Expression of theta antigen on mouse thymocytes during the cell cycle

Summary Cells from normal mouse thymus were subdivided according to size and analysed for surface antigen expression, protein content and DNA expression and content. It could be shown, that the expression of theta-antigen followed protein accumulation in individual cells. Protein content on the other hand, was correlated with DNA synthesis even if it varied over a greater range.Only one subgroup of small non-DNA-synthesizing cells was calculated to have a higher density indicative of a more condensed state together with an increased surface antigen content. These cells might represent the G₀ state of the remnant proliferating population and most resembled the small cortical cells.     

Asymmetric selection of T cell antigen receptor alpha- and beta-chains in HLA-B27 alloreactivity.

Endogenous peptides constitutively bind to class I MHC Ag and are thought to be integral parts of allospecific T cell epitopes. However, allospecific TCR can recognize structural features of the alloantigen as foreign. To define some crucial parameters determining HLA-B27 allorecognition, the structure of TCR alpha- and beta-chains from HLA-B27-specific CTL was analyzed. A strategy, based on V alpha and V beta family-specific oligonucleotides, was used for specific amplification and direct sequencing of TCR-alpha and -beta cDNA. We observed nonrandom usage of V beta segments and recurrent structural motifs within beta-chain junctional regions. In contrast, no structural restrictions were apparent among alpha-chains, even from CTL clones of related fine specificity. These results indicate an asymmetric contribution of TCR alpha- and beta-chains to HLA-B27 allospecificity among the CTL clones analyzed. They suggest recognition of multiple peptides and involvement of beta-chain junctional regions in recognizing shared motifs among some of these peptides.     

T cell antigen receptor expression by subsets of Ly-2-L3T4- (CD8-CD4-) thymocytes

The V beta 8-specific mAb F23.1 and KJ16 were used as fluorescent stains to test for TCR expression on the surface of subpopulations of early, CD4-CD8- (L3T4-Ly-2-) thymocytes from adult CBA mice. A surprisingly high proportion (27%) of Ly-2-L3T4- thymocytes were strongly F23.1 and KJ16 positive. No positive cells were detected among Ly-2-L3T4- thymocytes from V beta 8-negative SJL mice. In contrast to the adult thymus, Ly-2-L3T4- cells from embryonic CBA thymus lacked F23.1-positive cells. Subsets of adult CBA Ly-2-L3T4- thymocytes were separated to determine which expressed V beta 8. The major subset, Ly-1 low B2A2-M1/69+Thy-1+Pgp-1-, representing a phenotype similar to embryonic Ly-2-L3T4- thymocytes and the phenotype commonly isolated from adult thymocytes as Ly-1 "dull," lacked cells strongly positive for F23.1. In contrast, a series of subsets of adult CBA Ly-2-L3T4- thymocytes which were B2A2-M1/69- and Pgp-1+ all included strongly F23.1-positive cells. A minor subset, negative for most markers except Pgp-1 and presumed on the basis of this phenotype and some reconstitution studies to include the earliest intrathymic precursors, contained 28% F23.1-positive cells. However, no F.23.1-positive cells were detected in equivalent "prethymic" populations from bone marrow or from athymic mouse spleen. The subsets of Ly-2-L3T4- thymocytes which were Ly-1 high, B2A2-M1/69-, and Pgp-1+ all contained about 70% F23.1-positive cells, indicating a V beta 8 usage much higher than the mature T cell average. These results indicate that a series of distinct developmental events have occurred within these CD4-CD8- thymocytes previously considered as a single group of early precursor cells, and that some aspects of repertoire selection may be occurring amongst thymocytes which lack CD4 or CD8.     

Kinetics of B cell receptor signaling in human B cell subsets mapped by phosphospecific flow cytometry

Differences in BCR signaling may govern outcomes as diverse as proliferation and cell death. We profiled BCR signaling kinetics in subsets of primary human B cells using flow cytometry. In the predominant population expressing IgM, BCR cross-linking led to a quick burst of Syk, ERK1/2, and p38 signaling. In contrast, IgG B cells sustained higher per-cell ERK1/2 phosphorylation over time. This dichotomy suggested a mechanism for dampening signals transmitted by IgM. Regulatory phosphatase activity in IgM B cells was BCR-mediated and initiated more slowly than kinase activity. This BCR-mediated phosphatase activity was sensitive to inhibition by H(2)O(2) and required to attenuate IgM BCR signaling. These results provide the first kinetic maps of BCR signaling in primary human B cell subsets and enable new studies of signaling in B cell disorders, such as autoimmunity and cancer.     

Differential expression of H-Y antigen on lymphocyte subsets: analysis by flow cytometry

Expression of H-Y antigen in human white blood cells was measured using flow cytometry with monoclonal antibodies. In this system, lymphocytes were stained preferentially in the male, and to a lesser extent in the female. Analysis of the lymphocyte subsets with biotinylated H-Y antibody conjugated with streptavidin-fluorescein isothiocyanate (FITC) and subset-specific antibody conjugated with phycoerythrin derivative (RD1) revealed differential expression of H-Y among the subsets of the male. In samples from eight men, 41.1% +/- 21.7% of B cells (B1) were stained, compared with 20.7% +/- 12.8% of cytotoxic-suppressor T cells (T8) and 5.4% +/- 3.0% of helper-inducer T cells (T4). In samples from seven women, 12.4% +/- 10.9% of B cells were stained, but staining of T cells was negligible.     

