Analysis of hapten-specific T suppressor factors: genetic restriction of TsF1 activity analyzed with synthetic hybrid suppressor molecules

We have analyzed the first-order suppressor factor secreted by an azobenzenearsonate (ABA)-specific T suppressor cell (Ts) hybridoma. Treatment of the factor with 5 mM dithiothreitol (DTT) yields two fragments with distinct phenotypes and functional capabilities. One fragment is bound by a monoclonal anti-I-J antibody, the other is not. Further, although neither molecular fragment by itself is sufficient to suppress an ABA response, a mixture of the two reconstitutes the suppressive activity. The I-J- portion of the first-order suppressor factor (TsF1) presumably guides the antigen specificity; activity of the ABA-specific Ts I-J- TsF1 factor can be reconstituted with an I-J+ subunit of a TsF molecule of either sheep red blood cell (SRBC) or ABA specificity. The genetic restriction for Igh-linked determinants of the ABA/SRBC hybrid TsF molecules is influenced by the I-J+ portion, regardless of the original antigen specificity of that molecule. The data support a two-subunit TsF model. Polyclonal ABA-specific TsF1 molecules appear to resemble the monoclonal factor in structure.     

Isolation of functional polyclonal TsF from MRL-lpr spleens using monoclonal antibody absorbance

We have previously described a monoclonal antibody, B16G, which has been found to be specific for T-cell derived suppressor factors (TsF). B16G has been shown to react with T-suppressor cells, TsF in the spleens of normal or tumor-bearing mice, the TsF produced by a tumor-specific T-cell hybridoma, and with polyclonal whole human TsF isolated from tonsilar tissue. This panreactivity inherent to the B16G MAb has made it clear that it recognizes some common, shared epitope of the TsF molecule. In this study we have used B16G as a probe to isolate TsF from the spleens of MRL-lpr mice and compare the activity with these factors isolated from the spleens of an MHC compatible nonautoimmune strain, CBA. We find that equivalent quantities of functional TsF are isolable from both strains and thus, it can be concluded that the associated oligoclonal B-cell activation characteristic of MRL-lpr mice is not due to a polyclonal T-suppressor cell deficit, nor to the ability of TsC in these mice to produce soluble, functional TsFs. The molecular and biochemical characteristics of these TsFs are discussed.     

Suppressor T cell circuits in contact sensitivity. II. Induction and characterization of an efferent-acting, antigen-specific, H-2-restricted, monoclonal T cell hybrid-derived suppressor factor specific for DNFB contact hypersensitivity

This report defines a methodology for the production and characterization of an antigen-specific, monoclonal T cell hybrid-derived suppressor T cell factor (TsF) that suppresses the passive transfer of 2,4-dinitrofluorobenzene (DNFB) contact hypersensitivity. Fusion of T cells from BALB/c (H-2d) mice tolerized with syngeneic DNP-spleen cells to BW 5147 thymoma cells resulted in several hybrids that constitutively produce a soluble regulatory molecule. One of these hybrids, 26.10.2, was subsequently cloned, and its soluble factor was characterized with respect to its antigen specificity, biochemical nature, MHC restriction pattern, and identity of its target cell. 26.10.2 TsF suppresses the passive transfer of delayed-type hypersensitivity (DTH) mediated by DNP- but not trinitrochlorobenzene- or oxazalone-primed DTH T cells (TDH) after a 1 hr incubation at 37 degrees C. In contrast, 26.10.2 TsF had no suppressive effect on secondary in vitro DNP-specific T cell proliferative responses. 26.10.2 TsF therefore represents an antigen-specific factor with effector (efferent-acting) function. The monoclonal TsF was shown to consist of a two-chain, disulfide-bonded molecule, and to bear a receptor(s) specific for DNP and determinants encoded by the I region of the H-2 complex. Effector suppressive activity of 26.10.2 TsF was restricted by Class I H-2Dd determinants. One cellular target of this monoclonal factor was shown to be the DNP-specific TDH cell, because DNFB-primed lymph node cells from cyclophosphamide-pretreated donors (lacking Ts-auxiliary (Ts-aux) cells) were efficiently suppressed. The TsF appears to focus on passively bound, TDH receptor-associated, DNP-Class I determinants, as suggested by the observation that freshly prepared, but not overnight cultured, DNP-specific TDH cells were susceptible to suppression.     

