Activated human T cells can present alloantigens but cannot present soluble antigens

Human T cells, when activated by antigen or mitogen, express Ia antigens. We have examined the capacity of activated T cells to stimulate autologous and allogeneic T cells and their ability to present soluble antigen. Interleukin 2-dependent T-cell lines (TCL), free of accessory cells, were used for antigen-presenting cells. These activated T cells were potent stimulators in an autologous mixed lymphocyte reaction (AMLR), more so than autologous irradiated non-T mononuclear cells. Activated T cells were also able to stimulate proliferation of allogeneic T cells in the absence of any other accessory cells, and this stimulation was blocked by anti-Ia antibodies. Resting unstimulated T cells were unable to stimulate autologous or allogeneic responses. Thus, activated T cells were able to present self antigens and alloantigens. However, activated T cells could not present soluble antigens to autologous T cells or to antigen-specific TCL even if exogenous interleukin 1 was added to cultures. The ability of activated T cells to stimulate an AMLR in vitro may reflect an important immunologic amplification mechanism in vivo. The ability of activated T cells to present alloantigens but not soluble antigens suggests an inability to process antigen, and this may provide further insights into the complexities of antigen presentation.     

The mechanism of antigen presentation by dendritic cells and splenic adherent cells in the induction of an allogeneic cytotoxic T-lymphocyte response to H-2Kk liposomes

Induction of an allogeneic cytotoxic T-lymphocyte (CTL) response to purified alloantigen is partially dependent on uptake and processing of the class I alloantigen by antigen-presenting cells (APC) followed by recognition of the alloantigen and self Ia by helper T cells (TH). The activated TH provides the helper signal(s) to the alloantigen-specific CTL for proliferation and differentiation into an active effector CTL. The role of antigen processing and presentation of major histocompatibility complex alloantigens was examined and the ability of different types of APC to present purified H-2Kk liposomes was investigated. Splenic adherent cells (SAC), splenic dendritic cells (DC), and B-cell lymphoblastoid lines were all shown to be effective in the presentation of H-2Kk liposomes. The relative ability of these cells to serve as APC was determined to be DC greater than B-cell tumors greater than SAC. The role of processing of H-2Kk liposomes by SAC and DC was examined by investigating the effect of weak bases on pulsing of the APC. These experiments suggest that presentation of alloantigen by both SAC and DC involves a step which is sensitive to inhibition by weak bases. We examined whether the TH were activated by similar mechanisms when stimulated by the various APC. The functional involvement of the T-cell surface marker L3T4 was demonstrated in the induction of TH. In contrast, L3T4 was not involved in the subsequent generation of CTL since monoclonal antibody (MAb) specific for L3T4 was not effective in blocking CTL function in the presence of nonspecific T helper factor (THF). Similarly, Ia on the APC was shown to be involved in the stimulation of the TH pathway but not directly in the differentiation of the CTL. Thus, DC and B cells in addition to SAC can present H-2Kk to TH. The presentation of alloantigen by both cell types may involve an intracellular route as demonstrated by the blocking of the TH response by weak bases. Both Ia and L3T4 are required on the APC for induction of the TH response. The minimal requirements for activation of the CTL were H-2Kk liposomes and a source of THF.     

Expression of class II antigens by subsets of activated T cells

Gene products coded for within the HLA complex play an important role in the control of immune responses. Class I antigens, coded for by the HLA-A, B, and C loci, are expressed by virtually all mononuclear blood cells. Class II antigens, coded for by the DR, DQ, and DP loci, have a more limited tissue distribution. They are expressed by B cells, monocytes, and by activated, but not by resting, T cells. The class II molecules of B cells and antigen-presenting cells have long been of interest to immunologists, since they are involved in the presentation of antigen, in communication between T cells and B cells and between T cells and adherent cells, and in susceptibility to certain diseases. The class II antigens expressed by activated T cells, however, remain largely uncharacterized in terms of their specificity, functional significance, and molecular nature. We have studied the expression of DR and DQ antigens by activated T cells and then examined the expression of DR versus DQ antigens by Leu 2a and Leu 3a subsets of mitogen-activated populations. Our results demonstrated that, as for class II-positive macrophages, the intensity of staining with monoclonal antibodies directed against DR antigens was much greater than that obtained with those directed against DQ antigens. Interestingly, the percentages of Leu 2a- and Leu 3a-positive cells which expressed DR antigens were quite similar, as were the percentages of Leu 2a and Leu 3a cells which expressed DQ. Thus, there does not seem to be preferential expression of DR versus DQ antigens by mitogen-activated T-cell subsets. Finally, though both DR-positive-DQ-positive and DR-positive-DQ-negative populations were detected, few or no DR-negative-DQ-positive cells were observed in these populations.     

