Identification and partial characterization of surface antigens specific for human normal and leukemic T cells

Rabbit antisera to membrane preparations of human thymocytes (anti-HTLA), T lymphocytes of the MOLT 4 cell line or B lymphocytes of the LIK cell line, have been serologically characterized and used for the identification of surface membrane antigens specifically expressed by human lymphocyte subpopulations. By immunoprecipitation of surface ¹²⁵I- or NaB (³H)₄-labeled protein followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the precipitates, the anti-HTLA antiserum was shown to detect on thymocytes three glycoproteins of apparent molecular weights 160000, 110000 and 45000. The first two only appeared to be sialylated. A similar pattern of antigens could be detected on cultured T lymphocytes, except for the 45 kDalton glycoprotein which could not be detected on the surface of these cells. None of these proteins were detected on ¹²⁵I-labeled Raji cells. The serological specificity of the anti-MOLT 4 antiserum for T lymphocytes was more restricted since it only precipitated the 160 kDalton antigen. In addition, the 45 kDalton protein was shown to be sensitive to trypsin treatment as was the sheep red blood cell (SRBC) receptor. The results shown suggest a possible activity as SRBC receptor for this particular protein. In contrast, the anti-LIK antiserum which behaves as an anti-HLA-DR reagent in cytotoxicity testing, only precipitates from cultured B lymphocyte lysates the typical Ia-like 28 and 33 kDalton polypeptides.     

Surface antigens specifically expressed by activated T cells in humans

By immunizing BALB/c mice with human T cells derived from a secondary mixed lymphocyte reaction (MLR) and successive fusion of the spleen cells with P3X63Ag8U1 myeloma cells, several monoclonal antibodies to activated T cells were obtained. Four of them (MLR 1-4) were shown to be specific for activated T cells only, showing no reactivity with peripheral blood cells. The antigens recognized by these antibodies are differently distributed and appear on T cells at different times after stimulation. Thus, MLR 2 stains nearly all the responder T cells present in a secondary MLR, while MLR 1, 3 and 4 stain only a fraction. MLR 2 and 3 are also present on mitogen-stimulated cells. At least 3 of these MLR antigens may define different sets of activated T cells, and are likely to become useful in monitoring T cell activation also in the course of diseases involving the immune system.     

Differential expression of T cell differentiation antigens and major histocompatibility antigens on activated T cells during the cell cycle

In this report we have analyzed cell cycle-related fluctuations of both quantity and density of the T cell differentiation antigens, CD3 (T3), CD4 (T4) and CD8 (T8), as well as the major histocompatibility complex (MHC) antigens on the cell surface of activated T cells. Phytohemagglutinin-activated T cells cultured for 3 days with or without conditioned medium or for 10 days with conditioned medium and mixed lymphocyte culture-derived T cell clones were used for the analysis. Correlated measurements of the surface antigen quantity (immunofluorescence), DNA content (dye Hoechst 33342), and cell size (light scatter), not influenced by synchrony induction methods and cell fixation, were performed by dual-beam flow cytometry. Our results demonstrate that the T cell differentiation antigens, CD3, CD4 and CD8, and class I MHC antigens are increased in density in the G1 phase for all activated T cells tested. In contrast, class II MHC antigens are increased in density in the G2 phase of activated T cells maintained with conditioned medium. Since it is known that the T cell differentiation antigens and class I MHC antigens on activated T cells are necessary for proliferation of T cells, our study suggests that this effect is more significant in the G1 phase. The cell cycle changes in expression of class I and class II MHC antigens, but not of the T cell differentiation antigens, appear to be mediated by soluble factors, probably including interferon-gamma, which could produce a differential increase of class I and class II MHC antigens on G2 phase cells.     

Detection of cytoplasmic CD antigens within normal human peripheral blood leucocytes

