Developmental sequence of T200 antigen modifications in murine T cells

The T200 glycoproteins of T cells were analyzed at different stages of T cell development. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Lyt-2-L3T4-, and Lyt-2+L3T4+ thymocytes had similar T200 proteins, whereas Lyt-2+L3T4- and Lyt-2-L3T4+ thymocytes expressed a distinct set of T200 molecules. This result indicated a molecular switch in regulation of T200 protein expression upon differentiation of thymocytes to mature phenotype T cells. Further modifications were evident when the T200 proteins of peripheral T cell subsets were examined. In particular L3T4+ T cells expressed T200 proteins of m.w. 220,000, 200,000, and 175,000, whereas Lyt-2+ lymph node T cells expressed an additional T200 protein of m.w. 235,000. Antigenic differences in the T200 glyco-proteins of peripheral L3T4+ and Lyt-2+ T cells were also detected. The anti-B220 monoclonal antibody, 14.8, reacted with lymph node Lyt-2+ T cells but did not react with lymph node L3T4+T cells or with Lyt-2+L3T4- thymocytes. This finding demonstrated a lineage-specific modification of the T200 protein of Lyt-2+ T cells that occurred after exit of these cells from the thymus into peripheral lymphoid organs. This modification apparently occurred on the m.w. 235,000 and 220,000 proteins since these species were precipitated by 14.8, whereas the others were not. In vitro growth and activation also resulted in further T200 antigen alterations. The monoclonal antibody, RA3, which reacts with the B220 antigen of B cells but, unlike 14.8, does not react with any peripheral T cells, showed significant reactivity with Lyt-2+ cytotoxic T cell (CTL) clones but not with L3T4+ T helper cell clones. CTL clones were also 14.8+ but T helper cell clones were not. Immunoprecipitation by 14.8 and RA3 of T200 proteins from CTL clones yielded a single protein of m.w. 240,000 that co-migrated with the B cell form of T200. Overall, the results indicate the presence of developmentally regulated mechanisms that control T200 glycoprotein expression during T cell differentiation in the thymus and in peripheral lymphoid organs.     

Role of lipomodulin, a phospholipase inhibitory protein, in immunoregulation by thymocytes

The treatment of murine thymocytes with anti-lipomodulin antibody during Con A stimulation causes selective loss of suppressor activity, but not of helper activity on PFC assay, when co-cultured with T cell-depleted spleen cells. Interaction of the antibody with responder cells in thymocyte culture were necessary in the early stage rather than in the later stage of lymphocyte activation by Con A, which suggests that anti-lipomodulin antibody acts in the stage of suppressor T cells generation. When thymocytes were cultured with purified lipomodulin for 48 hr, suppressor activity was induced. Lipomodulin as detected by radioimmunoassay was found to be released from T cells with the phenotype of I-J+, Lyt-1-, Lyt-2+. The immunoprecipitates from the media of Con A-stimulated thymocyte with anti-I-Kk antibody and anti-lipomodulin antibody were analyzed on SDS-gel electrophoresis. I-J products had m.w. 36,000 and 24,000, whereas lipomodulin had m.w. 36,000, 24,000, and 15,000. Because anti-I-Jk antibody could precipitate 125I-labeled lipomodulin purified from rabbit neutrophils, these results suggest that lipomodulin is a product of I-J genes that induces suppressor T cells.     

Rat T lymphocyte antigens comparable with mouse Lyt-1 and Lyt-2,3 antigenic systems: characterization by monoclonal antibodies