Presentation of MUC1 tumor antigen by class I MHC and CTL function correlate with the glycosylation state of the protein taken Up by dendritic cells.

We previously reported that the glycosylated MUC1 tumor antigen circulating as soluble protein in patients' serum is not processed by dendritic cells and does not elicit MHC-Class II-restricted T helper responses in vitro. In contrast, a long synthetic peptide from the MUC1 tandem repeat region is presented by Class II molecules, resulting in the initiation of T helper cell responses. Here we addressed the ability of dendritic cells to present various glycosylated or not glycosylated forms of MUC1 by MHC Class I. We found that three different forms of MUC1, ranging from glycosylated and underglycosylated protein to unglycosylated synthetic peptide, were able to elicit MUC1-specific, Class-I-restricted CTL responses. The efficiency of processing and the resulting strength of CTL activity were inversely correlated with the degree of glycosylation of the antigen. Furthermore, the more efficiently processed 100mer peptide primed a broader repertoire of CTL than the glycosylated protein.     

T helper cell lines that augment in vivo cytotoxic T-cell responses to minor alloantigens

We have investigated the ability of long-term cultured T helper (Th) cell lines to help an in vivo cytotoxic T lymphocyte (CTL) response to non-H-2 alloantigens (minor antigens). Th cell lines specific for various single or undefined minor antigens were selected by regular restimulation with antigen in vitro. They were antigen specific and H-2 restricted in proliferation assays and were found to be able to help primary CTL responses to multiple minor antigens and secondary CTL responses to single minor antigens. Although the Th were antigen specific they did not determine the specificity of the CTL. Th cells were both necessary and limiting for an effective CTL response indicating that "helper-independent" CTL are not in themselves sufficient to generate a strong in vivo response. Under conditions where a CTL response was clearly H-2 restricted, Th cells were not. Thus, the Th cells appeared to be activated by reprocessed antigen rather than antigen on the surface of the injected antigenic cells even though the CTL themselves reacted directly to the injected antigen.     

An autoreactive T cell clone that can be activated to provide both helper and suppressor function

To understand further the biologic significance of the autologous mixed lymphocyte reaction, we determined the functional properties of autoreactive T cell lines and clones. Initially, we found that cells in an uncloned autoreactive Leu-3+ T cell line helped immunoglobulin production when added to cultures containing fresh T and non-T cells in the absence of pokeweed mitogen (PWM) but suppressed immunoglobulin production in the same cultures in the presence of PWM. To explain this phenomenon, we studied the immunoregulatory potential of an autoreactive T cell clone termed MTC-4. This clone bore the phenotype Leu-3+, 2-, 8-, 11-, DR+ and underwent proliferation when co-cultured with autologous, but not allogeneic non-T cells. Of interest, the immunoregulatory potential of the MTC-4 cells varied according to how the cells were activated. When MTC-4 cells were cultured with autologous non-T cells in the absence of antigen or mitogen (unactivated non-T cells), polyclonal immunoglobulin production (detected by reverse PFC assay) was observed. This helper activity was MHC-restricted in that it was elicited only by autologous non-T cells or MHC-matched allogeneic non-T cells; however, once activated by autologous non-T cells, it could also help allogeneic non-T cells. In contrast, when MTC-4 cells were cultured with autologous non-T cells in the presence of PWM (activated non-T cells), immunoglobulin production was greatly suppressed. This suppression was also observed when MTC-4 cells were added to cultures containing exogenous T cell help (such as that provided by autologous fresh T cells) and was not due to a direct effect of PWM on the T cell clone, because preincubation of MTC-4 cells with PWM before culture with non-T cells did not result in suppression. On the basis of these data, we conclude that autoreactive T cells can have dual immunoregulatory function that is manifest, at least in part, at the single cell level. Moreover, these regulatory functions are differentially elicited depending on the state of activation of the stimulating autologous non-T cells: when stimulated by MHC antigens present on unactivated B cells, they provide helper activity; and when stimulated by MHC antigens present on activated B cells, they act as suppressor cells. Autoreactive T cells with dual regulatory potential appear to make up a substantial proportion of all autoreactive T cells and are cells that are uniquely adapted to maintain immunologic homeostasis.     