Polymorphonuclear neutrophils (PMNs) are capable of synthesizing various pro-inflammatory cytokines which may indirectly influence specific immune responses. PMNs may also have the capacity to present foreign peptides to helper T cells (Th cells). In support of this hypothesis, recent studies have shown that neutrophils, when activated by the correct combination of cytokines, can be induced to express cell surface major histocompatibility complex (MHC) Class II (DR) antigen, CD80 (B7.1) and CD86 (B7.2): molecules required for antigen presentation and subsequent T-cell activation. In this study we have used normal "resting" human peripheral blood neutrophils and demonstrated, using a mild fixation and permeabilization protocol, significant cytoplasmic "stores" of these molecules known to be important in antigen presentation. Cytoplasmic MHC Class II antigen was found with two out of 20 normal donors tested whereas cytoplasmic CD80 and CD86 were found to a variable extent within all normal donors. Surprisingly, we also found several other neutrophil cytoplasmic CD antigens more commonly associated with B cells, i.e. CD20, CD21 (CR2/EBV-R) and CD22 (BL-CAM). All of these antigens were confined to the "resting" cell cytoplasm and were never found to be expressed on the cell surface. To exclude the possibility that these antigens were absorbed from plasma and to provide evidence for active synthesis, we used a novel whole blood in situ hybridization flow cytometry assay method to detect mRNA specific for these antigens within normal PMNs. We also conducted real-time polymerase chain reactions to confirm these findings using CD22 as a good example of an "inappropriately expressed" CD antigen. These observations therefore provide support for the hypothesis that human PMNs have the potential to express molecules required for antigen presentation and cell signalling.     

Comparative expression of T9, T10, and Ia antigens on activated human T cell subsets

In the present study, the expression of three surface molecules T9, T10, and Ia, which are found on activated T lymphocytes, was examined utilizing monoclonal antibodies and indirect immunofluorescence. These antigens were shown to appear on T lymphocytes in a defined temporal sequence which was dependent on the specific triggering stimulus. Moreover, when T cells were fractionated into individual subsets of T4+ inducer and T8+ cytotoxic/suppressor lymphocytes, the expression of all three molecules was restricted to the T4+ subset following soluble antigen stimulation. By contrast, both subsets expressed these surface determinants following mitogen or alloantigen stimulation. The above results suggest that there may be successive stages in the T cell activation process associated with the appearance of unique cell surface antigens and further support the view that various triggering stimuli activate T cell populations differently.     

"Ia-like" antigens on human T cells.

Human T lymphocytes have been tested for cell surface p. 28,33 "Ia-like" heteroantigen and DRw alloantigens. Small numbers (1--5%) of sheep (E) rosette or T antigen-positive, surface immunoglobulin-negative (E+, T+, smIg-) T cells were Ia+; these cells appeared to be restricted to the TG subset. Following activation by allogeneic lymphocytes or sperm, or by purified protein derivative of tuberculin (PPD), the proportion of positive T cells increased substantially. DRw typing indicated that Ia specificities on activated T cells were not acquired passively from the stimulator cells, suggesting therefore that either "selection" of a small DRw+ cell subset or derepression and/or exposure of DR locus gene products occurs during T cell activation.     

Simultaneous detection of T6 and HLA-DR antigens distinguishes three cell subpopulations in dispersed normal human epidermal cells

Normal human dendritic epidermal cells (EC) show specific surface membrane markers (T6 and DR antigens). This study presents evidence that, in dispersed EC suspensions, these membrane antigens may distinguish three antigen-positive EC subsets by means of double immunofluorescence labelling: 97.3 ± 1.2% of the labelled cells were DR(+) T6(+) while 2.2 ± 0.9% were DR(+) T6(?) and 0.5 ± 0.6% were DR(?) T6(+). Immunoelectron microscopy with gold particles confirms the co-existence of T6-positive and T6-negative epidermal cells.     

Signal transduction by HLA class II antigens expressed on activated T cells.

Human T cells express HLA class II antigens upon activation. Although activated, class II+ T cells can present alloantigens under certain circumstances, the functional role of class II antigens on activated T cells remains largely unknown. Here, we report that cross-linking of HLA-DR molecules expressed on allospecific, CD4+ T clones and cell lines can function as transduction elements that trigger rapid cellular responses including tyrosine phosphorylation of cellular proteins and mobilization of Ca2+ from internal stores. The proteins phosphorylated on tyrosine were distinct from those observed after cross-linking CD4. Ligation of CD4 and class II molecules generated a synergistic effect of the intracellular free Ca2+ concentration response that required an interaction between the molecules on the cell surface. Since class II is the natural ligand for CD4, the present data suggest that class II is induced on activated T cells to regulate CD4 function, possibly by specific interaction with the CD4-associated p56lck protein tyrosine kinase.     