Mapping T cell epitopes by flow cytometry

Epitope mapping by flow cytometry is a very modern approach that not only identifies T-cell epitopes but simultaneously allows for detailed analysis of the responding T-cell subsets including lineage, activation marker expression, and other markers of interest. The most frequently used approach is based on the identification of intracellular cytokines in secretion-inhibited activated T cells following stimulation with peptides or peptide pools. A more recently developed assay analyzes T-cell proliferation by measuring the decrease in carboxyfluorescein diacetate succinimidyl ester staining in proliferated cells. This article includes information on peptide configuration, a section on the design and efficient application of peptide pools, and working laboratory protocols for both assays.     

Ontogeny and expression of non-MHC-restricted T cell antigen-binding molecules by thymocytes and peripheral T cell subsets

The ontogeny of T cells which express major histocompatibility complex (MHC)-unrestricted T cell antigen-binding molecules (TABM) on the cell membrane was investigated. We used a rabbit anti-mouse TABM antiserum to investigate the expression of TABM by subsets of adult thymocytes, peripheral T cells, and thymocytes during gestation. TABM are expressed by CD4+, CD8-, CD4+, CD8+ thymocytes and single-positive thymocytes. During gestation, TABM are expressed as early as Day 16, and at birth the expression of TABM on thymocytes has reached adult levels. Data are also presented which suggest that the expression of membrane TABM (mTABM) on peripheral T cells can be upregulated during T cell activation. The results suggest that TABM are expressed by different T cell subsets and that TABM+ cells may utilize the same intrathymic developmental pathway as that of T cells which express the alpha/beta T cell receptor.     

Expression and tyrosine phosphorylation of the T cell receptor zeta-subunit in human thymocytes.

Recent evidence suggests that the zeta-subunit of the TCR complex plays a critical role in transducing signals initiated by the Ag receptor heterodimer. Because thymic maturation involves specific interactions between the TCR complex and thymic stromal cells, the zeta-subunit has been postulated to also play a role in this process. To assess the potential for zeta to contribute to thymocyte maturation, we have used an anti-zeta mAb (TIA-2) to quantitate its expression in mature (CD3bright) and immature (CD3dim and CD3-) populations of human thymocytes. Using both flow cytometric and immunoblotting analysis, we found that the relative expression of TCR-zeta varied directly with the surface expression of CD3. Importantly, TCR-zeta was detected in the majority of CD3- thymocytes, indicating that its expression precedes the surface appearance of CD3:TCR. In thymocytes, TCR-zeta was found to be constitutively phosphorylated on tyrosine residues. The relative expression of phospho-zeta varied directly with the maturational stage of the thymocyte, with the mature (CD3bright), single positive cells accounting for most of the phospho-zeta found in the human thymus. The expression of phospho-zeta could be significantly increased by activating thymocytes with mAb reactive with either CD3 or CD2. These results suggest that TCR-zeta is functionally linked to the major thymocyte activation receptors.     

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.     

Cell kinetic analysis of mixed populations using three-color fluorescence flow cytometry.

The development of antibodies to DNA-incorporated thymidine analogs has in turn led to the development of flow cytometric techniques for rapidly measuring cell kinetics parameters. More recently, these techniques have been applied to clinical tumor material. One problem with such measurements has been the difficulty of distinguishing malignant cells from coexistent normal cells in the biopsy material. In the present study, the feasibility of selecting out the desired malignant cell population for kinetic analysis from a mixture of cells was tested in vitro. An anticytokeratin antibody was used to discriminate between a mixture of tumor cells (cytokeratin positive) and normal cells (cytokeratin negative). The cell lines chosen for the study, A549 human lung carcinoma cells and Chinese hamster ovary (CHO) cells, were pulse labeled with iododeoxyuridine (IdUrd) and sampled every hour up to 16 hours. Selecting out cells from the mixture required the application of three-color fluorescence flow cytometry, which was carried out using the fluorochromes FITC (fluorescein isothionate, green fluorescence, IdUrd-DNA antibody), PE (phycoerythrin, orange fluorescence, cytokeratin antibody), and PI (propidium iodide, red fluorescence, DNA). This allowed single laser excitation. The staining procedure involved incubation with the IdUrd antibodies (specific antibody plus FITC-conjugated second antibody) followed by the cytokeratin antibodies (specific antibody plus PE-conjugated second antibody) and lastly by the DNA stain containing RNase. Two analysis methods of the IdUrd/DNA cytograms were applied: a mid-S window analysis and a relative movement (RM) analysis. Results of the analyses for cells selected out of mixtures were compared with results of cells stained and analyzed separately. A clear separation of the two cell lines could be obtained on the basis of orange fluorescence (cytokeratin content) despite a large overlap of their DNA histograms. By gating on high or low orange fluorescence, almost pure populations of the individual cell types could be selected out for further kinetic analysis. Little difference was seen, with both the mid-S and RM analyses, between cells gated from mixtures or stained separately. It is concluded that this technique is feasible for use on clinical material, provided good cell suspensions can be obtained, leading to the hope of increasing the accuracy of kinetic measurements on human tumors.     

