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.     

Antigen presentation by interferon-gamma-treated endothelial cells and fibroblasts: differential ability to function as antigen-presenting cells despite comparable Ia expression

The effect of interferon-gamma (IFN-gamma) on endothelial cell (EC) and fibroblast (FB) class II major histocompatibility complex (MHC) gene product expression and antigen presenting ability was examined. Control FB did not express class II MHC gene products, whereas a small (less than 1%) population of passaged EC expressed class II gene products. IFN-gamma induced a comparable density of HLA-DR expression on nearly all EC and FB. IFN-gamma-treated EC and FB also expressed HLA-DP but at a lower density, whereas HLA-DQ expression was barely detectable on either cell type. Control FB were not able to stimulate allogeneic T4 cell DNA synthesis or function as antigen-presenting cells (APC). Control EC were also unable to stimulate allogeneic T4 cell DNA synthesis unless large numbers of stimulator cells were used. Small numbers of IFN-gamma-treated EC were able to stimulate allogeneic T4 cell DNA synthesis, whereas larger numbers were markedly more effective than control EC. In contrast, IFN-gamma-treated FB were ineffective stimulators of allogeneic T4 cell DNA synthesis. IFN-gamma-treated FB were able to present the exogenous antigen SKSD to autologous but not allogeneic T4 cells, but they were extremely inefficient APC. The inability of IFN-gamma-treated FB to function as APC could not be explained by FB-mediated immunosuppression, Ia density, or HLA-DQ expression. This limited capacity of IFN-gamma-treated FB to participate in Ia-restricted functional interactions with T4 cells correlated with a similar diminished capacity to support nonspecific mitogen-induced proliferation of T4 cells before IFN-gamma-induced Ia expression. This accessory cell function was not enhanced by IFN-gamma treatment. Monocytes syngeneic to the responding T4 cells but not interleukin 1 (IL 1) permitted IFN-gamma-treated FB but not control FB to stimulate allogeneic T4 cell DNA synthesis, but they remained markedly less effective stimulators than monocytes. Moreover, IFN-gamma-treated FB were effective stimulators of alloprimed T4 cells, in contrast to their inability to stimulate fresh T4 cells. Furthermore, monocytes and IFN-gamma-treated FB were comparably effective stimulators of alloreactive T cell lines. These data suggest that accessory cells perform functions unrelated to Ia and IL 1 that are necessary for mitogen-, alloantigen-, and antigen-induced proliferation of freshly isolated T cells. Monocytes and EC effectively perform this function, but FB do not. This accessory cell function does not seem to be as important for the activation of primed T cells.     

Dissection of defective antigen presentation by interferon-gamma-treated fibroblasts

The capacity of interferon-gamma (IFN-gamma)-treated HLA-DR expressing human dermal fibroblasts (FB) to function as antigen-presenting cells (APC) was examined. FB were cultured with 250 U/ml IFN-gamma for 4 days to induce HLA-DR expression. Peripheral blood monocytes (M phi), FB, or IFN-gamma-treated FB from the same donor were then cultured overnight with or without the recall antigen streptokinase streptodornase (SKSD), and their capacity to stimulate autologous T4 cell DNA synthesis was examined. SKSD-bearing M phi stimulated T4 cell proliferation, whereas antigen-bearing HLA-DR (+) FB did not. Even after fixation with paraformaldehyde to eliminate metabolic activity, SKSD-bearing M phi, but not FB, were able to function as APC. However, when HLA-DR (-) endothelial cell (EC) or autologous or HLA-D-mismatched M phi were added to the cultures, antigen-pulsed IFN-gamma-treated FB and M phi were comparably effective stimulators of autologous T4 cell DNA synthesis. Antigen recognition by the T4 cell was restricted by the class II major histocompatibility complex (MHC)-encoded gene products expressed by the IFN-gamma-treated FB and was unrelated to the class I or II MHC-encoded gene products expressed by the additional cell type. EC-promoted T4 cell DNA synthesis induced by antigen-bearing IFN-gamma-treated FB was inhibited by 60.3, a monoclonal antibody directed at an epitope common to LFA-1, CR3, and the p150,95 molecule. Inhibition caused by 60.3 was completely reversed by the addition of IL 2 to the cultures. Antigen presentation by IFN-gamma-treated FB was also enhanced somewhat by IL 1, IL 2, or monoclonal antibody directed at Tp44 (9.3). However, each of these additions alone promoted T cell proliferation less effectively than EC and resulted in responses that were smaller than those triggered by antigen-bearing M phi. The data suggest that IFN-gamma-treated FB take up and process antigen effectively, but lack an accessory cell property necessary for antigen-induced T4 cell IL 2 production and proliferation.     