Rat T lymphocyte antigens were defined by using two distinct monoclonal antibodies (R1-3B3 and R1-10B5). R1-3B3 antibody, when tested for its reactivity with rat lymphoid cells by immunofluorescence, stained almost all of thymus and T cells but not the majority of B cells and bone marrow cells. The antigen defined by R1-3B3 existed more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Immunochemical data showed that R1-3B3 antibody recognized a single glycoprotein with a m.w. of 67,000, showing marked electric charge heterogeneity with isoelectric points ranging from 5.4 to 7.3. R1-10B5 antibody, on the other hand, had more restricted reactivity with rat T cells and labeled approximately 85% of thymus cells and 30% of the peripheral T cells but neither B cells nor bone marrow cells. These T cells positive for R1-10B5 appeared to be negative for W3/25 antigen, which has been shown to be the marker for the rat T cell subset associated with helper function. R1-10B5 antibody detected a basic glycoprotein complex consisting of sulfhydryl-linked subunits with 30,000 and 34,000 m.w. Although the antigen defined by R1-3B3 was resistant to trypsin digestion, the one detected by R1-10B5 was much more sensitive to trypsin cleavage. All of these data obtained with either R1-3B3 or R1-10B5 are quite comparable to those reported for mouse Lyt-1 or Lyt-2,3 antigens, and thus suggest that the antigens defined by R1-3B3 and R1-10B5 antibodies represent rat homologues of Lyt-1 and Lyt-2,3 antigens in the murine system, respectively.     

Genetic control of T-Cell subset representation in inbred mice

Lyt-2+ T cells constitute a significantly greater proportion of the total peripheral T-cell population in C57BL mice than in BALB/c and other mouse strains. The inheritance of this differential representation of Lyt-2- vs. Lyt-2+ T cells was studied by two-color immunofluorescence analysis of peripheral T cell subsets in BALB/c, C57BL, F1 and F2 generations, and in CXB recombinant inbred strains. It was shown that the C57BL phenotype (low Lyt-2-/Lyt-2+ ratio) is a dominant Mendelian character. Studies of subpopulations of thymocytes and of early thymus emigrants indicate that the representation of mature Lyt-2- and Lyt-2+ T cells is influenced by mechanisms of selection or differential turnover in the peripheral lymphoid organs, but that thymic and prethymic influences may also play a role.     

Subpopulations of mouse Lyt-2+ T cells defined by the expression of an Ly-6-linked antigen, B4B2

The production and characterization of a rat mu,kappa monoclonal anti-mouse T cell subset antibody, B4B2, is reported in this paper. B4B2 typing of lymphoid tissues of commonly used inbred mouse strains revealed two types of reactivity patterns. They can be characterized as C57BL/6-like (B6-like) or C3H/He-like (C3H-like). Among B6-like strains, B4B2 recognizes 5 to 10% of spleen cells, 30 to 50% of bone marrow cells, and less than 2 to 3% of thymocytes. In C3H-like strains, B4B2 reacts with less than 1% of spleen cells, 2 to 8% of bone marrow cells, and less than 1% of thymocytes. B4B2 recognizes a T cell subset differentiation antigen expressed by B6-like strains but not by C3H-like strains. Typing of BXH recombinant inbred strains showed linked expression of B4B2 and the Ly-6 antigen. The expression of B4B2 antigen appears to be under codominant control as the median fluorescence distribution of B4B2+ cells in C57BL/6 was approximately twice that of (C57BL/6xC3H)F1. B4B2 was shown to react with approximately 40 to 50% of Lyt-2+ T cells and less than 1% of L3T4+ T cells. No staining of resting or activated B cells by B4B2 was detected. The ratio of B4B2+:Lyt-2+ cells was similar for resting T cells and activated T cells obtained from mitogen-stimulated cultures or mixed lymphocyte cultures. In neonatal spleen, substantially more B4B2+ than Lyt-2+ cells were found. With increasing age, however, a rapid decline in B4B2+ cells and a corresponding increase of Lyt-2+ cells was observed. By approximately 1 mo of age, the relative proportion of these subsets had reversed so that Lyt-2+ cells became more numerous than B4B2+ cells.     

