Nature of the antigenic complex recognized by T lymphocytes. IV. Inhibition of antigen-specific T cell proliferation by antibodies to stimulator macrophage Ia antigens.

We have previously demonstrated that nonimmune guinea pig T lymphocytes could be specifically sensitized with TNP-modified allogeneic macrophages after eliminating the alloreactive T cells with bromodeoxyuridine (BUdR) and light treatment. This procedure allowed the unique opportunity to use anti-Ia sera directed against the Ia antigens of only the stimulator macrophages or responder T cells to determine against which cell type anti-Ia would block TNP-specific stimulation. It was found that the TNP-specific DNA synthetic response of BUdR and light-treated T cells stimulated with TNP-modified allogeneic macrophages was totally eliminated by anti-Ia sera directed solely against the allogeneic stimulator macrophage. In contrast, anti-Ia sera directed only against the responder T cells had no effect on their response to TNP-modified allogeneic macrophages. These findings indicate that macrophage Ia antigens are required for efficient T cell-macrophage interactions and raise the possibility that T cell Ia antigens may not be required for collaboration with macrophages. This latter possibility was substantiated by experiments in which we show that treating T cells with anti-Ia sera and complement to remove the Ia-positive cells either before or after priming, or both, had no effect on their ability to be primed and restimulated with TNP-modified macrophages.     

Functional differentiation in the genetic control of murine T lymphocyte responses to human fibrinopeptide B

The ability to generate proliferative and helper T lymphocyte responses in mice was compared by using the 14 amino acid peptide, human fibrinopeptide B (hFPB). Lymph node or peritoneal exudate T cells from mice immunized with hFPB were assessed for in vitro proliferation to soluble hFPB as determined by the uptake of 3H-thymidine. The T cell proliferative response to hFPB was found to be under MHC-linked Ir gene control; mice possessing the H-2a,k haplotypes were responders, whereas H-2b,d,q,s mice were nonresponders. The influence of non-H-2 genes on these responses was not investigated, so exclusive regulation by H-2 is provisional. The absence of a detectable lymph node and peritoneal exudate T cell proliferative response persisted in H-2b,d,q,s mice after immunization and boosting with several doses of hFPB. In addition, the capacity to produce a T cell proliferative response was inherited in an autosomal dominant manner and gene(s) controlling responsiveness to hFPB mapped to the I-A subregion of the H-2 complex. To measure peptide-specific helper T cell activity, an in vitro microculture assay in which hFPB-primed lymph node T cells and normal spleen B cells and macrophages were used was developed measuring anti-fluorescein isothiocyanate (FITC) IgM and IgG plaque-forming cell (PFC) responses after culture with FITC-conjugated peptide. Immunization of B10.BR, C57BL/10, B10.D2, and B6AF mice with hFPB primed for significant helper T cell activity as assessed by the ability to augment a primary in vitro IgM response to FITC. The normal B cell IgM responses were completely dependent on hFPB-primed T cells and required that hapten (FITC) and carrier (peptide) be linked. In addition, immunization with FITC-conjugated peptide elicited positive in vivo PFC responses to FITC in B10.BR and C57BL/10 mice, indicating similar genetic control of helper activity in both the intact animals and the in vitro microcultures. Thus, B10.BR mice show both T help and T proliferative responses to hFPB, whereas C57BL/10 mice show only T help and no T proliferative responses. In contrast to B10.BR mice, C3H and CBA mice immunized with hFPB were completely unresponsive when assayed for helper T cell activity in vitro despite their ability to generate positive lymph node T cell proliferative responses. These results indicate responsiveness to hFPB by T helper and proliferating cells is different and is under separate genetic control.     

