Effector function of resting T cells: activation of synovial fibroblasts

Synovial tissue in rheumatoid arthritis is characterized by infiltration with large numbers of T lymphocytes and APCs as well as hyperplasia of synovial fibroblasts. Current understanding of the pathogenesis of RA includes the concept that synovial fibroblasts, which are essential to cartilage and bone destruction, are regulated by cytokines derived primarily from monocyte-macrophage cells. Recently it has been found that synovial fibroblasts can also function as accessory cells for T cell activation by superantigens and other stimuli. We have now found that highly purified resting T cells, even in the absence of T cell mitogens, induce activation of synovial fibroblasts when cocultured for 6-24 h. Such activation was evident by induction or augmentation of mRNA for stromelysin, IL-6, and IL-8, gene products important in joint inflammation and joint destruction. Furthermore, increased production of IL-6 and IL-8 was quantitated by intracellular cytokine staining and flow cytometry. This technique, previously used for analysis of T cell function, was readily adaptable for assays of synovial fibroblasts. Resting T cells also induced synovial fibroblasts to produce PGE(2), indicating activation of expression of the cyclooxygenase 2 gene. Synergy was observed between the effects of IL-17, a cytokine derived from stimulated T cells that activates fibroblasts, and resting T lymphocytes. Various subsets of T cells, CD4(+), CD8(+), CD45RO(+), and CD45RA(+) all had comparable ability to induce synovial fibroblast activation. These results establish an Ag-independent effector function for resting T cells that is likely to be important in inflammatory compartments in which large numbers of T lymphocytes and fibroblasts can come into direct contact with each other.     

Antigen presentation by EBV-B cells to resting and activated T cells: role of interleukin 1

We have previously demonstrated that Epstein Barr virus-transformed human B lymphocytes (EBV-B cells) present antigen to activated T cells (lines and clones) in a MHC-restricted manner. In the present study, using EBV-nonimmune donors, we demonstrate that EBV-B cells are unable to trigger tetanus toxoid (TT) antigen-specific proliferation in autologous highly purified resting T cells. EBV-B cells from these same individuals were able to present TT to autologous TT-specific activated T cell blasts (Tbl). The inability of EBV-B cells to present TT to resting T cells was not caused by defective antigen processing by EBV-B cells. Thus, paraformaldehyde treatment of antigen-pulsed EBV-B cells did not impair their ability to trigger proliferation of antigen-specific Tbl, and EBV-B cells pulsed with antigen in the presence of autologous TT-specific T cell blasts did not present antigen to resting T cells. Furthermore, antigen-specific proliferation of resting T cells triggered by monocytes was enhanced rather than suppressed by EBV-B cells. The addition of partially purified human IL 1 allowed EBV-B cells to present TT antigen to resting T cells, suggesting that failure to secrete IL 1 contributed to the failure of EBV-B cells to present antigen. IL 1 could not be detected in supernatants of EBV-B cells stimulated with Staphylococcus epidermidis, concanavalin A, and TT antigen in the presence or absence of up to 5% autologous T cells. The differential capacity of EBV-B cells to present antigen to resting T cells vs activated T cells correlated with the T cell requirement for IL 1, because a rabbit antibody to human IL 1 inhibited the monocyte-supported proliferation of resting T cells but not that of activated T cells. These results suggest that the inability of EBV-B cells to present antigen to resting T cells is related to their inability to secrete detectable IL 1.     

Surface glycoproteins of resting and activated human T lymphocytes

Summary This review summarizes some recent studies on the surface glycoproteins of human thymocytes and T lymphocytes. Purified cells were surface labeled by the galactose oxidase-NaB³H₄ or periodate-NaB³H₄ techniques. The radioactive membrane glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. Thymocytes and T lymphocytes show characteristic surface glycoprotein profiles which are easily distinguishable from those of the other main groups of human leukocytes. We observed specific changes in the surface glycoprotein patterns which correlate with the degree of maturation and functional activation of T cells. Surface molecules carrying T cell specific antigens were identified by immune-precipitation from lysates of surface labeled thymocytes and T lymphocytes using rabbit anti-human T cell antibodies. Finally we describe a leukocyte membrane glycoprotein which is a precursor of serum ₁ acid glycoprotein (orosomucoid).     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

