A human autoreactive T cell line specific for minor histocompatibility antigen(s) isolated from a bone marrow-grafted patient

A human autoreactive T cell line named Bur-1 has been obtained from a woman 4 mo after an allogeneic bone marrow transplantation (BMT) from one of her HLA-identical brothers. The phenotype of the cell line is 100% T11+ and over 90% T4+, and the karyotype confirms its donor (male) origin. These donor T cells proliferate specifically in the presence of donor's peripheral blood monocytes (PBM) but not recipient's cells, and they kill specifically donor's but not recipient's Epstein-Barr virus (EBV)-induced lymphoblastoid cell lines (LCL). PBM from another HLA-identical brother and from several unrelated donors also stimulate Bur-1 cells, and EBV-induced LCL from the same donors are killed in cytotoxicity assays. All of these donors share HLA-DR5 or HLA-DRw11 (the major split of HLA-DR5) with Bur-1 cells. However, some but not all of the PBM sharing HLA-DR5 with Bur-1 cells are recognized. Therefore, in contrast with the previously described autoreactive T cells, Bur-1 cells are not directed against self-MHC antigens but rather recognize autologous minor histocompatibility (mH) antigens in the context of autologous HLA class II molecules. Because both male and female cells can be recognized, the reacting minor antigen could not be the male-specific HY antigen. It is suggested that autoreactivity against mH antigens can be observed in bone marrow-grafted patients due to the education of bone marrow donor precursors in the recipient thymus not allowing tolerance to autologous (donor) mH antigens not shared by the recipient.     

Nonrandom selection of T cell specificities in anti-HLA-DR responses. Sequence motifs of the responder HLA-DR allele influence T cell recruitment

The self-restriction of Ag-specific T cell responses is interpreted as the result of a positive selection of the individual's T cell specificities for their compatibility with self-MHC molecules. If the T cell receptor (TCR) specificities in any given individual have an affinity for syngeneic MHC molecules, it is unclear how they interact with allogeneic MHC structures. To approach this question, we analyzed 123 alloreactive HLA-DR4 Dw4 or Dw14 specific T cell clones that were generated from responder/stimulator combinations with defined disparities in the HLA-DR beta 1-chain. Sets of T cell clones were established from three different HLA-Dw4+ responders and compared for their fine specificities. The majority of HLA-DR4 Dw14 specific T cell clones co-recognized HLA-DR1 Dw1+ (33 to 36% of all T cell clones) or HLA-DRw14 Dw16+ (26 to 33%) stimulators, both of which share very similar sequences in the third hypervariable region of the HLA-DR beta 1-chain with the HLA-DR4 alleles Dw4 and Dw14. These data suggest that sequence and structural similarities in the alpha-helical portions of the HLA-DR molecule impose a strong bias on the recognition of allotargets. The second haplotype of the responder did not appear to affect the typical fingerprint of T cell recognition except for the deletion of self-reactive TCR specificities. Nonrandom usage of TCR specificities in anti-HLA-DR responses was also found for HLA-DRw11/DRw13+ and HLA-DRw11/DR7+ T cell donors who did not share any obvious polymorphic sequence stretches with the allostimulators HLA-DR4 Dw4 or Dw14. T cell clones from an HLA-DRw11/DRw13+ responder functionally resembled the TCR specificities derived from the HLA-DR4 Dw4+ donors. T cell clones derived from an HLA-DRw11/DR7+ individual were characterized by a distinct cross-reactivity pattern preferring HLA-DRw13 Dw19+ (50 to 60%) and HLA-DR3+ (43 to 57%) stimulator cells. These findings suggest that the responder HLA-DR alleles influence the structural constraints in the recognition of allo-HLA-DR molecules in closely related and in completely disparate responder/stimulator combinations.     

Defective antigen presentation by human melanoma cell lines cultured from advanced, but not biologically early, disease

