Expression of non-MHC-restricted T cell antigen-binding molecules by thymic lymphocytes

Heterologous antisera which recognize non-major histocompatibility complex (MHC)-restricted T cell antigen-binding molecules (TABM) were used to characterize the expression and structure of TABM on thymic lymphocytes. Approximately 70% of thymocytes express membrane molecules bound by anti-TABM antibodies (mTABM). Antibody activity for thymocyte TABM could be removed by adsorption to splenic T cells, but not by adsorption to splenic B cells. Similarly, adsorption of the antiserum to thymocytes or splenic T cells removed antibody activity to a purified TABM whereas adsorption with B cells had no effect. Radioiodinated thymic and splenic T cell mTABM were resolved by 2D-polyacrylamide gel electrophoresis and when reduced, both populations of mTABM migrated primarily as Mr 23,000 proteins with an isoelectric point range of 6.8-7.8. Multimers of this protein were also observed at Mr 85-97,000 and 130-150,000 on both thymocytes and splenic T cells. These data indicate that MHC-unrestricted antigen-binding molecules are expressed by a majority of thymocytes and these thymic TABM are structurally and antigenically similar to mTABM on peripheral cells. This suggests an ontogenic relationship between thymic TABM and peripheral TABM.     

T cell non-MHC-restricted antigen-binding molecules secreted or associated with the cell membrane are antigenically distinct

Some T cells produce membrane-associated or soluble molecules which bind nominal antigen specifically (TABM) and effect immunoregulation or events similar to cell-mediated hypersensitivity. We have used polyclonal antisera raised against an azobenzene arsonate (ABA)-specific TABM secreted by an ABA-specific T cell hybrid or against TNP-specific polypeptides produced by immunoregulatory T cells to identify the expression of soluble (secreted) or membrane-associated TABM. Ascites fluid or culture medium containing a T cell hybrid or T cell lines, respectively, contain TABM recognized only by an antiserum specific for the secreted T cell hybrid (ABA-specific) derived TABM. Conversely, an antiserum that recognized the TNP-specific polypeptides detected cell-membrane associated TABM but did not bind TABM secreted by the T cell hybrid or cell lines.     

Cell cycle-dependent expression of antigen-binding and I-J-bearing molecules on suppressor T cell hybridomas

The I-J and antigen-binding chains with constant region determinant (Ct) that compose an antigen-specific suppressor T cell factor were found on the surface of suppressor T cell hybridomas, serologically and morphologically demonstrated by a fluorescence-activated cell sorter (FACS) and immunoelectron microscopic analyses. Moreover, the surface expression of the I-J and Ct-bearing chains fluctuating with the same kinetics depended entirely upon the cell cycle. The maximum expression of these two chains was observed in the early stage of the M phase, and the minimum in the S phase. Similarly, the magnitude of the suppressor activity was maximal in the late stage of the M phase, and was minimal in the S phase. The results therefore demonstrated that there exists good correlation between the cell surface expression of the I-J and Ct-bearing chains and the magnitude of the suppressor activity produced. The antigen recognition units on suppressor T cell hybridomas have serologically and morphologically been characterized by using radiolabeled antigens or monoclonal antibodies against the I-J or Ct on the antigen-binding molecule. Cell-binding assay and radioautographic analysis demonstrated that the suppressor T cell hybridoma possesses the capacity to bind native antigen in an antigen-specific fashion as does the hybridoma-derived, antigen-specific suppressor factor composed of the I-J and the Ct-bearing chains, indicating that the recognition unit on the cell surface is composed of a structure similar to the factor.     

Flow sorting of antigen-binding B cell subsets

Antigen binding was used as a probe in the definition of functional B cell heterogeneity. Unprimed, anti-Thy 1 and complement-treated spleen cells were stained with fluorescent trinitrophenylated bovine serum albumin (FL-TNP-BSA). These cells were sorted, fluorescence negative from fluorescence positive, by using a multiparameter cell sorter and assayed for precursor frequency in antibody responses to TNP by limiting dilution analysis. The cells that bound FL-TNP-BSA were demonstrated to be enriched for antibody-forming precursors to the antigens TNP-lipopolysaccharide (LPS), TNP-sheep red blood cells (SRBC), and TNP- or DNP-Ficoll, whereas the fluorescence negative cells were depleted for these responses. B cells that bound FL-TNP-BSA were then sorted into populations that bound a moderate or high amount of FL-TNP-BSA. The B cells responsive to TNP-LPS and TNP-SRBC were present in both the moderate and high binding populations. In contrast, the B cells responsive to TNP- or DNP-Ficoll were present only in the cells that bound a moderate amount of FL-TNP-BSA. These experiments suggest that there is a population of B cells in adult mouse spleen that binds large amounts of antigen, and that can respond to antigen carried on LPS or SRBC but not carried on Ficoll.     