Inability of human alveolar macrophages to stimulate resting T cells correlates with decreased antigen-specific T cell-macrophage binding

Alveolar macrophages (AM) from the majority of human volunteers are defective antigen presenting cells (APC) in T cell proliferation assays despite the display by the cells of HLA-D region antigens. We have confirmed that AM secrete relatively little interleukin 1 (IL 1), but addition of exogenous IL 1 did not improve the capacity of AM to initiate antigen-induced T cell proliferation. Thus, the presence of HLA-D region antigens and IL 1 is not sufficient to enable an accessory cell to act as an APC. We developed a T cell-accessory cell binding assay to investigate early events in T cell activation. AM demonstrated a diminished capacity as compared with monocytes to bind antigen-specific T cell clones. Nevertheless, AM often induced proliferation of T cell clones as effectively as monocytes, indicating that antigen display was intact. The inefficiency of AM in bind T cell clones correlated with their reduced capacity to induce resting T cells to express IL 2 receptors, secrete IL 2, and proliferate in response to antigen. Indirect immunofluorescence established that similar percentages of AM and monocytes expressed LFA molecules, but the density of the molecules was greater on monocytes than AM. A role for LFA antigens in the physical binding of T cells to monocytes was demonstrated by blocking antigen-specific binding with a monoclonal antibody to LFA-1 antigen. LFA-1 antibody also blocked the low levels of specific binding between AM and T cell clones, indicating that LFA-1-ligand interactions were operative between these two cell types. These studies indicate that there are critical cell membrane characteristics that promote binding of T cells to APC in addition to T cell receptor-antigen interactions. This combination of nonspecific and specific interactions leads to avid T cell-APC binding that may be essential for activation of resting T cells. Furthermore, we postulate that the failure to AM to act as effective APC results from an inability to bind T cells efficiently.     

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.     

Induction of IgG and IgE synthesis in normal B cells by autoreactive T cell clones

During the course of generating tetanus toxoid (TT)-specific T cell clones frm an HLA-DR2,7 donor, four clones were obtained which proliferated in the presence of autologous monocytes alone without the addition of TT antigen. This proliferation was specifically inhibited by anti-HLA-DR framework mouse monoclonal antibody, and appeared to be HLA-DR-restricted. Two of the clones proliferated in response to HLA-DR2-bearing monocytes, and the other two clones proliferated in response to HLA-DR7-bearing monocytes. The capacity of these four autoreactive human T cell helper clones to induce IgE synthesis in B cells was studied. All four clones stimulated autologous peripheral blood B cells to synthesize IgE and IgG antibody. Induction of IgE synthesis in B cells by the autoreactive T cell clones followed the same pattern of HLA-DR restriction which governed the proliferative response of these clones. These results suggest that the interaction of autoreactive helper T cells with B cell HLA-DR antigens may be important in the activation of IgE immune responses in humans.     

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.     

Antigen-specific and -nonspecific mitogenic signals in the activation of human T cell clones