The present studies were undertaken to characterize Ag presentation by cultured human melanoma cell lines. Cell lines established from "biologically early" lesions of malignant melanoma were able to present the soluble Ag tetanus toxoid (TT) to autologous and HLA-DR-matched allogeneic, TT-immune T cell clones. Proliferation of T cell clones in response to Ag presented by primary melanoma peaked on day 2 of culture with Ag. Ag presentation was blocked by pretreatment of TT-pulsed and fixed melanoma cells with mAb against HLA-DR, but not HLA-DQ, HLA-DP, or HLA-ABC. Ag processing and presentation were inhibited by treating the melanoma cells with ammonium chloride. In parallel with previous findings from this laboratory demonstrating the inability of cell lines cultured from "advanced" primary or metastatic melanoma to induce autologous T cell proliferation, such cell lines also failed to present this exogenous Ag despite the presence of cell-surface HLA-class II molecules. Thus, in contrast to the finding in biologically early melanoma, none of the multiple TT-immune, T cell clones from autologous patients or HLA-DR matched donors was able to respond to TT presented by melanoma cells cultured from advanced disease. Co-incubation studies revealed that metastatic melanoma cells did not secrete inhibitory substances during the APC assay, however, they were able to process TT, rendering it "immunogenic" in the presence of fixed, autologous non-T cells. When fixed, autologous melanoma cells were assayed for their ability to present processed Ag; fixed cells of early but not advanced disease were able to present Ag in this setting, indicating that the presenting limb becomes flawed in the evolution of the metastatic phenotype. Finally, studies of chloroquine inhibition of the capacity of melanoma cells derived from early primary disease to stimulate autologous peripheral blood T cells suggest that such cells process and present tumor-associated Ag in the same fashion as the "model" Ag TT.     

Human T cell receptor-gamma delta-expressing T-cell lines recognize MHC-controlled elements on autologous EBV-LCL that are not HLA-A, -B, -C, -DR, -DQ, or -DP.

HLA-loss variants of an EBV-transformed B lymphoblastoid cell line (EBV-LCL) 721 were used to investigate whether human MHC molecules other than known class I or class II were involved in autologous T cell responses. Bulk lymphocyte cultures of purified T cells primed to an autologous variant EBV-LCL that fails to express HLA-class II and has reduced cell surface HLA-class I expression, and oligoclonal TCR-gamma delta-bearing lines derived from them, could lyse both this EBV-LCL and an independently derived, class II expressing autologous variant EBV-LCL that bears no HLA-A, -B, or -C, suggesting the presence of additional HLA-like restriction elements. Cold target inhibition of cytolysis mediated by these lines indicated that a shared or cross-reactive MHC controlled restriction element other than the known MHC determinants was retained by the EBV-LCL variants. Single-cell derived clones from these T cell lines which expressed only the TCR-gamma delta showed this same target cell specificity pattern, proving recognition of MHC-controlled determinants by autologous gamma delta T cells. Anti-gamma delta antibody could inhibit cytolysis by the gamma delta-expressing lines, suggesting that the TCR-gamma delta was involved in recognition of the EBV-LCL targets. Flow cytometric analysis with separate HLA-reactive antibodies indicated that the restriction element for these cytolytic responses is a molecule serologically cross-reactive with HLA-B and -C Ag, yet is a determinant that cannot be HLA-A, -B, -C, -DR, -DQ or -DP.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. II. Bifunctional clones with cytotoxic and virus-induced proliferative activities exhibit herpes simplex virus type 1 and 2 specific or type common reactivities

Human cytotoxic T lymphocyte (CTL) clones directed against herpes simplex virus (HSV)-infected cells were generated after stimulation of peripheral blood lymphocytes (PBL) with HSV type 1 (HSV-1) and HSV type 2 (HSV-2). These CTL clones were studied with regard to HSV type specificity and with regard to whether they also express helper cell activity. Some clones, generated after stimulation with HSV-1, were cytotoxic for autologous cells infected with either HSV-1 or HSV-2 ("HSV type common clones"), whereas other clones lysed HSV-1-infected cells only ("type-specific clones"). Similarly, after HSV-2 stimulation, both HSV-2 specific and HSV type common clones were obtained, indicating the heterogeneity of human cytotoxic T cells to HSV. All CTL clones tested were found to be bifunctional in that they also proliferated in response to stimulation with HSV. The HSV type specificity of the proliferative response was identical to that of the cytotoxic activity of the clones. An HSV type common clone, when stimulated with either HSV-1 or HSV-2, and an HSV-1 specific clone, when stimulated with HSV-1 but not with HSV-2, produced a factor, presumably interleukin 2 (IL 2), which induced proliferation of CTLL, an IL 2-dependent T cell line, providing evidence that our HSV-directed CTL clones also express helper cell activity. CTL clones that we previously reported were restricted in cytotoxic activity by HLA class II DR-1 or MB-1 antigens were found, in this study, to be restricted in proliferative response to HSV by these same HLA antigens. These results suggest that our bifunctional T cell clones directed against HSV may recognize the same viral antigenic determinants and the same HLA antigens for both cytotoxic and virus-induced proliferative activities. This is the first demonstration of human HSV type specific and HSV type common T cell clones and HSV specific T cell clones with both cytotoxic and helper cell activities.     