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.     

Information transfer between T cell subsets is directed by I-J+ antigen nonspecific molecules

T cell antigen-specific suppressor factors (TsF) consist of two distinct polypeptide chains: one that binds antigen (ABM) and one that bears I-J region markers (I-J+ chain). We studied the functional role of these two molecules in delivering the biologic message of suppression to its appropriate target cell. Two different biologically active TsF were used in these studies: TsiF, a T suppressor-inducer factor consisting of an ABM secreted by Ly-1 T cells (Ti-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Ji), which initiates the suppressor circuit by inducing an Ly-1,2 T cell; and TseF, a T suppressor-effector factor consisting of an ABM secreted by Ly-2 T cells (Te-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Je), which delivers the biologic message of suppression to the T helper (TH) cell. In both TsF, the ABM and I-J+ chain are noncovalently associated and can be easily separated. Both molecules must be present, however, for biologic activity of the TsF to be manifest. We studied the role of each chain in delivering these biologically active messages by constructing "hybrid" factors made from mixing the ABM from TsiF with I-J+ chains from either TsiF or TseF and determined which of these chains could reconstitute functional TsiF activity. Likewise, we mixed the AMB from TseF with I-J+ chains of TsiF or TseF to determine which I-J+ chain could reconstitute TseF activity. We found that I-J+ chain from TsiF (I-Ji) can reconstitute ABM from TsiF to form a functional TsiF capable of inducing suppression but cannot reconstitute ABM from TseF to form a functional TsiF capable of suppressing the activity of TH cells. Likewise, the addition of I-J+ chain from TseF to ABM from TseF can reconstitute its ability to suppress TH responses, but I-J+ chain from TsiF plus ABM from TseF has no effect on these TH cell responses. We did find, however, that this hybrid TsF composed of the ABM from TseF and the I-J+ chain from TsiF is capable of suppressing the Ly-1,2 Ttrans cell, the cell normally induced by the ABM + I-J+ suppressor inducer complex from T suppressor-inducer cells (TsiF).(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple cytolytic mechanisms displayed by activated human peripheral blood T cell subsets.

It has been proposed that CTL-mediated cytotoxicity may involve multiple lytic mechanisms. We have examined both the antibody-redirected cytolytic potential and the direct cytotoxicity of purified human peripheral blood high buoyant density CD4+ and CD8+ T cells activated with IL-2 and anti-CD3 mAb. TNF-sensitive and TNF-resistant targets and various metabolic inhibitors were used to compare the antibody-redirected cytotoxicity of T cell subsets and discern the role of potential lytic mediators. In a 4-h assay against several different nitrophenyl-modified targets, the heteroconjugated antibody (anti-CD3-anti-nitrophenyl) redirected cytolytic potential of 72-h activated CD4+ T cells was inhibited by the continuous presence of actinomycin D, cycloheximide, and EGTA, but not mitomycin C, cyclosporin A, or cholera toxin (CT). Conversely, only CT and EGTA inhibited the antibody-redirected cytolytic potential of activated CD8+ T cells. Despite both CD4+ and CD8+ T cell subsets expressing granzymes, pore-forming protein, TNF-beta, and TNF-alpha, these T cell subsets displayed distinct pathways of antibody-redirected lysis against TNF-sensitive and TNF-resistant targets, even in the presence of anti-TNF antibodies. In addition, these same effector T cell subsets were also directly cytotoxic (in the absence of heteroconjugated antibody) against TNF-sensitive targets in an 18-h assay. Indeed, this direct cytotoxicity was completely abrogated by anti-TNF-alpha antibody and was sensitive to the metabolic inhibitors (cyclosporin A, CT, cycloheximide, and actinomycin D), all of which blocked CD4+/CD8+ T cell TNF-alpha production. Therefore, both CD4+ and CD8+ T cells were demonstrated to utilize antibody and lymphokine-mediated lytic mechanisms. CD4+ and CD8+ effector subsets were demonstrated to lyse the same TNF-sensitive target by these two different mechanisms. Although it cannot be excluded that the redirected lytic mechanisms of both CD4+ and CD8+ effectors share common elements, it is likely that other important events in this cytolytic process are fundamentally distinct between these subsets of T cells.     