We have directly compared the signals required for: induction of the [Ca+2]i response, expression of Tac antigen, and proliferation in antigen-specific human T cell clones. We have previously shown that antigen-specific activation of cloned T cells under conditions leading to proliferation is accompanied by a rapid increase in [Ca+2]i. Cloned T cells showed increased [Ca+2]i, enhanced Tac expression, and proliferated in response to specific antigen in the presence of viable, genetically appropriate antigen-presenting cells. Paraformaldehyde fixation of antigen-presenting cells after "pulsing" with antigen prevented proliferation, but did not affect MHC-restricted [Ca+2]i or Tac responses. Treatment of cloned T cells with monoclonal anti-T3 antibody also increased [Ca+2]i and Tac expression but did not induce proliferation. Proliferation was restored by viable autologous or allogenic APC or exogenous IL 2, but not by IL 1. In contrast to resting T cells, T cell clones were insensitive to the mitogenic effects of lectins or of ionophores and phorbol esters. These results suggest that activation of antigen-specific T cells requires the sequential action of at least two signals. The first is MHC restricted and is mediated by interaction of antigen + MHC class II products with the T cell receptor (T3-Ti) complex. This leads to Tac expression and increased [Ca+2]i, but is not sufficient for proliferation. This signal can be bypassed by anti-T3 monoclonal antibodies. Proliferation requires a second, nonantigen-specific, non-MHC-restricted antigen-presenting cell signal, which cannot be replaced by IL 1 in our system. This signal can be bypassed, however, by the addition of exogenous IL 2 to cells that have received the first signal and express Tac, suggesting that it is required for IL 2 synthesis and secretion. T cell clones therefore provide a useful model for studying antigen-dependent and -independent events in cell activation.     

Mechanisms of human T cell response to mitogens: IL 2 induces IL 2 receptor expression and proliferation but not IL 2 synthesis in PHA-stimulated T cells

Human peripheral blood T cells were purified by a four-step procedure which included depletion of plastic-adherent cells, rosetting with sheep red blood cells, nylon wool passage, and treatment with mouse monoclonal antibodies to human Ia antigens plus complement. The purified T cells completely failed to proliferate to phytohemagglutinin (PHA). Bacterially derived recombinant human interleukin 2 (IL 2) reconstituted the proliferative response of resting T cells to PHA. The optimal concentration of IL 2 required was 100 to 200 U/ml. IL 2 alone caused no T cell proliferation. Both PHA and IL 2 needed to be present together for the proliferation of T cells to occur. Incubation of T cells with either PHA or IL 2 alone for up to 18 hr, followed by washing, then by the addition of the reciprocal reagent, resulted in no T cell proliferation. Expression of IL 2 receptors and of Ia antigens, as assessed by indirect immunofluorescent staining, revealed that both PHA and IL 2 needed to be present for Tac and Ia antigen expression by T cells. T cells incubated with PHA and IL 2 for 18 to 42 hr acquired responsiveness to IL 2. These T cells remained absolutely dependent on IL 2 for proliferation to occur. In contrast to T cells stimulated with PHA in the presence of monocytes, T cells stimulated with PHA and IL 2 released no detectable IL 2. The failure of IL 2 secretion was not caused by down-regulation of IL 2 production by IL 2 itself, because the addition of IL 2 to cultures of T cells stimulated with PHA in the presence of monocytes did not interfere with IL 2 production. These results indicate that IL 2 is a sufficient signal to induce the expression of its receptor in PHA-stimulated T cells and subsequent proliferation but is not sufficient to cause endogenous IL 2 release.     

Mutations affecting antigen processing impair class II-restricted allorecognition

Both exogenously derived and endogenously derived Ag generally require processing for their optimal binding and presentation by class I and class II major histocompatibility proteins. It is not known whether steps involved in Ag processing also affect the recognition of alloreactive T cells. We have recently described B cell mutants which have general defects in the processing and presentation of a variety of exogenous Ag to class II restricted T cells. In this report we have studied the ability of these processing mutants to stimulate a set of anti-DR3-specific alloreactive T cells clones. These processing/presentation mutants express normal MHC class II molecules, both in terms of primary sequence and cell surface abundance, but they appear unable to generate effective peptide-MHC complexes. When tested for their ability to stimulate MHC class II alloreactive T cell clones, only one of four T cell clones was stimulated by these mutants; the other three alloreactive T cell clones were not stimulated by either of two different mutants. Both of these mutants express normal levels of the accessory molecules, LFA-3 and ICAM-1. The inability of these mutants to stimulate three of four alloreactive clones indicates that the capacity to be recognized by many alloreactive T cells is linked to the Ag processing capacity of a stimulator cell.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