Xenogeneic monoclonal antibody to an Ly-6-linked murine cell surface antigen: differential reactivity with T cell subpopulations and bone marrow cells

The patterns of cellular and strain reactivity of a monoclonal antibody (6C3 MAb) derived from the fusion of SP2/0 cells with splenocytes from rats immunized against MRL/MpJ-lpr/lpr T cells were characterized by using flow cytofluorometry (FCF) analysis. This MAb was found to stain 70 to 90% of T cells of mice with the lpr/lpr genotype and 20 to 60% of T cells of congenic +/+ strains. Dual-parameter FCF analysis of Lyt-2 vs 6C3 expression revealed the existence of several Lyt-2- and Lyt-2+ T cell subsets, one of which (Lyt-2- bright 6C3+) was expanded in lpr/lpr-bearing mice. The 6C3 MAb stained only 2 to 5% normal thymocytes but reacted with 40 to 50% bone marrow (BM) cells. A strain survey demonstrated the expression of the 6C3 antigen on peripheral T cells (and BM cells) of all strains examined, with the exception of NOD, NZB/B1NJ, and ST/bJ. Interestingly, in the positive strains, two types of 6C3 staining patterns of T cells were observed: bimodal or trimodal. Study of BXH and CXB recombinant inbred (RI) strains demonstrated that the bimodal and trimodal 6C3 patterns are associated with the Ly-6.1 and Ly-6.2 phenotypes, respectively. Linkage of 6C3 expression with the Ly-6 locus was confirmed by using the congenic C3H.B6-Ly-6b strain. Moreover, the 6C3 staining of T cells in Ly-6.2 strains was reduced by preincubation with the H9/25 and SK-142-446 MAb, which are known to recognize Ly-6.2-associated antigens. Therefore, the 6C3 MAb appears to detect a frame-work determinant on an Ly-6-linked antigen that is absent from T cells of NOD, NZB, and ST/bJ mice. Analysis of (NZB x C58) NX8 RI strains demonstrated a correlation between the lack of 6C3 expression on T cells and unresponsiveness in autologous mixed lymphocyte reaction (a property of NZB/B1NJ mice). The 6C3 MAb should prove useful for further genetic and biochemical analysis of the Ly-6 locus and its product(s), and for the delineation of functional subsets of T cells and BM cells in normal and lpr/lpr-bearing mice.     

Up-regulation of interleukin 4 receptor expression on immature (Lyt-2-/L3T4-) thymocytes

In this report, the effect of interleukin 4 (IL-4) on the growth and differentiation of Lyt-2-/L3T4-(2-4-) thymocytes was investigated. It was found that these thymocytes proliferated extensively when cultured in the presence of IL-4 + phorbol myristate acetate without apparent differentiation to Lyt-2+ or L3T4+ cells. We also demonstrated that 2-4- thymocytes constitutively express a high affinity (dissociation constant of 20 to 40 pM) receptor for IL-4. Freshly isolated 2-4- thymocytes expressed on average about 100 IL-4 receptors per cell, but the number of receptors increased approximately 8-fold within 3 days after activation by IL-4 + phorbol myristate acetate. These findings suggest that IL-4 may play an important role in T cell ontogeny by promoting self-renewal of stem cells within the thymus.     

Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule

Monoclonal antibody GK1.5 recognizes a previously undescribed murine T cell surface molecule, designated L3T4, which migrates on SDS-PAGE under reducing conditions as a single band with an apparent m.w. of 52,000. L3T4 is expressed by approximately 80% of thymocytes and by approximately 20% of spleen cells. There appears to be poor correlation between expression of L3T4 by functional T cell clones and expression of Lyt-2, expression of the cytolytic phenotype, and class I MHC antigen reactivity. On the other hand, both a class II MHC antigen-reactive HTL clone and an Lyt-1- Mls-reactive HTL clone express L3T4. Analysis of the effect of mAb GK1.5 on PFC responses in adoptive transfer suggests that L3T4 is expressed by the helper/inducer subset of murine T cells. Expression of L3T4 by murine T cells, however, may correlate primarily with class II MHC antigen reactivity rather than with functional phenotype; mAb GK1.5 profoundly blocks antigen-specific cytolysis by the cloned class II MHC antigen-reactive CTL line A15-1.17. Antigen-specific cytolysis by A15-1.17 is blocked by mAb GK1.5 at a step before the lethal hit. Collectively, the flow cytometric, functional, and biochemical data indicate that L3T4 is similar to the human Leu-3/T4 molecule.     