Characterization of an anti-idiotypic T cell hybridoma involved in the regulation of the immune response to the P815 mastocytoma

We report the isolation and characterization of a T cell hybridoma (A29) which secretes a factor that exhibits anti-idiotypic and immune-modulating characteristics. The A29 cell line is thought to represent the hybrid analog of the Ts2 suppressor cell population in the cascade regulating the immune response to the P815 tumor in DBA/2 mice. The putative TsF2 molecule is reactive with the monoclonal antibody B16G, shown previously by us to bind a public specificity of T suppressor factors (TsF). A29 TsF also exhibits specific binding to a TsF1 secreted by another T cell hybridoma, A10, which shows specificity for antigen from the P815 tumor (this has been described previously). A29 itself does not exhibit binding to P815 antigens. Affinity-purified material from A29 appears to share characteristics with A10 molecules in that the predominant material has an apparent m.w. of 70,000. Studies with calcium flux of A29 cells showed that they respond significantly and specifically on exposure to A10 TsF stimulus. We showed further that affinity-purified A29 TsF molecules can specifically suppress the in vitro generation of syngeneic CTL to the P815 tumor, and that panning of DBA/2 splenocytes over A29-TsF-coated plates renders cell populations capable of generating a higher in vitro CTL response to P815 than appropriately treated controls.     

Isolation of an antigen-specific T suppressor factor that suppresses the in vivo response of DBA/2 mice to ferredoxin

A T cell hybridoma (Fd11) has been produced from B10.D2 mice that secretes a putative antigen-specific T suppressor factor (TsF). The TsF is isolable from culture supernatants of Fd11 by affinity purification over columns containing either a monoclonal antibody (B16G) shown previously to be capable of binding murine TsF or ferredoxin (Fd), the nominal antigen to which the Fd11 TsF binds. Specificity of the Fd11 TsF for Fd was established by comparing it to another TsF isolated by us (A10 TsF) in a sandwich ELISA, and by demonstrating the specific reactivity to Fd of the hybridoma in calcium flux studies. The Fd11 affinity-purified TsF was shown to contain two major unique components with m.w. in the region of 80,000 and 35,000 when run on reducing polyacrylamide gels in the presence of sodium dodecyl sulphate. Specific immunosuppressive properties of Fd11 were demonstrated when Fd11 TsF (10 micrograms) was injected i.v. into Fd-primed syngeneic mice at the time of antigen boost. Fd11 TsF specifically and significantly diminished the secondary antibody response to Fd in DBA/2 mice.     

Nonspecific inhibitor of contact sensitivity made by T-acceptor cells: triggering of T cells armed with antigen-specific T-suppressor factor (TsF) requires both occupancy of the major histocompatibility complex recognition site by soluble I-J product and cross-linking of the antigen recognition sites of the TsF

The phenomenon of associative recognition, i.e., the recognition of antigen together with major histocompatibility complex products (MHC) was studied in a model system. T-acceptor cells armed with antigen-specific T-suppressor factor (TsF) released a nonspecific inhibitor of the transfer of contact sensitivity when exposed to antigen together with MHC. The MHC product occurred in a KCl extract of cells and behaved genetically and serologically as I-J. Cells armed with anti-picryl or anti-"oxazolone" TsF could be triggered by the corresponding "bis-picryl-L-lysine" and "bis-oxazolone-L-lysine" together with MHC. This suggested that cross-linking of antigen recognition sites on separate molecules of TsF might be required. To investigate this possibility the bifunctional "mixed" hapten "N alpha-picryl-N epsilon-oxazolone-L-lysine," which is univalent with respect to the picryl and oxazolone haptenic groups, was synthesized. This triggered cells armed with a mixture of anti-picryl and anti-oxazolone TsF but not cells armed with either TsF alone. It was concluded that both occupancy of the I-J recognition site and the cross-linking of separate molecules of TsF was required for triggering. Moreover the hapten and the KCl extract could be given sequentially and in either order. This finding suggested that the triggering of the release of nonspecific inhibitor was due to the separate recognition of I-J and antigen and not to new antigenic determinants produced by their interaction.     

A monoclonal antibody raised to tumor-specific T cell-derived suppressor factors also recognizes T suppressor inducer factors of the 4-hydroxy-3-nitrophenyl acetyl hapten suppressor network

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.     