Characterization of murine T cell activation signals produced by B lymphoma cells

The activation requirements of alloreactive and antigen reactive murine T cells were examined by stimulating class II restricted T cell clones with monoclonal B lymphoma cells. One B lymphoma cell line (T27A) was found to stimulate IL 2 release from some alloreactive T cell clones without stimulating any significant T cell proliferation response. The same B lymphoma cells are capable of stimulating IL 2 release and proliferative responses from other T cell clones. Evidence is presented suggesting that B lymphoma cell stimulation of these T cell clones is largely IL 1 independent and that at least some T cell clones may require activation signals other than Ia, antigen, and IL 1. The addition of exogenous, purified IL 1 to the T cell activation assays was found to have a wide range of stimulatory effects on the proliferative responses of different T cell clones. The absence of comparable IL 1-induced stimulation of IL 2 secretion suggests that IL 1 primarily enhances antigen specific T cell proliferation through mechanisms other than acting as a co-stimulant for IL 2 release.     

The evolution of MHC restrictions in antigen recognition by T cells in a haploidentical bone marrow transplant recipient

We have longitudinally followed the major histocompatibility complex (MHC) restrictions that govern the response of T lymphocytes to specific Ag in a child with severe combined immunodeficiency who was successfully transplanted by using T cell depleted haploidentical maternal bone marrow cells and immunized shortly afterwards with tetanus toxoid (TT) Ag. In the first year post-transplant, monocytes were of both donor and recipient origin whereas T and B cells were of donor origin. Three years after transplant, all monocytes and T and B cells were of donor origin. T lymphocytes taken from the child at that time and depleted in vitro of alloreactivity to paternal Ag proliferated in response to TT presented by maternal as well as paternal monocytes. A TT-specific T cell line established from these cells in the presence of maternal monocytes cooperated with maternal but not with paternal monocytes, whereas a TT-specific T cell line established in the presence of paternal monocytes cooperated with paternal but not with maternal monocytes and with monocytes derived from a paternal uncle who shared the haplotype inherited by the recipient from her father. These results show that long-term memory T cells restricted to recipient MHC Ag not shared with the bone marrow donor continue to circulate long after the disappearance of accessory cells of recipient origin. These T cells could potentially participate in a secondary immune response because they were shown to recognize TT presented by recipient fibroblasts induced to express class II MHC molecules following treatment with IFN-gamma.     

The relationship between immune interferon production and proliferation in antigen-specific, MHC-restricted T cell lines and clones

The release of immune or gamma interferon (IFN-gamma) by major histocompatibility complex (MHC)-restricted pigeon cytochrome c-specific Lyt 1+2-, interleukin 2 (IL 2)-producing proliferative T cell clones when cultured with antigen and antigen-presenting cells (APC) is a sensitive measure of the state of activation of the cell. In general, the fine specificity of T cell activation was similar when activation was measured either by IFN-gamma production or by proliferation. In response to antigen and the correct Ia molecule, the T cell clones produced both high titered IFN-gamma and a strong proliferative response. However, IFN-gamma production and the degree of proliferation of the T cell clones differed at high antigen concentrations. As antigen concentration increased, the magnitude of proliferation became submaximal whereas the IFN-gamma response became maximal suggesting that IFN-gamma produced by the cells might act as an autoregulatory molecule inhibiting the proliferative response. Stimulating the T cell to divide via its IL 2 receptor by adding exogenous IL 2 produced high levels of proliferation but only low titers of IFN-gamma activity. In addition, irradiation of the clone eliminated the IFN-gamma release induced by IL 2 but did not affect the IFN-gamma release induced by antigen and Ia. Thus proliferation is not essential for IFN-gamma production and unlike antigen and Ia, IL 2 functions predominantly as a proliferative signal and not as a signal for factor release. Two T cell clones showed a dissociation of IFN-gamma production and proliferation. In one case, a clone that proliferated in response to both allogeneic and antigenic stimuli released IFN-gamma in response to antigen but failed to produce IFN-gamma in response to the allogeneic stimulus. A second clone that showed a strong proliferative response to pigeon cytochrome c but no proliferative response to a species variant of cytochrome c, tobacco hornworm moth (THWM) cytochrome c, produced IFN-gamma when stimulated with either of these antigens. Thus, the sensitivity of detecting activation of T cell clones as measured by the release of an individual lymphokine varies from one clone to another.     