Proliferating and helper T lymphocytes display distinct fine specificities in response to human fibrinopeptide B

The fine specificity of T cell responses involved in the generation of help for antibody production and proliferation was examined by using the 14 amino acid peptide human fibrinopeptide B (hFPB, B beta 1-14) and its synthetic peptide homologues B beta 1-14(Lys14), B beta 1-13, and B beta 3-14. Peritoneal exudate or lymph node T cells from C57BL/10 and B10.BR mice immunized with hFPB or its synthetic homologues were used to measure in vitro proliferative responses. T cells from hFPB-immunized B10.BR mice showed specific proliferation to hFPB, but were unresponsive to B beta 1-14(Lys14), B beta 1-13, and B beta 3-14. B10.BR mice immunized with B beta 1-14(Lys14), B beta 1-13, or B beta 3-14 were unresponsive to all peptides tested. T cells from C57BL/10 mice showed no specific proliferation after immunization and challenge with any of the peptide antigens. In contrast to the patterns of T cell proliferation, immunization of both B10.BR and C57BL/10 mice with hFPB, B beta 1-14(Lys14), B beta 1-13, or B beta 3-14 primed for significant helper T cell activity, as assessed by the augmentation of a primary in vitro B cell IgM anti-FITC plaque-forming cell response after culture with B beta 1-1(Lys14)-FITC. Significant peptide-specific helper activity was observed when the FITC moiety was conjugated to the carboxyl terminal lysine (B beta 1-14(Lys14)-FITC) as well as FITC substitution at the amino terminus (FITC-B beta 3-13 or FITC-B beta 3-14). These results suggest that the fine specificity of T cell responses to peptide antigens are different for helper and proliferating T cells and that responsiveness by one T cell subpopulation does not predict the response pattern of other functional subpopulations.     

Lymphocyte responses to syngeneic antigens. I. Enhancement of the murine autologous mixed lymphocyte response by polyethylene glycol

The normally weak murine T-cell proliferative response against autologous non-T stimulator cells (the autologous mixed lymphocyte culture (MLC) was enhanced markedly by inclusion of the hydrophilic polymer, polyethylene glycol (PEG), into the culture medium. Potentiation of the autologous MLC was indicated on the basis of increased [³H]TdR incorporation by responding cells, as well as by the numbers of viable cells recovered from mixed cell cultures. PEG is not a polyclonal activator of T and/or B lymphocytes, since nylon wool nonadherent lymphoid cells (T cell-enriched fraction), nylon wool adherent cells (B cell-enriched fraction) and T cell-deficient “nude” spleen cells were not stimulated into DNA synthesis when cultured separately with PEG. Inclusion of 4% PEG into the culture medium was found to optimally enhance autologous MLC, although concentrations between 2 and 5% also significantly elevated responsiveness. At a responder/stimulator ratio of 1:2, autologous MLC yielded peak [³H]TdR incorporation after 5 days of culture. At lower ratios (1:1 and 2:1), however, Δ cpm of autologous MLC continued to increase over a culture period of 7 days. Enhanced responsiveness in the presence of PEG was observed in strains of mice representing a variety of H-2 haplotypes, indicating that at least the potential for autoreactivity of this type is a naturally occurring and widespread characteristic of murine species. An absolute requirement for purified T responder cells was necessary in the autologous MLC, since unseparated lymphoid cell responder LN or spleen cells demonstrated marked proliferation when cultured alone in medium containing PEG. The proliferation of T cells to autologous non-T cells within the same unseparated lymphoid cell preparation appears to be responsible for this phenomenon. Ia antigens expressed by the stimulator cells are involved in the induction of T-cell response, since anti-Ia sera added directly to the cultures inhibited the autologous MLC, but did not affect other T-cell responses to alloantigens or mitogens. Despite the marked proliferation observed in the autologous MLC performed in the presence of PEG, there was no generation of cytotoxic effector cells. Thus, PEG does not appear to add, or alter determinants on stimulator cells to an extent that they are recognized as foreign by precursor cytotoxic T cells. Although the mechanism of enhancement of autologous MLC by PEG is not totally defined, it appears, at least functionally, to promote cellular interactions that occur normally between T cells, B cells, and macrophages. In this respect, PEG will be a powerful and useful probe to dissect the cellular interactions that take place in autologous responses.     