Functional modulation of hepatitis B core antigen-specific T lymphocytes by an autoreactive T cell clone

Hepatitis B core (HBc)Ag-specific T cells present in the peripheral blood of a patient with chronic active hepatitis B were expanded by co-cultivation for 7 days with rHBcAg. After cloning at 1 cell/well in the presence of PHA and IL-2, five HBcAg-specific CD4+ cloned lines were obtained. All five lines proliferated and produced IL-2, IFN-gamma, and TNF in a dose-dependent fashion in response to HBcAg, but not to HBV envelope Ag. The cloned lines and derivative clones were HLA class II (DR1) restricted. All T cell clones were able to induce anti-HBc production by autologous B cells in response to HBcAg (helper effect). The proliferative response and the helper effect of the HBcAg-specific T cell lines and clones were augmented by co-cultivation with an autologous, autoreactive (HLA-DQ1 specific) T cell clone, even in the absence of HBcAg, and the autoreactive T cells directly stimulated anti-HBc secretion by autologous B cells, presumably due to the release of Ag-nonspecific factors. These findings define a model immunoregulatory circuit the physiologic significance of which remains to be determined.     

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destruction of B16 melanoma cells mediated by bystander cytolysis in vitro

Two Lyt-1+, L3T4a+ autoreactive T cell clones specific for self-class II major histocompatibility complex (MHC) gene products were established from lymph node cells and spleen cells of C57BL/6J mice, respectively, by different methods. They were stimulated to proliferate in culture in response to I-Ab antigen-bearing syngeneic spleen cells in a class II MHC-restricted manner. This stimulation was inhibited completely by the addition of anti-L3T4a (GK1.5) or anti-I-Ab (3JP) monoclonal antibodies. The autoreactive T cell clones lysed syngeneic I-Ab+ target cells such as lipopolysaccharide (LPS) blasts. They also lysed I-A- bystander cells such as Cloudman and B16 melanoma and lymphoid tumor cells in the presence of I-Ab+ stimulator cells but not I-Ad+ cells. This bystander killing was most likely mediated by soluble factors released from the autoreactive T cells in response to I-Ab antigens, because culture supernatants from activated autoreactive T cells inhibited the proliferation of B16 melanoma cells in vitro and also had significant cytolytic activity. Both lymphotoxin and interferon-gamma were released from activated autoreactive T cells, suggesting that these cytotoxic lymphokines were responsible for autoreactive T cell-mediated cytolysis. The finding that the two clones, established independently and by different methods, show self-class II MHC antigen-restricted cytolysis, and bystander cytolysis suggests that these properties are not restricted to a unique population of autoreactive T cells. These results favor the concept that in vivo, autoreactive T cells may express not only regulatory activity in regard to antibody responses, but also anti-tumor activity via bystander cytolysis.     

Heterogeneity of allogeneic restriction of human cytotoxic T cell clones specific for Epstein Barr virus

Clones of human cytotoxic T cells (Tc) specific for Epstein Barr virus (EBV) were isolated from peripheral blood lymphocyte (PBL) cultures stimulated repeatedly with autologous EBV-transformed lymphoblastoid cell line (LCL) cells in vitro. The method employed to clone EBV-specific Tc was a limiting dilution technique utilizing T cell growth factor (TCGF). The EBV specificity of Tc clones was determined by showing that they were significantly cytotoxic for autologous LCL cells but not for either autologous PBL or (natural killer-sensitive) K-562 cells. Eight EBV-specific Tc clones derived from a single donor exhibited distinct cytotoxic patterns against allogeneic LCL targets. Two clones were cytotoxic to LCL targets sharing both HLA-A26 and B15 antigens with effectors, and killing by two other clones was strongly restricted to autologous LCL cells. The four remaining clones showed cytotoxicities against various allogeneic LCL targets irrespective of HLA antigen expression. Eight EBV-specific Tc clones derived from a second donor also exhibited a wide spectrum of cytotoxicity to allogeneic LcL targets. We conclude that EBV-specific Tc, induced in vitro, consist of a number of clones with respect to restrictions imposed by the major histocompatibility complex. The determinants regulating these restrictions may include not only private HLA antigenic determinants that are defined by the HLA serotyping, but also undefined HLA antigenic determinants.     

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.     

Presentation of autoantigen by human T cells.