A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones

Hybridoma H129 .19 was derived by fusion between spleen cells of a Lou / Ws1 rat immunized with an Lyt-1+,2- anti-I-Ak cytolytic T lymphocyte (CTL) clone and the nonsecreting myeloma X63-Ag8.653. The monoclonal antibody (mAb) H129 .19 (IgG2a, kappa) was selected for its capacity to inhibit the lytic potential of the immunizing clone. H129 .19 identified a monomorphic determinant on a 55 m.w. murine T cell differentiation antigen, which appeared to be homologous to the human T4 molecule in that: 1) H129 .19 reacted with 80% adult thymocytes, with a subset of splenic T cells, and with the interleukin 2 (IL 2)-producing EL4 thymoma; 2) The mAb bound to and inhibited the IL 2 production and the proliferation of various allo- or soluble antigen-reactive T cell clones that recognized restriction or activating determinants on the I-A or I-E molecules, respectively; 3) H129 .19 did not inhibit the proliferation and/or cytolysis of Lyt-2,3+ T cells specific for class I MHC antigen; and 4) Among six anti-Iak CTL clones examined in this study, the mAb H129 .19 reacted with two I-Ak-specific, Lyt-2,3- clones on which it exerted strong cytolysis inhibiting effect at the effector cell level. By contrast, two other anti-I-Ak and two anti-I-Ek CTL clones were found to express the Lyt-2,3+,T4- cell surface phenotype. The cytolytic potential of the latter clones was not inhibited by anti-Lyt-2,3 mAb. These studies strongly suggest that the mouse T4 molecule facilitates the recognition of class II MHC antigen by most but not all T cells.     

Helper cell-independent clonal expansion of H-2D-restricted cytolytic T cells

Cytotoxic T lymphocyte (CTL) clones of different phenotypes have been described: some need extrinsic growth factors for proliferation whereas others can be expanded by antigenic stimulation. Up to 40% of CTL clones obtained early during in vitro culture can be stimulated by antigen in the absence of extrinsic interleukin 2 (IL 2) whereas all late clones require exogenous IL 2. Early clones regularly change their phenotype, i.e., their growth becomes dependent on exogenous IL 2. We propose that the growth of Lyt-2+ cells restricted by class I major histocompatibility complex (MHC) antigen is essentially independent of growth factors produced by class II MHC antigen-restricted T helper cells but can be augmented by such factors, especially at later stages of antigen-induced differentiation.     

Alloantigenicity of human endothelial cells. 1. Frequency and phenotype of human T helper lymphocytes that can react to allogeneic endothelial cells.

To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.     

Expression of interleukin 1 receptors on human peripheral T cells

The expression of interleukin 1 receptors (IL 1R) on human peripheral T cells was studied by the binding assay with 125I-labeled recombinant human interleukin 1 (IL 1) alpha and IL 1 beta and by the flow cytofluorometry with the fluorescein isothiocyanate (FITC)-conjugated IL 1 alpha. Peripheral blood lymphocytes expressed only few IL 1R without any stimulations. When they were stimulated with concanavalin A (Con A), IL 1R-positive cells began to increase by 4 hr, reached the maximum level at 48 hr, and then gradually decreased. The kinetics of the expression of IL 1 alpha R and IL 1 beta R showed the same pattern. Furthermore the binding of 125I-labeled IL 1 alpha to IL 1R on T cells was inhibited by the addition of either cold IL 1 alpha or IL beta, but not by interleukin 2 or interferons. The similar results were observed in the binding of 125I-labeled IL 1 beta. These results suggest that IL 1R on human peripheral T cells reactive for IL 1 alpha and IL 1 beta were identical. By Scatchard plot analysis, the numbers of IL 1R were estimated as 40 and 350 molecules per cell before and after Con A stimulation, respectively, and their Kd values were 3.1 X 10(-10) M and 2.8 X 10(-10) M. When purified T cells alone were stimulated with Con A, IL 1R were only marginally expressed. However, by the addition of monocytes, IL 1R were expressed on T cells in a dose-dependent manner. The maximum response was induced in the presence of 10% monocytes. The maximum IL 1R-positive T cells were approximately 30% by the detection of the flow cytofluorometry with FITC-conjugated IL 1 alpha. This enhancing activity of IL 1R expression on T cells by monocytes was inhibited by the addition of an anti-HLA-DR antibody or by the treatment of monocytes with the anti-HLA-DR antibody and complement. Furthermore T cell proliferative responses induced with IL 1 and Con A were also enhanced by the addition of HLA-DR-positive monocytes. These results suggest that IL 1R are expressed as the result of monocyte-T cell interaction in the early stage of T cell activation, and the expression of IL 1R on T cells and the responsiveness of T cells for IL 1 require the accessory function of HLA-DR-positive monocytes.     