Differential antigen-presenting cell requirements of hapten-specific T cell lines restricted to class II determinants

We used a panel of class II-restricted T cell lines (TCL), generated against trinitrophenyl (TNP)-modified autologous peripheral blood mononuclear cells (PBMC), to examine the antigen-presenting functions of various PBMC-derived class II-positive cell types, including adherent cells, B + null cells, and activated T cells. However, activated T cells and transformed or activated B cells differed in their ability to present TNP to the TCL; TNP-modified activated lymphocytes stimulated only a subset of the class II-restricted TCL that responded to class II-positive resting cells. Moreover, certain antigen-specific TCL distinguished between antigen presented on activated T cells and transformed B cells. The differences in stimulatory capacity for particular TCL did not appear to reflect differences in the expression of class II molecules or in the ability of these cells to deliver hormonal signals or process antigen. Instead, the data suggest that differences in the ability of the cells to recognize antigen on the surface of different class II-positive cells may be a function of a secondary cell surface interaction.     

Requirement for recognition of class II molecules and processed tumor antigen for optimal generation of syngeneic tumor-specific class I-restricted CTL

The roles of Class II-restricted L3T4+ T cells and of accessory cells (AC) during the in vitro generation of Class I-restricted Lyt-2+ cytotoxic T cells (CTL) specific for a Class II-negative syngeneic tumor cell line, FBL, was examined. Treatment of responder FBL-immune spleen cells with alpha L3T4 plus complement before culture, as well as the direct addition of alpha L3T4 to cultures, diminished the generation of FBL-specific CTL. The contribution of L3T4+ cells could be completely replaced by the addition of exogenous cytokines. The data demonstrate that the optimal generation of FBL-specific Lyt-2+ CTL requires the presence of L3T4+ cells, presumably to provide necessary lymphokines. FBL-specific CTL could not be generated from purified FBL-immune T cells in the absence of AC. Syngeneic Ia+ macrophages (M phi), added at the initiation of culture, restored the response of purified T cells. Pretreatment of M phi with ammonium chloride or chloroquine, or the addition of monoclonal alpha I-Ab antibody at the initiation of culture, inhibited the ability of M phi to reconstitute the CTL response. Finally, the addition of exogenous helper factors could replace M phi and reconstitute the FBL-specific response of AC-depleted immune T cells. These results suggest that during the generation of Lyt-2+ CTL to a syngeneic tumor expressing only Class I MHC antigens, Ia+ AC are required to biochemically process antigen released from the tumor cells and present this modified antigen to Class II-restricted T helper cells.     

Human T cell clones use a CD3-associated surface antigen recognition structure to exhibit both NK-like and allogeneic cytotoxic reactivity

In the present study, we developed human non-MHC-restricted CTL clones from human peripheral blood mononuclear cells activated in vitro with recombinant IL 2 and subsequently expended with PHA. The CD3/Ti+ clones were selected for their ability to exhibit non-MHC-restricted CTL reactivity by killing various tumor cell lines in culture, including the line K562 which does not express MHC antigens. We report that, at least for some of the NK-like T cell clones, it is possible to establish an allo-CTL activity, and that the CD3-associated surface antigen recognition structure might be involved in both reactivities.     