Bromodeoxyuridine and light treatment enhances responsiveness of pokeweed mitogen-stimulated human lymphocytes to autologous and allogeneic determinants

We have investigated a novel system whereby lymphocytes from normal human subjects can be induced to develop exaggerated reactivity to histocompatibility antigens in vitro. Peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM) showed increased and accelerated subsequent proliferation to both autologous and allogeneic stimulators. Addition of bromodeoxyuridine (BUdR) during the period of maximal PWM-induced DNA synthesis followed by light exposure caused unexpected, but marked enhancement of this secondary proliferation. While untreated cultures contained a preponderance of T8⁺ cells after PWM activation, BUdR plus light-treated cultures were largely T4⁺ cells. Because removal of suppressor cells in nonsuicided cultures with anti-T8 and complement just before restimulation failed to unmask enhanced autoreactivity, events critical in the induction of the enhanced response must have occurred during priming. Cultures of PBMC with medium alone or concanavalin A, as well as purified T cells cultured with PWM, gave no enhanced autoproliferation after BUdR and light; thus T and non-T cells must be acted on by a T- and B-cell mitogenic stimulus to prime T cells for enhanced responsiveness. The interactions between T cells and activated B cells in this in vitro system may be relevant to regulatory mechanisms important in the induction of pathological autoimmune responses.     

T lymphocyte recognition of insolubilized peptide antigen

To study the role of antigen-presenting cells (APC) in T lymphocyte responses, the stimulation requirements of a murine T cell hybridoma specific for the peptide antigen human fibrinopeptide B (hFPB)/I-Ak was examined. The fine specificity of T cell recognition of this peptide was determined by using several hFPB homologs and analogs, which indicated that the intact 14-amino acid peptide must remain intact to preserve the antigenic determinant, and that the carboxyl terminal Arg14 was important for T cell responses. Of particular interest was the finding that APC-associated hFPB failed to stimulate the T cells, and that activation was only observed with soluble peptide or by brief hFPB treatment of the T cells and APC mixed together. In addition, hFPB covalently bound to agarose beads was able to cause T cell activation, provided that I-Ak+ APC were also present in the culture. A number of control experiments were performed that showed that hFPB was not released from the bead and that the antigenic peptide involved in T cell responses remained bound to the beads. These results indicate that the form of the hFPB peptide antigen recognized by this T cell can be provided separately from APC.     

Nonrandom catabolism of proteins in the formation of antigenic peptide fragments

To examine the role of protein catabolism in the formation of antigenic peptide fragments, human fibrinopeptide-immune guinea pig T cells were stimulated with the large native molecule, human fibrinogen. Two different systems were tested. In the first, we determined responses by human fibrinopeptide B (hFPB)-immune T cells, to which strain (St.) 2 guinea pigs are responders and St. 13 are nonresponders, and by human fibrinopeptide A (hFPA)-immune T cells to which St. 13 are responders and St. 2 are nonresponders. Of interest in this comparison is that both hFPA and hFPB are amino terminal peptides on the A and B chain of fibrinogen, respectively, and are readily cleaved by thrombin during fibrin formation and by other trypsin-like enzymes, leaving a carboxyl terminal Arg. Thus, if fibrinogen catabolism occurred, both antigenic peptides should be equally represented for availability in T cell responses. It was found that hFPB-immune St. 2 T cells responded to fibrinogen, but no response was observed with hPFA-immune St. 13 T cells cultured with fibrinogen. To rule out that there was a general catabolic defect in St. 13 antigen-presenting cells, fibrinogen was presented by (2 X 13)F1 macrophages to fibrinopeptide-immune parental T cells. Again it was found that F1 macrophages could present fibrinogen to hFPB-immune T cells but failed to present hFPA. In another comparison, responses with fibrinogen were also determined with des-ARg-hFPB, which lacks the carboxyl terminal Arg of hFPB, to which St. 13 are responders and St. 2 are nonresponders. The advantage of this comparison is that both antigenic determinants are contained within the same small peptide. St. 13 des-Arg-hFPB-immune T cells failed to respond in vitro by culture with human fibrinogen, suggesting that these antigenic determinants are not produced from larger peptides or proteins containing those determinants. To rule out the possibility that this was only an in vitro phenomenon, guinea pigs were immunized with the larger protein, the B chain of fibrinogen, and the immune T cells were examined for responses to fibrinopeptides derived from the B chain. Immune St. 2 T cells responded to hFPB but not to des-Arg-hFPB, whereas St. 13 T cells remained unresponsive with both peptides. These results indicate that proteolysis of larger proteins to form small antigenic peptides is not a random event and that not all potential antigenic determinants contained in a protein are produced during antigen processing.     

