CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta 2-microglobulin-dependent and independent forms.

We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and beta 2m-/- mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23- CD43-) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in beta 2m-/- mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both beta 2m-/- and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the beta 2m-dependent form of CD1. They further suggest that a beta 2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.

Comparison of membrane-associated proteins of murine cytolytic and helper cloned T-cell lines: identification of a protein, p24, prominent in membrane fractions from cytolytic but not helper T-cells.

It has recently been reported that liposomes containing membrane components from cytolytic T-cell (TC) clones could transfer lytic activity to noncytolytic T- and B-cell lines, strongly suggesting that TC possess membrane-associated molecules which noncytolytic lymphocytes lack and which play a critical role in the lytic mechanism. It was thus of interest to compare the membrane-associated proteins from TC-lines to those of noncytolytic helper T-cell (TH) lines to determine whether any membrane-associated proteins unique to TC could be identified. Cells from three TC-lines and four TH-lines were internally labelled with [35S]methionine and then disrupted by hypotonic lysis. Low-density (plasma membrane enriched) and high-density (endoplasmic reticulum enriched) membrane fractions were isolated from each cloned cell line and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two proteins were identified which were prominent in the membrane fractions from each of the three TC-lines but not in the membrane fractions from any of the four TH-lines. One of these, p215, migrated as a broad band with an apparent mol. wt of 215,000. The other, p24, migrated as a sharp band, or tightly spaced doublet, with an apparent mol. wt of 24,000. Immunoprecipitation studies using monoclonal antibodies to T200, LFA-1, Thy 1 and Lyt 2 suggested that p215 was a variant of T200 found on TC-lines but not on TH-lines. Treatment of solubilized membrane proteins from TH-lines with anti-T200 precipitated a 185-kD protein seen on each of the TH-lines but on none of the TC-lines. In contrast, p24 was not precipitated by any of these monoclonal antibodies. It therefore appears that p24 represents a previously unidentified protein which is strongly expressed by TC but not by TH and is thus deserving of further study as to its functional significance.

p215 and p24: two membrane-associated proteins expressed on cloned cytolytic T-cells but not on cloned helper T-cells.

Cloned lines of murine alloreactive cytolytic and helper T cells were derived from secondary mixed leukocyte cultures. Cells from three TC lines and four TH lines were internally labeled with 35S-methionine and then disrupted by hypotonic lysis. Low density (plasma membrane-enriched) and high density (endoplasmic reticulum-enriched) membrane fractions were isolated from each cloned cell line and analyzed by SDS-PAGE under reducing conditions. Two proteins were identified which were associated with membrane fractions from each of the TC lines but none of the TH lines. One of these, p215, migrated as a broad band with an apparent molecular weight of 200-220 kD. The other, p24, migrated as a sharp band or closely spaced doublet with an apparent molecular weight of 24 kD. Immunoprecipitation studies using monoclonal antibodies to T200, LFA-1, Thy 1, and Lyt 2 revealed that p215 was a variant of T200 found on TC lines but not on TH lines. Treatment of solubilized membrane proteins from TH lines with anti-T200 precipitated a 180-195 kD protein band seen on each of the TH lines but none of the TC. In contrast, p24 was not precipitated by any of these monoclonal antibodies. It appears that p24 represents a previously unidentified protein which is unique to TC and thus deserving of further study as to its functional significance.

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones.

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.

Mechanisms by which human gliomas may escape cellular immune attack.

Whereas substantial evidence indicates that the majority of glioma patients make humoral immune responses to their own tumours, the evidence that glioma patients make significant cellular immune responses is more tenuous and controversial. In order to study those properties of human gliomas that might contribute to their ability to escape cell-mediated immune attack, we have examined the ability of cultured human glioma cells to elicit allogeneic cytolytic lymphocyte responses in vitro. Five of ten glioma lines were unable to elicit allogeneic cytolytic lymphocyte responses in mixed lymphocyte-tumour cultures, despite the presence of serologically detectable alloantigens on the surface of the glioma cells. Analysis of the reasons why certain glioma lines failed to stimulate cytolytic lymphocyte responses revealed three distinct mechanisms by which human gliomas may escape cellular immune attack: 1. a defect in immunogenicity which can be overcome by "help" from an allogeneic mixed lymphocyte reaction, 2. the secretion of a protective mucopolysaccharide coat, and 3. the production of macromolecular immunosuppressive substance(s). The implications of these findings for the immunotherapy of human gliomas are discussed.