Activated human T cells express MHC class II and have been shown to present foreign Ag to autologous T cells. We now demonstrate that MHC class II+ T cell clones can present myelin basic protein (MBP) peptide autoantigen in the absence of traditional APC to autologous MBP reactive T cell clones. MBP peptide-pulsed T cell clones specifically stimulated autologous MBP-reactive T cell clones to flux calcium and proliferate. Activation responses were peptide epitope specific and blocked by mAb to MHC class II, indicating a TCR-mediated response. In addition, mAb to the adhesion molecules LFA-3, CD2, LFA-1, CD29, and to the tyrosine phosphatase CD45 also inhibited proliferation, indicating the involvement of T to T cell interactions. In contrast to peptide Ag, T cell clones did not respond to autologous T cells pulsed with HPLC-purified MBP, suggesting that T cells are unable to process whole MBP. However, batch-purified MBP Ag preparations containing lower m.w. breakdown products were presented by T cells, indicating that naturally occurring breakdown products of autoantigens could be presented by activated T cells in vivo. These results raise the possibility that T cell presentation of autoantigen at inflammatory sites may be important in regulation of immune responses to self Ag.     

Human cytotoxic T cell clones directed at autologous virus-transformed targets: further evidence for linkage of genetic restriction to T4 and T8 surface glycoproteins

Human cytotoxic T cell clones were generated against autologous EBV-transformed B lymphocytes. Whereas the majority of the clones expressed the T8 surface glycoproteins and showed a specificity for class I MHC gene products on the target cell, a minority expressed the T4 surface glycoprotein and demonstrated a class II specificity. Monoclonal antibodies to T4 and T8 inhibited cytotoxic effector function of reactive clones in a fashion analogous to their effect on alloreactive CTL clones. Each autoreactive T cell clone was cytotoxic for EBV-transformed B lymphocytes but not pokeweed mitogen-activated or resting autologous lymphocytes, suggesting a dual specificity for an MHC gene product as well as an antigen induced and/or encoded by virus. Taken together, the present findings provide further support for the notion that T4 and T8 serve as associative recognition elements on T lymphocytes for MHC gene products.     

EBV-specific CD4+ T cell clones exhibit vigorous allogeneic responses

Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4(+) T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4(+) T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-alpha production by the EBV-specific CD4(+) T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.     

Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated. The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones. In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined. The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Highly polymorphic products of both HLA-DR and HLA-DQ genes contribute to the polymorphism of the HLA-DR w13 haplotype

We studied the polymorphisms of HLA-DR and HLA-DQ products from HLA-DRw13 haplotypes by analyzing the restriction of influenza A-specific cloned T cells from an HLA-DRw13,DQw1,Dw19 homozygous individual. The results show that (1) some functional epitopes, which can be borne by either HLA-DR or HLA-DQ molecules, are strictly correlated with the HLA-Dw19 subtype of HLA-DRw13. This clearly indicates that both HLA-DR and HLA-DQ products contribute to the HLA-Dw19 subdivision of HLA-DRw13. (2) At least two different restricting epitopes are borne by DR products: one is correlated with the HLA-DRwl3 serologically defined specificity, which includes Dw19 and Dw18 haplotypes; the other is correlated with the only HLA-Dw19 subtype of HLA-DRwl3. (3) Restricting epitopes borne by DQ molecules have been found on Dw19 cells only. (4) DQ-restricted clones were unable to react with DQwl APC of any other haplotypes tested, including DR1, DR2-long, DR2-short, and DRw14, demonstrating a high degree of functional polymorphism among the serologically defined DQw1 specificities.Abbreviations used in this paper: APC antigen-presenting cells - cpm count per minute - HAU hemagglutinin units - IL-2 interleukin 2 - MHC major histocompatibility complex - mAb monoclonal antibody - PBM peripheral blood mononuclear cells - PHA phytohemagglutinin - pl isoelectric point - PMA phorbol myristic acetate - SD standard deviation     

Analysis of insulin-specific T cell clones. I. Strategy for production of clonotypic antibody

A novel strategy for production of T cell clone-specific antiserum is described. Clones that are both antigen-specific and alloreactive can be injected in small numbers i.v. into a strain which expresses the appropriate alloantigens, inducing consistently high-titered antisera containing antibodies to the unique T cell receptor molecules on these cells. The antisera characterized in this report block activation of these cells by either antigen plus autologous class II products or alloantigen. Furthermore, in the absence of antigen, the antiserum strongly stimulates the specific clones to divide, and to synthesize and secrete various proteins. Consistent with the findings of other investigators, the antiserum immunoprecipitates a disulfide-linked dimer of 90,000 m.w. from the surfaces of cells with the appropriate specificity.     