Professional presentation of antigen by activated human T cells

Activated human T cells express class II molecules, but their capacity to present soluble antigens and stimulate T cells has been repeatedly questioned. Two lines of evidence indicate that T cells may indeed function as professional antigen-presenting cells. First, T cells that have been recently activated can efficiently capture, process and present tetanus toxoid to class II-restricted T cell clones. This capacity correlates with the rate of class II synthesis. Second, activated T cell clones express high levels of B7, are powerful stimulators in mixed lymphocyte reactions, and their stumulatory capacity is inhibited by soluble CTLA4 or anti-B7 antibody. Furthermore, expression of B7 can be detected in vivo on T cells from biopsies of patients with liver disease. Presentation of soluble antigen by activated T cells may play a role in the amplification of the specific response, and possibly in immunopathological states.     

Stimulation of activated rat T cells in vitro by Listeria monocytogenes antigens.

A soluble extract of Listeria monocytogenes bound firmly and in similar amounts to a variety of rat cells. Cells that bound this material differed in their capacity to stimulate the in vitro proliferation of lymphocytes obtained from the thoracic duct of Listeria-immune donors. The capacity of cells to serve as antigen-presenting cells in this system coincided or closely overlapped the expression on these cells of an Ia antigen-like structure. Three lines of evidence indicate that T cells respond to L. monocytogenes antigen: the responder cells are members of a nylon-wool nonadherent population that lacks readily detectable surface immunoglobulin; they express determinants recognized by the W3/25 monoclonal antibody (a surface marker of rat peripheral T cells); and they are stimulated optimally by L. monocytogenes antigen when the latter is displayed on cells that share a haplotype with the responder lymphocytes.     

Suppression of antibody formation by bone marrow T cells activated by histocompatibility antigens

The suppressive action of bone marrow T cells activated by histocompatibility antigens on antibody formation was studied. The bone marrow of CBA mice was shown to contain thymus-dependent lymphocytes which, on hyperactivation by repeated transplantation into F₁ recipients, have a suppressive action on the development of the cooperative immune response to sheep's red cells. Preliminary treatment of the bone marrow cells with antithymocytic globulin and complement abolished the suppressive effect.Institute of Biophysics, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Sciences of the USSR, P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 3, pp. 300–302, March, 1977.     

Immune induction of Ia antigens in activated T cells and in kidney epithelial cells in mice.

A rat monoclonal antibody against mouse Ia antigens was used to investigate the induction of Ia expression by activated T cells and renal epithelial cells during immune responses. The induction of low levels of surface Ia on activated helper and cytotoxic/suppressor T-cell phenotypes was directly demonstrated by differential fluorescence staining. Renal epithelial cells were observed to become strongly Ia-positive after primary immunization with alum-precipitated protein antigen, or after secondary challenge with the soluble antigen used for priming.     

Virus-specific CD8+ T lymphocytes within the normal human liver

The frequency and phenotype of human antiviral memory CD8(+) T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8(+) T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature 'effector memory' phenotype of antigen-specific CD8(+) T cells within the liver. An enrichment of an activated subset of NKT cells (V alpha 24/V beta 11) was also observed, a finding which may be relevant to the regulation of the antiviral populations.     

Do carbohydrate antigens stimulate human T cells? Review article

T cells fail to recognize free antigenic determinants. What the T-cell receptor recognizes is a complex consisting of a peptide fragment cleaved from antigen and self-MHC structures on the surface of antigen-presenting cells. While extensively investigated with protein antigens, only limited information is available on the capability of T cells to recognize carbohydrate antigens in a specific way. Therefore, we have investigated the specificity of human T-cell lines and clones reactive to streptococcal A (Strep A) vaccine. It was found that neither soluble streptococcal A carbohydrate (A-CHO) nor synthetic oligosaccharides deduced from bacterial carbohydrates could stimulate Strep A-reactive T cells, although A-CHO stimulates specific antibody production in B cells very effectively. In conclusion, Strep A-specific T cells seem to recognize other structures of the bacterial vaccine than A-CHO. This was confirmed by retained stimulation after removal of carbohydrate epitopes by periodate treatment. Such Strep A-reactive T cells are frequently (greater than 10(-3] found in CD4+ T cells of healthy donors. Implications of this finding with regard to anti-carbohydrate immune responses are discussed.     