Nature of the antigenic complex recognized by T lymphocytes. V. Genetic predisposition of independent F1 T cell subpopulations responsive to antigen-pulsed parental macrophages.

Nonimmune (2 x 13)F1 guinea pig T lymphocytes initially stimulated with trinitrophenyl- (TNP) modified macrophages from one parental strain and then treated with bromodeoxyuridrine (BUdR) and light are unable to be primed subsequently with TNP-modified macrophages of the same parental strain. In contrast, these BUdR and light treated F1 T cells can be primed with TNP-modified macrophages of the other parental strain. These results demonstrate that in a nonimmune F1 animal two T cell subpopulations exist before priming that are genetically predisposed to respond to antigen associated with macrophages derived from one or the other parent.     

Nature of T lymphocyte recognition of macrophage-associated antigens. IV. Inhibition of peptide antigen presentation by brief treatment of macrophages with anti-Ia serum

To examine the role of macrophage la antigens in T-lymphocyte stimulation, guinea pig macrophages were briefly treated with anti-Ia serum before or after antigen pulsing with the peptide antigen human fibrinopeptide B (hFPB). To assess their antigen-specific stimulatory capacity, the variously treated macrophages were added to culture with hFPB-immune guinea pig T cells and stimulation was determined by the incorporation of [³H]thymidine. Macrophages treated with anti-Ia serum before antigen pulsing stimulated T-cell responses equivalent to those observed with antigen-pulsed macrophages treated with normal serum. By contrast, brief anti-Ia treatment of macrophages immediately following a 2-hr antigen pulse reduced subsequent T-cell responses by 45 to 70%. Similar treatment of macrophages pulsed with antigen for only 1 hr produced only modest inhibition of T-cell responses. However, if macrophages pulsed for 1 hr with antigen were incubated several hours before brief anti-Ia serum treatment, the subsequent T-cell responses were reduced by 40 to 60%. This inhibition was specific for antiserum directed against Ia antigens of the guinea pig MHC, since brief macrophage treatment with antiserum directed against B.1 antigens, the guinea pig equivalent of murine H-2K and H-2D antigens, produced no inhibition of their T-cell stimulatory capacity. These results are discussed with respect to the formation of the immunogen presented by macrophages for T-cell recognition.     

Splenocyte blastogenesis suppressed in rats implanted with an osmotic pump.

Rats implanted subcutaneously with an empty osmotic pump connected by a polyethylene catheter to a jugular vein for 5 to 10 days evinced a decreased splenocyte responsiveness to blastogenic stimulation in vitro to bacterial lipopolysaccharide, a known B cell stimulator, as well as to the plant mitogens pokeweed mitogen (PWM), a known stimulator of T and B cells, and Concanavilan A, a known T cell stimulator. The surface of the implanted pumps became infiltrated with lymphoid cells, especially macrophages. Suppression of blastogenic responsiveness after implantation for 10 days with an empty pump or even a pump dispensing pyrogen free saline only in a continuous manner was nearly as marked as that which occurred at 2-5 days after continuous infusion with endotoxin. These depressed blastogenic responses, although less, were also evident when rats were implanted with a catheter into a jugular vein connected by means of a swivel to either an empty pump or one dispensing pyrogen free saline. Suppression of blastogenic responsiveness was not related to alteration in serum complement or corticosteroid levels. Since administration of immunomodulatory substances systemically to individuals often involves implantation of an osmotic pump, investigation into the mechanisms of lymphoid cell suppression associated with an implanted pump itself has potential significance.     