Heterogeneity in the response of T cells to antigens presented by B lymphoma cells

Proliferation of antigen-specific T-cell populations was induced in cultures stimulated with antigen and a suitable source of antigen-presenting cells. Soluble (keyhole limpet hemocyanin) and particulate (horse red blood cells) antigens were presented by irradiated spleen cells and by a variety of B-lymphoma-cell lines, providing support for antigen-specific H-2-restricted T-cell responses. A marked heterogeneity was demonstrated, however, in the capacity of T-cell lines to proliferate in response to antigen presented by the B-lymphoma cells. T-cell populations were prepared from the lymph nodes of antigen-primed mice and restimulated in vitro in the presence of antigen and irradiated spleen cells. During the first six in vitro restimulations, these T-cell populations maintained the capacity to respond to antigen presented either by irradiated spleen cells or by B-lymphoma cells. Continued growth of these T-cell populations, again in the presence of antigen and irradiated spleen cells, resulted in the generation of T-cell lines which had lost the ability to respond to antigen presented by B-lymphoma cells. These lines however, fully retained the capacity to proliferate in the presence of antigen and irradiated spleen cells. T-cell clones derived from one of these lines were also unable to respond to antigen presented by B-lymphoma cells but again proliferated in the presence of antigen and irradiated spleen cells. Supernatants containing high levels of IL-1, IL-2, or IL-3 activity failed to reconstituted the antigen-specific response of T-cell lines which had lost the capacity to respond to antigen presented by B-lymphoma cells. Furthermore, titrated numbers of irradiated spleen cells, while having the capacity to support T-cell proliferation themselves, failed to synergize with B-lymphoma cells in the support of antigen-specific T-cell proliferation. Thus we have defined populations of antigen-specific, H-2-restricted T cells which do not recognize antigen presented by B-lymphoma cells and can therefore discriminate between different antigen-presenting cell types.     

Regulation of the growth of Epstein-Barr virus-infected B cells: temporal profile of the in vitro development of three distinct cytotoxic cells

The time course of the appearance of cytotoxic cells was examined in cocultures of E-rosetting (E+) cells and EBV-infected non-T cells (4:1 ratio) from the blood of VCA-positive healthy adults. Classical HNK-1+ NK cells were present at the initiation of the cultures and they produced 76 +/- 2% specific 51Cr-release from K-562 cells, but they did not effectively lyse the NK-resistant Daudi cells, nor did they kill autologous EBV-induced lymphoblasts (LCLEBV). The NK activity decreased during the first week in culture to 40 +/- 7% cytotoxicity. At the same time, nonspecific cytotoxic cells capable of killing Daudi as well as K562 developed and persisted into the third week in culture when it declined. This later nonspecific cytotoxicity was mediated by 4F2+, T8-, HNK-1- activated E+ cells. After 10 days in culture, killing of autologous LCLEBV increased continuously, from 4 +/- 3% at Day 10 to 38 +/- 4% by Day 22. The cytotoxicity to LCLEBV was mediated by classical T8+ CTL, and it was antigen specific and at least partially HLA Class I restricted. The regression of BEBV growth that occurs in E+/BEBV cocultures coincides with the development of this CTL-mediated cytotoxicity.     

Bronchoalveolar cells from sarcoid patients demonstrate enhanced antigen presentation

The recognition of foreign antigens by T lymphocytes in association with lung antigen-presenting cells may be critical in the initiation of the mononuclear alveolitis and granuloma formation of pulmonary sarcoidosis. However, it has been shown that bronchoalveolar cells (BAC) from normal volunteers function poorly as antigen-presenting cells. Therefore, the ability of sarcoid BAC to serve as accessory cells for antigen-dependent autologous T cell proliferation, as measured by tritiated thymidine uptake, was compared with that of normal BAC. Although irradiated sarcoid BAC supported antigen-induced T cell proliferation, normal BAC did so poorly (p less than 0.005). Because it has been shown that sarcoid BAC produce more interleukin 1 (IL 1) than normal BAC, it was considered that the enhancement of antigen-induced proliferative responses could result from an increased amount of IL 1, and that contaminating monocytes in the peripheral blood T cell preparations displayed the antigen for T cell recognition. Therefore, it was necessary to establish that antigen-induced T cell responses required HLA-D region compatibility between the sarcoid BAC and T lymphocytes. BAC from sarcoid patients stimulated antigen-specific proliferation in T cells lines matched for at least one HLA-D-region antigen, but failed to stimulate T cell lines that were unmatched for both antigens. This finding indicates that cells in bronchoalveolar lavage fluids from sarcoid patients were fully capable of acting as antigen-presenting cells. The identification of antigen-presenting cells in the lungs of patients with sarcoidosis together with the previous findings of activated T cells, enhanced IL 1 production, and spontaneous interleukin 2 release in sarcoid patients is compatible with the hypothesis that local cell-mediated immunity is involved in the pathogenesis of pulmonary sarcoidosis.     