Recognition mechanism of non-peptide antigens by human gammadelta T cells

The majority of gammadelta T cells in adult human blood exhibit Vgamma2/Vdelta2-TCR and specifically respond to various kinds of non-peptide antigens. In this study, we comparatively analyzed the CDR3 repertoires of Vgamma2-gamma and Vdelta2-delta chain genes in the adult and cord blood. It was confirmed that the vast majority of adult gammadelta T cells exhibited Vgamma2-gamma chains bearing a Jgamma1.2 segment with no or short N-region and Vdelta2-delta chains with a conserved hydrophobic residue (leucine, valine or isoleucine) at position 97 encoded by N-region of Vdelta/Jdelta junction (deltaL97). The cord blood cells stimulated with pyrophosphomonoester antigen in vitro showed preferential expansion of the gammadelta T cells expressing Vgamma2- and Vdelta2-TCR chains with these structural features as compared with those stimulated with a polyclonal mitogen phytohemagglutinin. TCR gene transfer studies indicated that alanine substitution of lysine at position 108 in Jgamma1.2 (gammaK108) or deltaL97 abrogated the responsiveness of Vgamma2/Vdelta2-TCR to all kinds of the non-peptide antigens without affecting the response to anti-CD3 antibody. Furthermore, alanine substitution of arginine at position 51 in Vdelta2 segment (deltaR51) adjacent to gammaK108 in the Vgamma2/Vdelta2-gammadelta TCR also abolished the antigen responsiveness. These results strongly suggested that a hydrophobic and two cationic residues (deltaL97, gammaK108 and deltaR51) clustered in a particular topology at the surface edge of the pocket structure of Vgamma2/Vdelta2-gammadelta TCR played essential roles in the recognition of non-peptide antigens.     

Expression of polymorphic B-cell antigens on human kidneys

We have examined the expression on a panel of 22 human kidneys of polymorphic B-cell determinants recognized by mouse monoclonal antibodies. Monoclonal antibodies from a mouse immunized with an antigenic preparation from a DR4 positive B-cell line reacted preferentially with kidneys from DR4 positive donors (p less than 0.005), and the pattern of reactivity with kidney tissues was similar to that of antibodies to monomorphic determinants of class II. However, these antibodies did not show clear specificity for DR4 on lymphocytes in standard serological analyses. These results provide evidence for the expression of polymorphic class II determinants on human kidneys. Reasons for the differences in the apparent specificities of the monoclonal antibodies when tested on kidney sections and lymphocytes are discussed.     

Structure and function of lipid rafts in human activated T cells

Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, have been shown to function as signaling platforms in T cells. Surface raft expression is known to be increased in human T cells upon activation, and this increased raft expression may account for efficient signaling capability and decreased dependency for co-stimulation in effector and/or activated T cells. However, raft-mediated signaling ability in activated T cells remains to be clarified. In this study, we analyzed the structure and function of lipid rafts in human activated T cells. We demonstrated that raft protein constituents are dramatically changed after activation along with an increase in lipid contents. T cells stimulated with anti-CD3 plus anti-CD28 antibodies showed an increase not only in surface monosialoganglioside GM1 expression but also in total amounts of raft-associated lipids such as sphingomyelin, cholesterol and glycosphingolipids. Raft proteins increased after activation include Csk, Csk-binding protein and Fyn, the molecules known to be involved in negative regulation of T cell activation. Consistent with the increase in expression of these proteins, TCR-mediated Ca(2+) response, a response dependent on raft integrity, was clearly inhibited in activated T cells. Thus, the structure and function of lipid rafts in human activated T cells seem to be quite distinct from those in naive T cells. Further, human activated T cells are relatively resistant to signaling, at least transiently, by TCR re-stimulation even though their raft expression is increased.     

Expression of B-cell antigens by Hodgkin''s and Reed-Sternberg cells.

Twenty frozen and 55 paraffin sections of lymphnode specimens from 55 patients with pretreatment Hodgkin's disease (nodular sclerosis Hodgkin's disease, n = 45; mixed cellularity Hodgkin's disease, n = 10) were studied by immunohistochemistry and molecular analysis to determine the phenotype of Hodgkin's and Reed-Sternberg cells (HRS). In all cases the HRS cells were CD45-, and CD30+, and in 43/55 (78%) cases they were CD15+. In 48/55 cases (87%) HRS cells were reactive with at least one B-cell marker (CD19, CD20, CD22, CDw75, MB2), 8/55 cases (14.5%) showed reactivity (mainly cytoplasmic) of a subpopulation of HRS cells with the T-cell markers CD3 and beta F1. All cases that expressed T-cell antigens were also reactive with at least one B-cell marker. In frozen sections, a minority of HRS cells in each case studied showed cytoplasmic positivity for bcl-2 protein. Rearrangement of immunoglobulin heavy chain genes was detected in one case and of T-cell receptor beta chain genes in none. The authors were unable to confirm previous reports of bcl-2 gene rearrangement in Hodgkin's disease. The results strongly support a B lymphocytic origin of HRS cells.