Staphylococcal enterotoxin-mediated human T-T cell interactions.

Staphylococcal enterotoxins (SE) are known to stimulate a large proportion of T cells. SE bind to MHC-class II molecules on APC and a particular segment of certain TCR V beta and V gamma gene products. Resting human T cells do not express HLA class II Ag and therefore cannot present SE to T cells. Activated human T cells, however, do express HLA-DR, -DP, and -DQ Ag and could consequently serve as APC for SE. As such, local immune responses to SE might be regulated and/or abrogated by SE-mediated T-T cell interactions leading to T cell destruction. We have investigated if such SE-mediated T-T cell interactions can occur in vitro using human cytolytic TCR-alpha beta+ and TCR-gamma delta+ T cell clones. We demonstrate that the TCR-alpha beta+ T cell clones can efficiently present staphylococcal enterotoxin A (SEA) to each other: T cell clones coated with SEA are lysed by SEA-reactive T cell clones but not by a SEA-nonreactive T cell clone. Furthermore, the SEA-reactive TCR-alpha beta+ clones (but not the SEA-nonreactive clone) destruct themselves in the presence of SEA at low concentrations of SEA (less than 0.01 microgram/ml). Also, SEA-coated T cell clones can induce proliferative responses although such responses are much weaker than those induced when B cells are used as stimulator cells. In contrast, the SEA-reactive TCR-gamma delta+ T cell clones are resistant to autokilling in the presence of SEA and they do not lyse SEA-coated TCR-gamma delta+ targets. However, such targets can be lysed by TCR-alpha beta+ effector cells. These results indicate that TCR-gamma delta+ cells are relatively resistant to lysis and that during local nonspecific immune responses triggered by SE, which induces HLA-class II expression by the responding T cells, SE-mediated T-T cell interactions may play a role in the regulation and/or abrogation of these immune responses.     

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 processing requirements for T cell activation: differential requirements for presentation of soluble conventional antigen vs cell surface MHC determinants

The present studies were undertaken to characterize the antigen-processing requirements involved in the responses to T cells to soluble antigen (antigen specific), to allogeneic cell surface MHC determinants (alloreactive), and to syngeneic MHC determinants (autoreactive). T cell clones were used that have dual cross-reactive specificities either 1) for self MHC plus soluble antigen and for allogeneic MHC products or 2) for syngeneic MHC and for allogeneic MHC, in order to permit comparison of the processing requirements for responses of the same T cell to distinct antigenic stimuli. The proliferative responses of antigen-specific, Ia-restricted T cell clones to soluble antigens were sensitive to treatment of antigen-presenting cells (APC) with 125 to 250 microM chloroquine, a lysosomotropic agent previously shown to inhibit the processing of soluble antigens. In contrast, the same T cell clones were only minimally affected in their ability to respond to similarly chloroquine-treated APC expressing allogeneic MHC products. The responses of autoreactive T cell clones to syngeneic stimulating cells and their cross-reactive responses to allogeneic cells were both resistant to chloroquine treatment of stimulating cells. The failure of chloroquine to inhibit antigen presentation to autoreactive T cell clones suggests that these clones are specific for self Ia not associated with in vitro processed foreign antigen. Thus, chloroquine sensitivity distinguishes the in vitro antigen-processing requirements for presentation of the soluble antigens tested from the requirements for presentation of syngeneic or allogeneic cell surface MHC determinants to the same T cells.     

IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.     

The net effect of costimulatory blockers is dependent on the subset and activation status of the autoreactive T cells

In this study, we investigated whether CD4 and CD8 autoreactive T cells have different costimulatory requirements for their activation in vitro by testing the effect of a panel of Abs specific for various costimulatory molecules. Our results showed that CD8 interphotoreceptor retinoid-binding protein-specific T cells are more dependent on costimulatory molecules for activation than their CD4 counterparts. Interphotoreceptor retinoid-binding protein-specific T cells are less dependent on costimulatory molecules in the secondary response than the primary response. We also showed that blockade of costimulatory molecules can either promote or inhibit the proliferation of autoreactive T cells, depending on the degree of activation of the cells. Our results show that anti-costimulatory molecule treatment can have diverse actions on autoreactive T cell subsets, the net effect being determined by the subset of immune cells affected and the type and dose of treatment used.