Characterization of antigen processing and presentation by resting B lymphocytes

The production of antibody to a thymus-dependent Ag requires cooperation between the B cell and an Ag-specific Th cell. MHC restriction of this interaction implies that the Th cell recognizes Ag on the B cell surface in the context of MHC molecules and that the Ag-specific B cell gets help by acting as an APC for the Th cell. However, a number of studies have suggested that normal resting B cells are ineffective as APC, implying that the B cell must leave the resting state before it can interact specifically with a Th cell. Other studies, including our own with rabbit globulin-specific mouse T cell lines and hybridomas, show that certain T cell lines can be efficiently stimulated by normal resting B cells. One possible explanation for the above contradiction is that our B cells have become activated before presentation. Here we show that presentation by size-selected small B cells is not the result of nonspecific activation signals generated by the T cells or components of the medium. Also, although LPS activation does increase the efficiency of presentation by small B cells, use of large cells in place of small cells or preincubation of resting B cells with mitogenic doses of anti-Ig does not. Another possibility that we considered was that small B cells are unable to process Ag and that we had selected T cell lines that were capable of recognizing native Ag on the B cell surface. In the majority of cases, experiments with B cell lines and macrophages have shown that Ag presentation requires Ag processing, a sequence of events that includes internalization of Ag into an acid compartment, denaturation or digestion of Ag into fragments, and its return to the cell surface in the context of class II MHC molecules. The experiments reported here show that our T cell lines require an Ag processing step and that small resting B cells, like other APC, process Ag before presenting it to T cells. Specifically, we show that an incubation of 2 to 4 h is required after the Ag pulse before Ag presentation becomes resistant to irradiation. Shortly after the pulse, the Ag enters a pronase-resistant compartment. Although efficient Ag presentation requires initial binding to membrane Ig, Ag is no longer associated with membrane Ig at the time of presentation and is not presented in its intact form, because removal of membrane Ig by goat anti-Ig blocks presentation before but not after the Ag pulse.     

Antigen presentation by hapten-specific B lymphocytes. V. Requirements for activation of antigen-presenting B cells

Splenic B cells specific for the haptens, 2,4,6-trinitrophenyl (TNP) or fluorescein isothiocyanate (FITC) were cultured with a range of concentrations of unmodified or TNP- or FITC-conjugated conalbumin and the conalbumin + I-Ak-specific, interleukin (IL) 1-dependent helper T cell clone, D10 . G4, in the presence and absence of IL-1. Lymphokine secretion, T cell proliferation, and antibody secretion by B cells all exhibited identical antigen dose responses. Thus, hapten-binding B cells presented low concentrations of haptenated conalbumin for activation of both the T and the antigen-presenting B cells. Whereas proliferation of D10 . G4 required the addition of IL-1, both lymphokine production and stimulation of B cells to antibody secretion occurred without exogenous IL-1. These results demonstrate that when B lymphocytes function as presenting cells for antigens that bind to their immunoglobulin receptors, activation of the responding T cells and the B cells themselves occur at similar concentrations of antigen. Moreover, for functional T-B interactions, antigen-presenting B and responding T lymphocytes constitute a complete system that requires no other accessory stimuli, whereas clonal expansion of T cells is dependent on accessory factors such as IL-1. Finally, since D10 . G4 secretes IL-4 but neither IL-2 nor interferon-gamma, our results demonstrate that differentiation of B cells as a consequence of direct ("cognate") interactions with helper T cells as well as of bystander B cells can occur in the absence of IL-1, IL-2, and interferon-gamma.     

Cytochalasins distinguish by their action resting human T lymphocytes from activated T-cell blasts

In the majority of resting human peripheral T lymphocytes obtained from separate individuals cytochalasin B (CB) and D (CD) cause a disappearance of microvilli and induce a rapid formation of prominent sac and bleb-like projections with a length of 1–10 μm randomly distributed over the cell surface. During mitogen stimulation the cells lose the tendency to develop such projections when subsequently exposed to CB and CD. By contrast, in activated T lymphocytes the cytochalasins provoke an asymmetric localization of microvilli including cell surface antigens and actin to a prominent protuberance often separated from the cell body by a constriction. This protuberance is distinct from conventional spontaneous uropods formed by conA-stimulated lymphocytes in relation to contact with other cells and with non-cellular surfaces. The cytochalasins therefore in their action distinguish resting small lymphocytes from activated T-cell blasts.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.     

Antigen receptor-mediated anergy in resting T lymphocytes and T cell clones. Correlation with lymphokine secretion patterns.