Hapten-specific responses to the phenyltrimethylamino hapten. V. A single chain antigen-binding I-J+ first-order T suppressor factor requires antigen to induce anti-idiotypic second-order suppressor T cells

We have previously shown that a single i.p. injection of the monovalent antigen, L-tyrosine-p-azophenyltrimethylammonium in complete Freund's adjuvant induces a Ly-1+2-, idiotype-bearing, and antigen-binding first-order T suppressor (Ts1) population. We showed that soluble factors extracted from these cells could suppress delayed-type hypersensitivity responses if administered at the induction phase of the response. In this paper we additionally characterize the suppressor factor, TsF1, with respect to its biologic, serologic, and chemical properties. The studies show that the TsF1 is neither allotype nor H-2 restricted and can induce anti-idiotypic T suppressor cells (Ts2), but it requires the presence of antigen to do so. The factor binds antigen, bears I-J encoded determinants, is resistant to reduction and alkylation, and elutes as a single chain factor after adsorption onto monoclonal anti-I-J antibody-coupled Sepharose beads in the presence of dithiothreitol (DTT). This is in marked contrast to TsF2 (derived from Id-specific Ts2-containing spleen cells), which lost its suppressive activity after reduction and alkylation, and behaves as a two chain factor after adsorption and elution from anti-I-J-coupled beads in the presence of DTT. The TsF1 is discussed with respect to the properties of it and those of TsF1 from other similar idiotype-dominated antigen systems.     

Insulin-specific suppressor T cell factors

Murine antibody responses to heterologous insulins are controlled by MHC-linked immune response genes. Although nonresponder mice fail to make antibody when injected with nonimmunogenic variants of insulin, we have recently shown that nonimmunogenic variants stimulate radioresistant, Lyt- 1+2- helper T cells that support secondary antibody responses. However, the helper activity can not be detected unless dominant, radiosensitive Lyt-1-2+, I-J+ suppressor T cells are removed. In this paper we report that extracts of primed Lyt-2+ suppressor T cells contain insulin-specific suppressor factors (TsF) that are capable of replacing the activity of suppressor T cells in vitro. The activity of these factors is restricted by MHC-linked genes that map to the I-J region, and immunoadsorption studies indicated that they bind antigen and bear I-J-encoded determinants. Insulin-specific TsF consists of at least two chains, one-bearing I-J and the other the antigen-binding site. Furthermore, mixing of isolated chains from different strains of mice indicates that the antigenic specificity is determined by the antigen-binding chain and the MHC restriction by the H-2 haplotype of the source of the non-antigen-binding, I-J+ chain. Moreover, mixtures containing antigen-binding chain from allogeneic cell donors and I-J+ chain from responder cell donors have activity in cultures containing responder lymphocytes. This suggests that preferential activation of suppressor T cells, rather than differential sensitivity to suppression, results in the nonresponder phenotype to insulin.     

Downregulation of helper T cells by an antigen-specific monoclonal Ts factor.

The findings of previous studies in this laboratory demonstrating that conjugates of human monoclonal (myeloma) IgG (HIgG) and monomethoxypolyethylene glycol (mPEG) were able to induce in mice antigen-specific tolerance and CD8+ suppressor T (Ts) cells were confirmed in the present study. An extract (TsF) of a nonhybridized clone of Ts cells (viz., clone 23.32), which had been derived from spleen cells of mice tolerized with HIgG(mPEG)26, was shown to possess antigen-specific suppressive activity. This monoclonal TsF was able to specifically suppress in vitro antibody formation only if it was present from the beginning of the culture. From the results of the cellular dissection of the system used it was concluded that (i) the TsF had no effect on fully differentiated primed B cells or plasma cells, and (ii) the TsF inactivated carrier-primed Th cells when the culture contained concomitantly naive CD8+ T cells, accessory cells, and antigen. These data support the view that the monoclonal TsF exerted its downregulating effect on Th cells only if it could first interact with a CD8+ T cell, in the presence of accessory cells and antigen.     

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.     

Regulation of immune responses by I-J gene products. V. Heterogeneity of I-J gene products as detected by anti-I-J monoclonal antibodies

The products of I-region genes of the murine major histocompatibility complex (H-2) are intimately involved in the regulation of immune responses. In a number of antigen systems, suppressor T (Ts) cells and their factors (TsF) bear determinants of gene(s) that map to the I-J subregion of the H-2 complex. Suppression in one such system, poly(Glu50Tyr50)(GT), involves the interaction of multiple Ts subsets. Three monoclonal I-Jk-bearing GT-TsF have been identified that functionally differ from one another. We report the binding of these monoclonal GT-TsF to different anti-I-Jk monoclonal antibody columns. We find that these anti-I-Jk monoclonal antibodies display differing degrees of efficiency of GT-TsF binding. These data suggest a greater degree of heterogeneity of I-Jk gene products than has been proposed before.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