Relationship between T cell receptors and antigen-binding factors. I. Specificity of functional T cell receptors on mouse T cell hybridomas that produce antigen-binding T cell factors

Upon antigenic stimulation with OVA-pulsed syngeneic macrophages, the mouse T cell hybridoma 231F1 produced glycosylation inhibiting factor (GIF) having affinity for OVA and IgE-suppressive factors, whereas another T cell hybridoma, 12H5, cells produced OVA-binding glycosylation enhancing factor (GEF) and IgE-potentiating factor. The OVA-binding GIF from the 231F1 cells is an Ag-specific Ts cell factor, whereas OVA-binding GEF from the 12H5 cells is an Ag-specific augmenting factor. Both hybridomas express CD3 complex and functional TCR-alpha beta. Cross-linking of TCR-alpha beta or CD3 molecules on the hybridomas by anti-TCR-alpha beta mAb or anti-CD3 mAb and protein A resulted in the formation of the same factors as those obtained by the stimulation of the cells with OVA-pulsed syngeneic macrophages. It was also found that both the 231F1 cells and 12H5 cells formed IgE-binding factors upon incubation with H-2d and H-2b APC, respectively, with a synthetic peptide corresponding to residues 307-317 in the OVA molecules (P307-317). Six other synthetic peptides, including those containing the major immunogenic epitope, i.e., P323-339, failed to stimulate the hybridomas in the presence of APC. Indeed, all of the 10 T cell hybridoma clones, which could produce either OVA-binding GIF or OVA-binding GEF, responded to P307-317 and APC for the formation of IgE-binding factors. In contrast, GIF/GEF derived from six other hybridoma clones, whose TCR recognized P323-339 in the context of a MHC product, failed to bind to OVA-coupled Sepharose. The results indicate the correlation between the fine specificity of TCR and the affinity of GIF/GEF to the nominal Ag. The amino acid sequence of P307-317 suggested that TCR on the cell sources of Ag-binding factors are specific for an external structure of the Ag molecules.     

Expression of T cell antigen receptor beta-chains on subsets of mouse thymocytes. Analysis by three-color flow cytometry

The expression on adult mouse thymocytes of a T cell antigen receptor beta-chain epitope, recognized by the antibody F23.1, has been studied by three-color flow cytometry. Low density F23.1 staining was found mainly on CD4+8+ thymocytes. High density staining was mainly on CD4+8- and CD4-8+ cells. Variable proportions of CD4-8- cells were also F23.1+. Among CD4-8+ cells, F23.1 was expressed only on the J11d- subset with mature T cell function. We conclude that many subpopulations of thymocytes express antigen receptors and are candidates for the population subject to thymic selection, but at present no single subpopulation makes a convincing claim.     

Relationship between T cell receptors and antigen-binding factors. II. Common antigenic determinants and epitope recognition shared by T cell receptors and antigen-binding factors

The T cell hybridomas 231F1 and 12H5 constitutively secrete glycosylation-inhibiting factor (GIF) and glycosylation-enhancing factor (GEF), respectively, which lack affinity for OVA-coupled Sepharose. When the 231F1 and 12H5 cells were stimulated by OVA-pulsed syngeneic macrophages, however, GIF and GEF produced by the cells had affinity for OVA. Both the OVA-binding GIF from the 231F1 cells and OVA-binding GEF from the 12H5 cells bound to a mAb against TCR-alpha beta and a mAb against TCR-alpha, suggesting a serologic relationship between TCR and OVA-binding factors. However, the OVA-binding GIF and GEF bound to mAb 14-12 and 14-30, respectively. Because these mAb do not bind TcR alpha beta-chains, it appears that the Ag-binding factors are different from TCR itself. The OVA-binding factors from both 12H5 cells and 231F1 cells do not bind to urea-denatured OVA. The binding of the factors to OVA Sepharose was inhibited by a peptide corresponding to residues 307-317 (P307-317) in the native OVA, but not by the peptide corresponding to residues 323-339 (P323-339). Furthermore, the OVA-binding factors bound to P306-319-coupled Sepharose but not to P323-339-coupled Sepharose, and were recovered by elution of the former Sepharose at acid pH. The binding of OVA to anti-OVA antibodies was not inhibited by either peptide. Inasmuch as the 231F1 cells and 12H5 cells can be stimulated by P307-317 in the context of a MHC product, it appears that the Ag-binding factors and TCR-alpha beta on the cell sources of the factors may recognize the same epitope in the OVA molecules. The results also showed that Ag-binding factors and antibodies recognize distinct epitopes in the Ag molecules.     