Modified MHC restriction of donor-origin T cells in humans with severe combined immunodeficiency transplanted with haploidentical bone marrow stem cells

The choice of class II MHC determinants that serve as self-recognition elements for murine CD4+ T cells is thought to be determined by the environment in which T cells mature rather than their genotype. Patients with severe combined immunodeficiency (SCID) reconstituted with T cell depleted haploidentical parental stem cells provide an excellent model for studying this phenomenon in humans. After engraftment, the T cells that develop in these infants are all of donor origin. We sought to determine whether the successful immune reconstitution observed in two such SCID chimeras involved modification of the MHC restriction of Ag recognition by the genetically donor T cells as they matured to become competent T cells in the infants' microenvironment. A tetanus toxoid (TT)-specific T cell line and TT-specific T cell clones were established from the blood of two reconstituted SCID patients and from their maternal donors. T cell responsiveness was determined by [3H]thymidine incorporation after TT presentation by EBV-transformed B cell lines (EBV-B) from various donors. The TT-specific T cell line from patient 1 proliferated when presented Ag by patient, maternal donor, and paternal APC. A CD4+ donor origin clone that proliferated when presented TT by patient and paternal EBV-B, but not by maternal donor EBV-B, was isolated from each patient. TT recognition by these clones was shown to be restricted by the HLA DR determinant shared by patient and father, but not present in the donor. Four TT-specific clones isolated from maternal donors failed to proliferate when presented TT by the appropriate paternal EBV-B. These studies demonstrate that, in these human SCID bone marrow chimeras, engrafted donor-origin stem cells maturing to competent T cells in the recipient microenvironment are capable of utilizing recipient HLA determinants as restriction elements for Ag recognition. This suggests that human, as well as murine, MHC restriction patterns for Ag recognition by CD4+ T cells are environmentally determined.     

Accessory function and interleukin 1 production by human leukemic cell lines

Highly purified T lymphocytes do not proliferate in response to mitogens, unless adherent HLA-DR-positive monocytes are added to the culture. This accessory function (AF) of monocytes requires the release of interleukin 1 (IL 1). Cells from three human leukemic cell lines, K562, HL60, and U937, could very efficiently replace monocytes in a 72-hr mitogen-induced T cell proliferation assay. The AF was clearly related to precise maturational stages of these cells; the hematopoietic precursor K562 cells spontaneously exerted high AF, but lost this property when treated with differentiation inducers. On the contrary, the promyelocytic HL60 cells and the "histiocytic" U937 cells exhibited no spontaneous AF, but acquired this property when induced to differentiate along the granulocytic and/or monocytic pathway. Three leukemic cells could not only stimulate T cells to proliferate and produce IL 2 in the presence of mitogens, but also under appropriate culture conditions these cells could produce IL 1, which could not be distinguished from normal human monocyte derived IL 1 by gel filtration and isoelectric focusing. Moreover, analysis of phenotypic markers revealed that AF and production of IL 1 could be demonstrated in different cell types and therefore are not restricted to the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL60 cells. Thus, the expression of the DR antigens is not required for AF and IL 1 production in response to mitogens. These three human leukemic cell lines will provide convenient sources of human IL 1.