The goal of these studies was to define the stimuli and factors that control the induction of anergy in unimmunized resting T lymphocytes. Initial experiments, aimed at establishing the system, showed that exposure of Th1 but not Th2 clones to immobilized anti-CD3 leads to a block in autocrine growth factor production and proliferation upon subsequent restimulation with Ag+APC. Anergy is not prevented by accessory cells, suggesting that this model of T cell tolerance may be due to receptor-mediated inhibitory signals, independent of costimulatory molecules. Culture of small (resting) unimmunized T lymphocytes with anti-CD3 +/- IL-2 induces unresponsiveness to restimulation with anti-CD3, but culture with anti-CD3+IL-4, which stimulates the differentiation of resting cells into IL-4 producers, does not induce anergy. Thus, IL-4-producing clones and bulk populations of IL-4-producing T cells are resistant to Ag receptor-mediated inhibitory stimuli. These results provide experimental models for studying the mechanisms of anergy in normal, unselected, mature T cells, and demonstrate fundamental similarities between cloned cell lines and unimmunized T lymphocytes in the induction of anergy.     

Antigen presentation by resting B cells. Effectiveness at inducing T cell proliferation is determined by costimulatory signals, not T cell receptor occupancy

Resting B cells stimulated the proliferation of two T cell clones much less efficiently than T cell-depleted low-density APC. In contrast, low-density cells and resting B cells stimulated the clones to produce similar levels of inositol phosphates, a rapid biochemical event dependent only on occupancy of the TCR. The inefficient stimulation of T cell proliferation by resting B cell APC was dramatically improved by the addition of allogeneic low-density accessory cells incapable of being recognized by the TCR on the responding T cells. The results are most consistent with a model where low-density and resting B cell APC display similar amounts of Ag/Ia molecule complexes capable of being recognized by the TCR on the responding T cells but differ in the provision of costimulatory signals that, together with TCR occupancy, are required for IL-2 production.     

Antigen presentation by human B cells: T cell proliferation induced by Epstein Barr virus B lymphoblastoid cells

T cell proliferation in response to antigen requires the presence of an antigen-presenting accessory cell. The accessory cell most commonly associated with antigen presentation has been the macrophage (Mo). This study demonstrates that B cells in the form of Epstein Barr virus-transformed B lymphoblasts (EBV-B) are able to present tetanus toxoid (TT) to human T cells and induce proliferation of these cells. T cells freshly isolated from peripheral blood were shown to undergo blast transformation and proliferation in response to TT and irradiated EBV-B cells. Furthermore, TT-reactive T cell blasts were shown to proliferate in the presence of EBV-B cells and TT, but not with other antigens. There was a progressive increase to a plateau in T blast proliferation with increasing numbers of EBV-B cells added to the culture. The concentration of TT required for optimal antigen presentation was similar for EBV-B cells and for Mo. TT-specific cloned T cells also proliferated in response to TT and EBV-B cells and could be continuously grown in culture with TT, interleukin 2-containing supernatant, and EBV-B cells in place of autologous Mo. EBV-B cells pulsed with TT could also act as antigen-presenting cells. The proliferative response of T cell clones to TT in the presence of EBV-B cells was inhibited by antiserum to human p29,34 glycoprotein but not by anti-beta 2-microglobulin. This inhibition was shown to result from interaction with Ia-like determinants on EBV-B cells. These results indicate that B lymphoblastoid cells in man are able, like Mo, to present antigen to T cells in the context of Ia-like determinants.     

Stimulation by xanthine oxidase of 3T3 Swiss fibroblasts and human lymphocytes

Xanthine oxidase stimulates [3H]thymidine incorporation by 3T3 cells even in the absence of any added xanthine, but in the presence of, and synergistically with, insulin or various other growth-stimulating factors. Optimal stimulation was obtained with 2-3 mU enzyme/ml and higher concentrations were toxic. Xanthine oxidase also stimulated human peripheral blood lymphocytes in the presence of phytohemagglutinin and xanthine.     

Specific activation of human T lymphocytes by Robinia pseudoacacia seeds' lectin.

Human peripheral blood and tonsil lymphocytes were fractionated into T and B cells by centrifugation after rosetting with native sheep erythrocytes and tested with Robinia pseudoacacia lectin. The purity of B- and T-enriched populations was checked by E-rosette formation or heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to Robinia lectin from all the donors tested was not found to differ from that of unfractionated cells, whereas no response of highly purified B cells could be observed to the lectin even with different concentrations of the lectin and different culture periods. B cells, however, were found to bind as much ³H labeled Robinia lectin as unfractionated lymphocytes. In addition, treatment of cells by antihuman T-lymphocyte antigen (HTLA) serum and complement before addition of Robinia lectin completely abolished their response, whereas similar treatment by antihuman B lymphocyte and monocyte antigen (HBLMA) serum did not prevent the T cells from incorporating thymidine. The Robinia lectin, like the other phytomitogens, thus appears to be a specific T-cell activator.     