The requirements for antigen multivalency in class I antigen recognition and triggering of primed precursor cytolytic T lymphocytes

In vitro generation of a secondary cytolytic T lymphocyte (CTL) response to Class I alloantigen requires two signals: recognition of the Class I antigen by precursor CTL (Signal 1), and subsequent interaction with lymphokine(s) (Signal 2). Previous work using subcellular antigen stimulation has demonstrated that the required lymphokine(s) is produced as a result of adherent cell uptake, processing, and Ia-restricted presentation of alloantigen to helper T cells. This pathway could be bypassed by addition to the cultures of supernatant from Con A-stimulated rat spleen cells. When an optimal level of lymphokine(s) is provided by addition of Con A supernatant, the magnitude of the CTL response obtained is dependent on the effectiveness of alloantigen recognition and triggering of the primed precursor CTL (pCTL). By using this approach, we examined the cellular and molecular requirements for generation of Signal 1. Previous results had indicated that pCTL were able to directly recognize subcellular antigen, and that cellular presentation of the antigen to pCTL was not required. Further evidence for this was provided by the finding that pulsing of the responder population for short times with liposomes containing purified H-2Kk resulted in effective stimulation of the response. Exposure of cells to antigen for 1 to 2 hr at 4 degrees C generated responses of comparable magnitude to those obtained when antigen was continuously present in the cultures. Experiments were also done to directly examine the ability of alloantigen-pulsed splenic adherent cells (SAC) to deliver Signal 1. Although the antigen-pulsed SAC were very effective in presenting to helper T cells to result in factor production, they were found to be very ineffective in providing Signal 1 to the pCTL. Having obtained strong evidence for triggering of pCTL occurring via direct recognition of the subcellular alloantigen, we then examined the role of antigen multivalency in recognition and triggering. Purified H-2Kk was prepared in a variety of forms of differing multivalency, ranging from monovalent papain cleavage product to large, highly multivalent liposomes and plasma membranes. The magnitude of the CTL responses obtained was found to be critically dependent on the multivalency of the antigen preparation. Examination of the antigen dose-response curves and maximal responses obtained suggests that valency of the antigen may be important both in determining the avidity of interaction between the pCTL and the antigen-bearing structure, and in determining the extent to which localized receptor cross-linking occurs on the cell surface to result in triggering.     

Alteration of the metastatic potential of line 1 lung carcinoma cells: opposite effects of class I antigen induction by interferons versus DMSO or gene transfection.

Class I antigens are necessary for the recognition of tumor cells by cytotoxic T lymphocytes (CTL). The line 1 lung carcinoma is a spontaneous murine tumor deficient in class I antigen expression. Consistent with this, line 1 cells are highly metastatic in vivo. We investigated whether increasing class I antigen expression on line 1 cells could alter the metastatic potential of these tumor cells using an in vivo lung metastasis model. We used three methods to induce class I antigen expression on line 1 cells: gene transfection, treatment with dimethyl sulfoxide (DMSO), or treatment with interferon (IFN)-beta or -gamma. We found that line 1 cells expressing a transfected class I gene were significantly less metastatic than parental line 1 cells. DMSO-treated line 1 cells also formed significantly fewer metastases than parental line 1 cells. These results indicate that increased class I antigen expression decreases the metastatic potential of line 1 cells in vivo. However, we did not observe a significant decrease in the number of lung metastases in mice receiving line 1 cells treated with IFN-beta or -gamma, despite high levels of class I antigen expression. Thus, increasing class I antigen expression with IFN has an opposite effect on metastasis from class I antigen expression induced by transfection or DMSO. These results show that the method used to increase class I antigen expression is critical in terms of the in vivo effect observed. To investigate a possible mechanism for the differences observed in vivo between these class I expressing cells, we tested whether IFN alters or blocks susceptibility of line 1 cells to immune effector cells. We found IFN treatment increased the ability of line 1 cells to be recognized by CTL but concomitantly decreased the susceptibility of line 1 cells to NK cell lysis by a non-class I antigen-related mechanism. In contrast, transfected or DMSO-treated line 1 cells which were less metastatic in vivo were susceptible to both CTL and NK-mediated lysis. Taken together, these results suggest that immune intervention against metastasizing line 1 cells may involve NK cells and CTL.     