Antigen-specific murine T-cell proliferation: role of macrophage surface Ia and factors.

The proliferation of Mycobacterium-primed murine lymph node T cells to purified protein derivative of tuberculin (PPD), as measured by the uptake of tritiated thymidine, requires the obligatory participiation of macrophages which stimulate the T cells either directly with antigen in association with cell surface Ia (I region-defined antigens), or indirectly by means of soluble factors. We have examined the possibility that this functional dichotomy is due to heterogeneity within the macrophage population. Since the maturation of macrophages from the precursor monocytes is associated with cell enlargement, macrophage subpopulations differing in developmental stage are obtained by cell fractionation according to size by velocity sedimentation. Nylon-wool-purified T cells which have been depleted of macrophages and B cells are stimulated with PPD either in a free form or bound to macrophages which have been incubated for a short time (i.e., pulsed) with PPD. We found that for PPD-pulsed macrophages, only the smallest (and probably the most immature) are capable of inducing T-cell proliferation. This antigen presentation function is mediated by cell surface Ia since it is abolished by pretreatment of the macrophages with anti-Ia serum and complement. On the other hand, all macrophages, irrespective of sensitivity to anti-Ia serum, secrete factors which will stimulate T-cell proliferation in the presence of free PPD. Thus the maturation of macrophages is accompanied by a shift from Ia-dependent to Ia-independent mechanisms of immunostimulation.     

Accessory cell function of cells of the mononuclear phagocyte system isolated from mycobacterial granulomas

Epithelioid cells from BCG-induced granulomas and macrophages from Mycobacterium leprae-induced granulomas were examined for their ability to act as accessory cells for T-cell proliferation to mitogen (Con A) and antigen (PPD). The granuloma cells were separated on a FACS using monoclonal antibody specific to guinea pig macrophages. Epithelioid cells (which are Ia negative) were able to support proliferation to Con A but not to antigen. Cultures containing Ia positive granuloma macrophages from M. leprae sensitized animals did not show responsiveness to Con A or to PPD. Oil-induced peritoneal exudate macrophages from BCG or M. leprae immunized animals were able to act as accessory cells for both mitogen and antigen proliferation. The nonresponsiveness of cultures containing epithelioid cells stimulated with PPD or M. leprae granuloma macrophages stimulated with Con A was not due to suboptimal or supraoptimal accessory cell:lymphocyte ratios.     

Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

Growth factor responsiveness: role of MyoD and myogenin.

A differentiation-defective variant (DD-1) of the MM14 myoblasts acquired the ability to synthesize DNA in response to treatment with epidermal growth factor (EGF) (R. W. Lim and S. D. Hauschka, 1984, Dev. Biol. 105, 48) and no longer expressed myogenic determinant genes (i.e., MyoD and myogenin) (P.R. Mueller, and B. Wold, 1989, Science 246, 780). To determine the effect of expression of MyoD on EGF responsiveness, DD-1 cells were cotransfected with a MyoD expression vector and with pRSVneo. A clone, MyoDD-1 cells, which was G418 resistant, formed multinuclear syncitia, and also expressed MyoD and myogenin, was further characterized. EGF responsiveness, as assessed by DNA synthesis, was decreased 5- to 10-fold in the MyoDD-1 cells from that in G418-resistant control DD-1 cells, despite similar EGF receptor numbers and binding affinities of the receptors. Responsiveness of MyoDD-1 cells to fibroblast growth factor (FGF) was also diminished although to a lesser extent. To determine the effects of decreased myogenic determinant gene expression on mitogen responsiveness, MM14 myoblasts were grown in medium supplemented with 5 microM 5-bromo-2'-deoxyuridine (BUdR-MM14). BUdR-MM14 cells had decreased expression of MyoD and myogenin, did not fuse, and had an altered morphology, from round to flat. The BUdR effect on fusion and cell shape was reversed by growth in control medium. BUdR-MM14 cells were responsive to EGF and had enhanced responsiveness to FGF. The combined studies support the view that expression of MyoD and/or myogenin contributes to negative regulation of mitogen responsiveness.     