Antigen presentation to human T lymphocytes. II. Requirements for Mac-120+ macrophages and responsiveness to interleukin 2

To investigate mechanisms by which antigen, macrophages, and interleukin 2 (IL2) participate in the induction of secondary T-cell proliferative responses, trinitrophenyl (TNP) was presented in three distinct modes: (i) TNP-modified peripheral blood mononuclear cells (TNP-PBMC), (ii) TNP-PBMC cell sonicates, and (iii) TNP-ovalbumin (TNP-OVA). Stimulators were depleted of Mac-120+ macrophages using Mac-120 monoclonal antibody plus complement. TNP-Mac-120 macrophages stimulated primed T cells nearly as well as TNP-unfractionated macrophages (which were about 40% Mac-120+). In contrast, although greater than 70% DR+, Mac-120- macrophages plus either TNP-OVA or TNP-PBMC sonicate elicited minimal responses compared to unfractionated macrophages plus antigen. After 21-28 days of in vitro priming, macrophage-depleted T cells were not stimulated to proliferate by either IL2 alone or sonicates alone. IL2 plus TNP-PBMC sonicates, however, stimulated significant proliferation. Furthermore, this response was considerably greater than that to IL2 plus either TNP-T cell sonicates or TNP-mouse spleen sonicates. Thus, the Mac-120+ macrophage population may have an important antigen-presenting and/or accessory function in the stimulation of primed T cells by soluble or particulate antigen, although it is unnecessary for responses to intact TNP-Ia+ PBMC. In addition, the data suggest that Ia+ sonicates alone may suffice for induction of IL2 responsiveness, but not for endogenous IL2 production and subsequent proliferation by primed T cells.     

Class II MHC self-antigen presentation in human B and T lymphocytes

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.     

Effects of interferon-gamma on the activation of human T lymphocytes

The role of interferon (IFN)-gamma in the activation of human T cells was investigated. Addition of IFN-gamma to mixed-lymphocyte cultures (MLC) augmented both the proliferation and the development of T-cell-mediated cytotoxicity. IFN-gamma also augmented the early expression on CD8+ but not CD4+ lymphocytes of IL-2 receptor alpha chain (Tac antigen) and Class II major histocompatibility antigen (HLA-DR). This effect synergized with that caused by interleukin 2 and was not observed with IFN-alpha. The addition of neutralizing antibody against IFN-gamma to MLC suppressed the development of cytotoxicity and proliferation and the expression of activation antigens on CD8+ cells. In experiments in which highly purified CD8+ T cells were activated with cell-free stimuli, IFN-gamma slightly but significantly augmented proliferation, antibody to IFN-gamma suppressed proliferation, and excess IFN-gamma reversed this suppression. It is concluded that (i) IFN-gamma augmented activation of T cells in human MLC, (ii) IFN-gamma exerted effects directly on T cells, and (iii) IFN-gamma preferentially augmented CD8+ cell activation.     

Inhibition of DNA repair by deoxyadenosine in resting human lymphocytes

Profound lymphopenia is characteristic of immunodeficient children who lack adenosine deaminase (ADA). When ADA is inactive, deoxyadenosine (dAdo) is phosphorylated by immature T lymphoblasts and inhibits cell division. However, dAdo also causes the slow accumulation of DNA strand breaks in nondividing, mature human peripheral blood lymphocytes. To explore the basis for this phenomenon, we have assessed the effects of dAdo and other deoxynucleosides on the repair of gamma-radiation induced DNA strand breaks in resting normal lymphocyte cultures. As measured by a sensitive DNA unwinding assay, most DNA strand breaks were rejoined within 2 hr after exposure of lymphocytes to 500 rad. In medium supplemented with deoxycoformycin, a tight binding ADA inhibitor, dAdo retarded DNA rejoining in a dose and time dependent manner. The inhibition required dAdo phosphorylation. Over an 8-hr period, 10 microM dAdo gradually rendered peripheral blood lymphocytes incompetent for DNA repair. Among several other compounds tested, 2-chlorodeoxyadenosine, an ADA resistant dAdo congener with anti-leukemic and immunosuppressive activity, was the most powerful inhibitor of DNA repair, exerting significant activity at concentrations as low as 100 nM. Both dAdo and 2-chlorodeoxyadenosine blocked unscheduled DNA synthesis in irradiated resting lymphocytes, as measured by [3H]thymidine uptake. On the basis of this and other data, we suggest that quiescent peripheral blood lymphocytes break and rejoin DNA at a slow and balanced rate. The accumulation of dATP progressively retards the DNA repair process and thereby fosters the time-dependent accretion of DNA strand breaks. By inhibiting DNA repair, dAdo, 2-chlorodeoxyadenosine and related compounds may substantially potentiate the toxicity of DNA damaging agents to normal and malignant lymphocytes.