Analysis of DNA degradation in the thymocytes of irradiated rats by flow cytometry

The method of flow cytofluorometry of cells treated with probes specifically bound to AT- or GC-pairs of DNA was used to study DNA degradation in thymocytes of irradiated and hydrocortisone-treated rats. Death of thymocytes was shown to be accompanied by the decrease in the DNA content. The main regularities in the formation and accumulation of cells, the DNA content of which being lower than that of diploid cells, were the same as those of the internucleosome DNA fragmentation.     

Depletion of mouse alpha beta T cell antigen receptor bearing lymphocytes by neonatal monoclonal antibody treatment.

Neonatal treatment with a monoclonal antibody specific for the alpha beta TCR results in mice with a long term, severe depletion in the number of alpha beta T cells in the periphery. Significant numbers of T cells reappear in the periphery about age 65 days, but these cells tend to lack expression of CD4 or CD8. Splenocytes of antibody-treated mice are less sensitive to mitogen stimulation or stimulation with MHC allogeneic cells. The level of serum IgG but not IgM was decreased by the treatment. Anti-alpha beta TCR antibody treatment decreased single-positive T lymphocytes that express high levels of the CD3/alpha beta TCR complex from the thymus, suggesting that the treatment could act in part by affecting negative selection of alpha beta TCR+ thymocytes. This treatment does not, however, detectably affect either the homing or the numbers of gamma delta T cells which are abundant in the intestinal epithelium, but which remain a minor population in the spleen and lymph nodes. This supports the hypothesis that gamma delta T cells are developmentally autonomous from alpha beta T cells. These mice provide an excellent model system for assessing the developmental and functional role of gamma delta T lymphocytes in vivo.     

Expression of T cell receptors by thymocytes: in situ staining and biochemical analysis.

We have examined the in situ expression of T cell receptor (TCR) V beta 8 protein in murine thymus during ontogeny using the monoclonal antibody F23.1. Positive cells were first detected at day 15 of gestation (0.6%). By day 16 the frequency of positive cells increased dramatically (4.18%). From day 16 to day 17 positive cells doubled (8.17%). The first clusters of F23.1 positive cells were seen at day 17. In the cortex, positive cells decreased from 14% in the newborn mice to 9.8% in 8-week-old mice, whereas in the medulla the frequency remained unchanged at 20%. The antibody F23.1, as well as an antiserum raised against the constant region of the beta chain, immunoprecipitated receptor dimers from highly purified Lyt2+, L3T4+ thymocytes and from two thymic lymphomas of cortical phenotype which express full size alpha and beta mRNA. The receptor dimer could not be precipitated from Lyt2-, L3T4- thymocytes. The results are discussed with regard to intrathymic T cell repertoire selection.     

Cross-reactive antigen recognition by an encephalitogenic T cell receptor. Implications for T cell biology and autoimmunity.

The dominant immune response to rat myelin basic protein in H-2u mice is directed against the acetylated, N-terminal peptide Ac1-11 (AcASQKR-PSQRHG). This peptide causes encephalomyelitis on injection into mice of the H-2u haplotype. Only two residues of the peptide are required for ligation of the TCR from an Ac1-11-specific T cell hybridoma. Proline at position 6 could not be substituted by any other L-amino acid, whereas glutamine at position 3 could be replaced by phenylalanine, histidine, methionine, or tyrosine. Cross-reactive recognition of these residues appears to be specific, because increasing the affinity of each analogue for its MHC restriction element, by replacing lysine with tyrosine at position 4, did not alter the pattern of cross-reactivity. For the majority of substitutions at this position, a lack of stimulation could not be explained by failure to bind to I-Au. However, competition binding studies showed that introduction of proline at position 3 reduced the efficacy of binding to I-Au. Cross-reactive analogues of Ac1-11 were injected into H-2u mice to test the extent to which cross-reactive T cell activation might lead to autoimmune disease in this model. An analogue containing methionine at position 3 caused clinical experimental autoimmune encephalomyelitis in a small percentage of H-2u mice.     

Evaluation of NK-to-target cell binding and evidence for T cell conjugates by flow cytometry

A flow cytometric methodology was set up to assess the binding capability of peripheral blood NK and T cells to the K562 tumor cell line. Differential side scatter characteristics between effectors and targets were used to analyze conjugated and unconjugated cells. The previous labeling of NK and T cells with anti-Leu 11c and anti-Leu 4 monoclonal antibodies, allowed the distinction between unconjugated non-fluorescent and conjugated fluorescent targets and the percentual evaluation of bound anti-Leu 11c⁺ and anti-Leu 4⁺ cells.Abbreviations NK Natural Killer - MoAb Monoclonal Antibody - SSC Side Scatter - FLS Forward Light Scatter - PE Phycoerythrin - MHC Major Histocompatibility Complex - GVHD Graft Versus Host Disease