Thy.1lowCD3− Cells Sorted from Nylon Wool-Passed Bone Marrow Cells Can Augment the H-2 Identical but Not Non-Identical Cytotoxic T Lymphocyte Precursors in Mixed Lymphocyte Cultures

Thy. 1^lowCD3^– cells obtained from nylon wool-passed murine bone marrow (NW-BM) cells by cell sorting did not express CD4, CD8, or T cell receptor-α/β and -γ/δ on their cell surfaces. An extremely limited number of B10.BR (H-2^k) responder lymph node (LN) cells were stimulated with B10. D2 (H-2^d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1^lowCD3^– NW-BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H-2^b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy. 1^lowCD3^– NW-BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt-2⁺ cells or Thy.1⁺ L3T4^– (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat-TCGF and Thy.1^low CD3^– NW-BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat-TCGF could be replaced by Thy.1⁺ Lyt-2^– helper T (Th) cells sorted from spleen cells. These findings showed that NW-BM cells could augment CTL precursors in a self-major histocompatibility complex (self-MHC)-antigen restricted manner, and further that both NW-BM cells and Th cells had different and independent functions to induce CTL.     

Regulation of Mycobacterium-specific mononuclear cell responses by 25-hydroxyvitamin D3

The active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to be an important regulator of innate and adaptive immune function. In addition, synthesis of 1,25(OH)(2)D(3) from 25-hydroxyvitamin D(3) (25(OH)D(3)) by the enzyme 1α-hydroxylase in monocytes upon activation by TLR signaling has been found to regulate innate immune responses of monocytes in an intracrine fashion. In this study we wanted to determine what cells expressed 1α-hydroxylase in stimulated peripheral blood mononuclear cell (PBMC) cultures and if conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) in PBMC cultures regulated antigen-specific immune responses. Initially, we found that stimulation of PBMCs from animals vaccinated with Mycobacterium bovis (M. bovis) BCG with purified protein derivative of M. bovis (M. bovis PPD) induced 1α-hydroxylase gene expression and that treatment with a physiological concentration of 25(OH)D(3) down-regulated IFN-γ and IL-17F gene expression. Next, we stimulated PBMCs from M. bovis BCG-vaccinated and non-vaccinated cattle with M. bovis PPD and sorted them by FACS according to surface markers for monocytes/macrophages (CD14), B cells (IgM), and T cells (CD3). Sorting the PBMCs revealed that 1α-hydroxylase expression was induced in the monocytes and B cells, but not in the T cells. Furthermore, treatment of stimulated PBMCs with 25(OH)D(3) down-regulated antigen-specific IFN-γ and IL-17F responses in the T cells, even though 1α-hydroxylase expression was not induced in the T cells. Based on evidence of no T cell 1α-hydroxylase we hypothesize that activated monocytes and B cells synthesize 1,25(OH)(2)D(3) and that 1,25(OH)(2)D(3) down-regulates antigen-specific expression of IFN-γ and IL-17F in T cells in a paracrine fashion.     

Cellular requirements for antigen processing by antigen-presenting cells: evidence for different pathways in forming the same antigenic determinants