IFN-treated neuroblastoma cell lines remain resistant to T cell-mediated allo-killing, and susceptible to non-MHC-restricted cytotoxicity

Neuroblastoma cell lines can have very low MHC Ag expression. The cell lines are insensitive to allo-killing by primed CTL, but are sensitive to non-MHC-restricted cytotoxicity. IFN-gamma increased class I expression, but the cells remained insensitive to CTL. Susceptibility to nonrestricted effectors was preserved. Class I+ glioma cell lines behaved similarly. The CTL resistance was localized to the recognition phase. Neuroblastoma lines did not form conjugates with primed T cells, but were lysed if they were coupled to the effectors via lectins. The levels of class I expression, and resistance to CTL, were constant over a range of IFN doses. HLA-A,B,C structure and distribution were studied more intensively on one cell line, CHP-100. HLA-A2 and -A3 were present on greater than or equal to 99% of the cells, in a unimodal distribution. After IFN treatment, the levels were similar to B cell controls. In two-dimensional gel electrophoresis, the molecules co-migrated with those of B cell controls. The defect may thus be in accessory proteins that are necessary for T cell recognition or binding, rather than in the structure or distribution of the HLA-A,B,C proteins.     

Hodgkin’s lymphoma RNA-transfected dendritic cells induce cancer/testis antigen-specific immune responses

Cytotoxic T lymphocytes (CTL) can kill Hodgkin's lymphoma (HL) cells, and CTL have been used for the treatment of Epstein-Barr virus (EBV)-positive HL. For patients with EBV-negative HL, this strategy cannot be employed and alternative target structures have to be defined. In order to establish a system for the stimulation of HL-reactive T cells, we used dendritic cells (DC) as antigen-presenting cells for autologous T cells and transfected these DC with RNA from established HL cell lines. After stimulation of peripheral blood mononuclear cells (PBMC) with RNA-transfected DC, we analyzed the reactivity of primed PBMC by interferon gamma enzyme-linked immunospot. Our results suggest the presence of antigens with expression in HL cell lines and recognition of these antigens in combination with DC-derived human leukocyte antigen molecules. By the analysis of Gene Expression Omnibus microarray data sets from HL cell lines and primary HL samples in comparison with testis and other normal tissues, we identified HL-associated cancer testis antigens (CTA) including the preferentially expressed antigen in melanoma (PRAME). After stimulation of PBMC with RNA-transfected DC, we detected PRAME-reactive T cells. PRAME and other HL-associated CTA might be targets for HL-specific immune therapy or for the monitoring of HL-directed immune responses.     

Murine CD4+ T cell responses are inhibited by cytotoxic T cell-mediated killing of dendritic cells and are restored by antigen transfer