Qualitative and quantitative heterogeneity in Pgp-1 expression among murine thymocytes

A proportion of Pgp-1+ cells in the thymus have been shown to have progenitor activity. In adult AKR/Cum mice the total Pgp-1+ population in the thymus differs from that of the bulk of thymocytes and is antigenically heterogeneous when examined by flow cytometry. Pgp-1+ thymocytes are enriched for several minor cell populations compared to total thymocytes: B2A2-, interleukin-2-receptor+ (IL-2R+), and Lyt-2-, L3T4-. However, these subsets are still a minor proportion of the Pgp-1+ cells, the majority being Lyt-2+ and/or L3T4+ and B2A2+. Pgp-1+ thymocytes also differ from the bulk of thymocytes in having lower amounts of Thy-1 and in showing a higher proportion of single positive (Lyt-2+, L3T4- or Lyt-2-, L3T4+) cells. Populations of adult thymocytes that are enriched in progenitor cells can be isolated by cytotoxic depletion using either anti-Thy-1 antibody (Thy-1 depletion) or anti-Lyt-2 and anti-L3T4 antibody (Lyt-2, L3T4 depletion). Pgp-1+ cells in progenitor cell-enriched populations are also phenotypically heterogeneous. Pgp-1+ cells in both populations may be IL-2R+ or IL-2R- and B2A2+ or B2A2-. The population of Pgp-1+ cells in progenitor cell-enriched populations in the adult differs from that of the fetus at 14 days of gestation in that in the 14-day fetus, most Pgp-1+ cells are IL-2R+. By Day 15 of gestation, distinct populations of Pgp-1+, IL-2R-; Pgp-1+, IL-2R+; and Pgp-1-, IL-2R+ cells are observed. In the 15-day fetus, as in the adult, many Pgp-1+ thymocytes express low to moderate levels of Thy-1. The total percentage of Pgp-1+ cells in the thymus varies among different mouse strains, ranging from 4 to 35% in the thymus of young adult mice. Pgp 1.1 strains contain more detectably Pgp-1+ thymocytes than Pgp 1.2 strains; however, there is variability in the proportion of Pgp-1+ cells, even among Pgp 1.2 strains. In contrast to AKR/Cum mice, the Pgp-1+ thymocyte population in BALB/c mice, which contain a high proportion of Pgp-1+ thymocytes, closely resembles the total thymocyte population.     

A new mouse lymphoid alloantigen (Lgp100) recognized by a monoclonal rat antibody

A new genetically polymorphic cell surface antigen recognized by a monoclonal rat anti-mouse antibody is expressed on mouse lymphoid cells. Fluorescence analysis on the fluorescence-activated cell sorter (FACS) locates the antigen on thymocytes, lymph node cells, and both T and B cells in the spleen. It also appears on approximately 40% of cells in the bone marrow.Immune precipitations from surface iodinated spleen cells followed by 2-D gel electrophoresis demonstrate that the antigen is a glycoprotein of approximately 100,000 daltons. Since it is expressed in all lymphoid tissues and on both T and B cells, we designate it lymphoid glycoprotein 100 (Lgp100).Strains with Lgp100 include A/J, AKR/J, AKR/Cu, BALB/c, 129/J, CBA/N, C3H/HeJ, CBA/2J, and SJL/J. Strains with no detectable antigen include C57BL/6J, C57BL/10J, C57BR/cdJ, C57L/J, and C58/J. Intercrosses and backcrosses establish a pair of alleles, a positive and a negative one, at a single locus. Heterozygotes display about 50% as much antigen as homozygotes by quantitative membrane immunofluorescence on the FACS. Tests for Lgp100 in 35 recombinant inbred strains from three crosses (CXB, BXB, and BXH) locate this locus on chromosome 1, closely linked to theMls locus.