x 10(7) viable normal and tumoral cells before fixation, and up to 4 x 10(6) cells after FACS. The gels run before and after fixation showed no major differences. The rate of cytokeratin-positive cells in the samples was the following (mean, CI 5-95%): mucosa 29.5% (8.9-66.7%), tumor 44.3% (6.6-94.8%). The epithelial cell content in colorectal cancer and normal mucosa shows important intersample variations. This is important for any comparison of fresh samples, whether at DNA, RNA, or at the protein level. We propose a method allowing the preparation of pure epithelial cell samples from normal and tumoral colonic fresh mucosa.