Cytotoxic T lymphocytes (CTL) provide protection against pathogens and tumors. In addition, experiments in mouse models have shown that CTL can also kill antigen-presenting dendritic cells (DC), reducing their ability to activate primary and secondary CD8(+) T cell responses. In contrast, the effects of CTL-mediated killing on CD4(+) T cell responses have not been fully investigated. Here we use adoptive transfer of TCR transgenic T cells and DC immunization to show that specific CTL significantly inhibited CD4(+) T cell proliferation induced by DC loaded with peptide or low concentrations of protein antigen. In contrast, CTL had little effect on CD4(+) T cell proliferation induced by DC loaded with high protein concentrations or expressing antigen endogenously, even if these DC were efficiently killed and failed to accumulate in the lymph node (LN). Residual CD4(+) T cell proliferation was due to the transfer of antigen from carrier DC to host APC, and predominantly involved skin DC populations. Importantly, the proliferating CD4(+) T cells also developed into IFN-γ producing memory cells, a property normally requiring direct presentation by activated DC. Thus, CTL-mediated DC killing can inhibit CD4(+) T cell proliferation, with the extent of inhibition being determined by the form and amount of antigen used to load DC. In the presence of high antigen concentrations, antigen transfer to host DC enables the generation of CD4(+) T cell responses regardless of DC killing, and suggests mechanisms whereby CD4(+) T cell responses can be amplified.     

Antigen-presenting B cells: efficient uptake and presentation by activated B cells for induction of cytotoxic T lymphocytes against vesicular stomatitis virus

We have evaluated the efficacy of mitogen (LPS/DxSO4)-activated B cells (B lymphoblasts) to function as antigen-presenting cells (APC) for vesicular stomatitis virus (VSV). Our studies revealed that B lymphoblasts induced potent cytotoxic thymus (T)-derived lymphocyte (CTL) activity in VSV-immune splenic T cells depleted of adherent accessory cells. Dose-response curves indicated that B lymphoblasts were approximately 15-20 times more efficient APC than spleen cells for CTL induction against VSV. There was little evidence of reprocessing of viral antigens by the responder population because only CTL activity restricted to the parental haplotype of the B lymphoblast was generated following stimulation of VSV-immune F1 T cells. B lymphoblasts activated VSV-specific memory CTL which expressed the Lyt-1-23+, AsGM1+ phenotype without activating natural killer and/or lymphokine-activated killer cells. The ability of B lymphoblasts to function as efficient APC was not related to enhanced viral replication in these cells because potent VSV-specific proliferative and class I-restricted CTL responses were induced by B lymphoblasts infected with VSV rendered noninfectious by exposure to ultraviolet (uv) light. This indicates that activated B cells can efficiently process and present input virion protein. Purified splenic B cells that were not activated by mitogen stimulation did not function as APC for VSV even at high multiplicities of infection. The failure of B cells to function as APC for VSV was related to inefficient uptake of VSV and their inability to provide accessory cell signals required for T-cell proliferation; both these functions developed following mitogen stimulation. These data suggest that activated B cells may function as a potent APC population for virus independent of the specificity of their immunoglobulin antigen receptor.     

Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.     

Human Antigen-Presenting Cells: Characterization of the Cells in the T-Lymphocyte Proliferative Response

Human antigen-presenting cells (APC) which present the antigen to T lymphocytes resulting in a T-lymphocyte proliferative response were found among peripheral mononuclear cells (MNC), by employing purified protein derivative (PPD) as soluble antigen. To assess the adherence capacity of human antigen-presenting cells, MNC were separated by plastic Petri dishes or nylon wool columns. Plastic nonadherent cells were almost equivalent to unseparated cells in antigen-presenting ability. Plastic adherent cells, however, showed better antigen-presenting ability than unseparated cells. On the other hand, cells passed over nylon wool columns showed essentially no ability to present PPD to T lymphocytes. Removal of phagocytic cells by carbonyl iron resulted in about 50–70% reduction in antigen-presenting ability. Carrageenan, which is known to be toxic to macrophages, had no effect on APC. By using both rabbit anti-human Ia-like antiserum and alloantiserum specific for HLA-DR phenotype and complement, it was shown that APC possessed Ia-like antigens, whereas they did not bear surface immunoglobulins. These results indicate that the human APC is probably a cell in the monocyte-macrophage lineage. Allogeneic MNC were used as APC in order to determine whether any genetic restriction exists between MNC as APC and responding T lymphocytes. Optimal stimulation was shown to require identity of mixed leukocyte reaction (MLR)-activating determinants between APC and T lymphocytes. It is, however, obscure whether an HLA-D region restriction exists in these combinations because PPD-pulsed allogeneic MNC lost their ability to elicit even MLR. It is possible that this failure to elicit MLR was caused by T lymphocytes among the MNC used as APC.