In this report we examined the antigen-presenting cell (APC) requirements for activation of T-cell hybridomas specific for the protein antigen PPD (purified protein derivative of tuberculin). During the course of these studies we observed that glutaraldehyde fixation of Ia-positive A20.2JAD (A20) and P388D1 stimulator cells had different effects on T-cell activation. A20 cells fixed with glutaraldehyde stimulated the T cells in the presence of PPD as efficiently as nonfixed A20 cells. By contrast, glutaraldehyde treatment of Ia-positive P388D1 cells dramatically inhibited their ability to process and/or present PPD to T cells. This was not due to nonspecific effects on the P388D1 cells since cells prepulsed with PPD prior to glutaraldehyde treatment stimulated T cells as efficiently as non-glutaraldehyde-treated P388D1 cells. In addition, there was no apparent difference in "fixing" of the two cell types as determined by the uptake of radiolabeled thymidine. These observations suggested that P388D1, but not A20, cells required PPD internalization to form the relevant antigenic determinants. This was substantiated by showing that treatment of P388D1 cells with chloroquine prior to PPD pulsing eliminated their stimulatory capacity, but had no effect on P388D1 cells previously pulsed with PPD. Chloroquine treatment had no effect on stimulation by A20 cells. Since PPD internalization appeared not to be required for presentation by A20 cells, we next determined if isolated A20 plasma membranes would substitute for the intact cell. We observed that the isolated plasma membranes from PPD-pulsed A20 cells stimulated the T hybridoma cells, and that this stimulation was antigen-specific and was inhibited by anti-Ia monoclonal antibodies. Taken together, the results presented here suggest that for the PPD-specific T-cell responses examined here, different APC utilize distinct pathways to present the same antigenic determinant for T-cell recognition.     

Cytotoxic T cells generated in the autologous mixed lymphocyte reaction. I. Primary autologous mixed lymphocyte reaction

In the course of the culture of an autologous mixed lymphocyte reaction (AMLR), T cells proliferated in response to autologous non-T cells, and differentiated to cytotoxic T cells (AMLR killers). DNA synthesis was necessary to generate AMLR killers, as the elimination of autoreactive proliferating cells with BUdR and UV light completely abrogated AMLR killer cytolysis. Amlr killers lysed various lymphoid cell lines, including autologous B cell lines, autologous or allogeneic mitogen blasts stimulated by Con A, PHA, or pokeweed mitogen, variious nonlymphoid cell lines derived from human, mouse, or rat, and weakly normal autologous or allogeneic non-T cells. KMT-17, methylcholanthrene-induced rat fibrosarcoma, was the only resistant cell line to have been tested. AMLR killers had characteristics similar to NK cells, Major histocompatibility antigens were not the target antigens for AMLR killers. AMLR killers distinguished the blasts stimulated by alloantigens as self from the blasts stimulated by mitogens as non-self.     

Ia-independent binding of peptide antigen to the T cell receptor

We have investigated the potential for direct interaction between a peptide Ag, human fibrinopeptide B (hFPB), and the TCR on an hFPB-specific murine T hybridoma. Fluoresceinated hFPB binds specifically to hFPB-responsive T cells, but not to unrelated T hybrids. Among variant subclones of the original hybridoma, ability to bind hFPB correlates with hFPB-specific response and expression of the CD3/TCR complex, indicating that hFPB is binding to the TCR. This TCR-hFPB interaction has an affinity of approximately 6.6 microM, reflecting slow association and rapid dissociation of the Ag from its receptor. These findings confirm the potential for direct Ag-TCR interaction and indicate an Ag recognition mechanism that is not initiated by Ag-MHC interaction.     

Plasmodium berghei: The in Vitro immune response

Following primary in vitro Stimulation by Plasmodium berghei, IgM titers by the indirect fluorescent antibody test (IFAT) were negative in in vitro reconstituted syngeneic mouse spleen cultures containing T cells and macrophages, or B cells and macrophages, or macrophages alone, but IgM titers of 1:20 were obtained from cultures containing B cells, T cells, and macrophages. IFAT IgG titers were negative for cultures with T cells and macrophages together, or macrophages alone, but rose to 1:40 with cultures containing B cells and macrophages and 1,80 with cultures of B cells, T cells, and macrophages together. After a second in vitro challenge, IgM and IgG titers were similar to the stimulated cultures from immunized mice; the IgM titer reached only 1:20 but the IgG titer rose to 1:160. Total immunoglobulin was higher in the cultures from immunized mice than from in vitro primed cultures.