RAG1 and RAG2 expression by B cell subsets from human tonsil and peripheral blood

It has been suggested that B cells acquire the capacity for secondary V(D)J recombination during germinal center (GC) reactions. The nature of these B cells remains controversial. Subsets of tonsil and blood B cells and also individual B cells were examined for the expression of recombination-activating gene (RAG) mRNA. Semiquantitative analysis indicated that RAG1 mRNA was present in all tonsil B cell subsets, with the largest amount found in naive B cells. RAG2 mRNA was only found in tonsil naive B cells, centrocytes, and to a lesser extent in centroblasts. Neither RAG1 nor RAG2 mRNA was routinely found in normal peripheral blood B cells. In individual tonsil B cells, RAG1 and RAG2 mRNAs were found in 18% of naive B cells, 22% of GC founder cells, 0% of centroblasts, 13% of centrocytes, and 9% of memory B cells. Individual naive tonsil B cells containing both RAG1 and RAG2 mRNA were activated (CD69(+)). In normal peripheral blood approximately 5% of B cells expressed both RAG1 and RAG2. These cells were uniformly postswitch memory B cells as documented by the coexpression of IgG mRNA. These results indicate that coordinate RAG expression is not found in normal peripheral naive B cells but is up-regulated in naive B cells which are activated in the tonsil. With the exception of centroblasts, RAG1 and RAG2 expression can be found in all components of the GC, including postswitch memory B cells, some of which may circulate in the blood of normal subjects.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Stimulation of murine T cell subsets with anti-CD3 antibody. Age-related defects in the expression of early activation molecules

Splenocytes from young (3 to 4 mo) and aged (24 to 26 mo) C57BL/6 mice were stimulated with anti-CD3 epsilon mAb in vitro. At the time of peak DNA synthesis (day 2), cells from aged mice incorporated congruent to 60% less [3H]TdR than cells from young mice. This age-related defect was not attributable to gross differences in anti-CD3 does optima, response kinetics, accessory cell function, numbers of T cells cultured, CD4+:CD8+ cell ratios or surface levels of CD3 epsilon molecules. In an attempt to analyze pre-S phase events in these responses, we monitored CD4+ and CD8+ cells in splenocyte cultures for the time-dependent expression of three T cell activation markers: RL388 Ag and IL-2R and transferrin R. Parallel analyses of mean T cell size and cell cycle phase distributions were performed. Non-activated T cells from both age groups similarly expressed moderate levels of RL388 Ag, low levels of transferrin R, and undetectable levels of IL-2R. Analysis of stimulated T cells revealed, in both age groups: 1) detectable increases in expression of all three markers by 6 h of culture, and continued increases associated with blastogenesis and G1 phase transit and 2) a preferential stimulation of the CD8+ subset to a state of high level marker expression. Age group comparisons of activation marker expression over time suggested that the age-related defect reflects proportionally smaller fractions of CD4+ and CD8+ cells that respond normally, rather than a general defect in all T cells or a subset-specific defect. Finally, we found that supernatants from aged donor cell cultures stimulated with anti-CD3 contained less Il-2 than those of young controls. Addition of an IL-2 containing supernatant to aged donor cell cultures increased, but did not restore, the S phase response on day 2; however, the response on day 3 was comparable to the peak (day 2) response of young controls. These data suggest that exogenous IL-2 can improve the aged response, perhaps by expanding the fraction of normally reactive T cells.     

Interleukin-9 and T cell subsets

Comment on: Nowak EC, et al. J Exp Med 2009; 206:1653-60.     

Quantitation of T cell antigen-binding molecules (TABM) in the sera of nonimmunized, immunized, and desensitized mice

Heterologous antibodies to non-MHC-restricted T cell antigen-binding molecules (TABM) were used in an ELISA to detect and quantitate TABM levels in the sera of nonimmune mice and in mice immunized and/or desensitized to methylated bovine serum albumin (MBSA). TABM were detected at low levels in sera from nonimmune and immune mice. Amounts of TABM rose 300-fold in MBSA-immune mice within 12 hr of a desensitizing injection of MBSA, and rapidly returned to pre-desensitization levels. Serum TABM were purified by precipitation with 50% (NH4)2SO4 and chromatography in carboxymethylcellulose. The purified TABM were Mr 30,000 to 37,000 reduced, 72,000 nonreduced, and had a pI range of 5.5 to 7.1. A portion of the elevated TABM in desensitized mice bound MBSA specifically, whereas TABM from the sera of nonimmune/nondesensitized mice did not bind MBSA. The timing of the appearance of TABM early in desensitization suggests the possibility that they may play a role in the subsequent appearance of suppressor cells in later stages of this phenomenon. In addition, the results presented here raise the possibility that fluctuations in TABM levels might prove to be a sensitive indicator of immunoregulatory status in general.