TMA-specific first-order T-suppressor hybridoma. I. Characterization of the hybridoma-derived single-chain inducer suppressor factor, TsF1

Experiments described in this report will characterize a monoclonal phenyltrimethylammonium (TMA) specific, first-order T-suppressor factor (TsF1) produced by a T-cell hybridoma, 8A.3. The hybridoma expressed the Thy-1, Lyt-1, Lyt-2 antigens as well as cross-reactive idiotypic (CRI) determinants but did not express I-J encoded epitopes. It was also found to bear determinants recognized by a monoclonal antibody raised against single-chain GAT-specific TsF1. The hybridoma-derived factor was capable of suppressing primary in vitro trinitrophenol (TNP)-specific responses induced with the Brucella abortus antigen, conjugated with TMA and TNP haptens (TMA-BA-TNP). In addition, in vivo administration of 8A.3 culture supernatant resulted in the specific suppression of TMA-specific delayed-type hypersensitivity (DTH) responses. Analysis of this factor revealed it to be an induction-phase, antigen-binding, CRI+, and I-J+ single chain polypeptide. Our results represent only the second such described single chain, antigen binding, I-J+ suppressor factor derived from a monoclonal T-cell hybridoma.     

Isolation and characterization of a tumor-specific T suppressor factor from a T cell hybridoma

In our laboratory we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T cell factors (TsF) in DBA/2 mice. Therefore, B16G was used as a probe to identify T cell hybridomas secreting putative TsF. Hybridomas were obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815 tumor membrane extracts with the thymoma BW5147. One such hybridoma, A10, was selected and used for additional studies. From both the supernatants and ascites fluid of this hybrid a factor could be obtained that could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and that scored positively with B16G in an ELISA after elution. Such reactivity could not be obtained from A10 supernatants or ascites absorbed over irrelevant columns, nor was it obtained from supernatants or ascites from other T cell hybrids that had scored B16G nonreactive in the original screening. In vivo studies indicated that affinity-purified A10 material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-I. Additionally, this material did not inhibit the in vitro mixed leukocyte reaction (MLR) between DBA/2 splenocytes and allogeneic B10.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which has been demonstrated to be suppressive in this type of MLR). Biochemical analysis of this tumor-specific TsF from A10 was undertaken; the native m.w. was found to be in the region of 80,000 and 90,000. Under reducing conditions, affinity-purified A10 TsF was found to resolve in SDS-PAGE as what appeared to be a heterodimer of 45,000 and 43,000. In most preparations, an associated molecule resolving at about 25,000 was observed. The implications of these observations are discussed.     

Identification of a mouse monoclonal antibody, LHLP-1, specific for human Lp(a)

Heretofore, immunologic reagents used to define and quantify human Lp(a) have been polyclonal in origin and therefore heterogeneous in antigenic specificity. We report here the isolation of a mouse monoclonal antibody, LHLP-1, monospecific for Lp(a). The antigen reactive with LHLP-1 was expressed in both lipoprotein Lp(a) as well as apolipoprotein Lp(a) delipidated by SDS treatment; however, disulfide reduction of apolipoprotein Lp(a) inhibited LHLP-1 reactivity. The antigen reactive with LHLP-1 on Lp(a), therefore, appears not to require lipid for expression of its conformationally dependent (disulfide-inhibitable) epitope. Antigen reactivity was virtually absent in the apoB and other proteins contained in very low density, low density, and high density lipoprotein particles. Immunologic quantification of Lp(a) in individual serum samples with a rabbit reference antiserum or LHLP-1 showed good correlation. We conclude that the monoclonal antibody LHLP-1 identifies an antigen unique to Lp(a) and that this antibody may therefore be useful in the further characterization and measurement of human Lp(a).     

Mechanism controlling the genetic restrictions of an NP-specific suppressor factor that inhibits B cell responses

The mechanism of B cell suppression by a T cell hybridoma-derived monoclonal effector suppressor factor (TsF3) was studied in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. The NP-specific effector suppressor cells that produce TsF3 are Lyt-1-, 2+, I-J+, NP-binding T cells and are induced by immunization with NP conjugates. Monoclonal TsF3 inhibits both T cell activity as measured by suppression of contact sensitivity responses and B cell function as measured by suppression of antibody production to both T-independent and T-dependent antigens. The present studies were designed to specifically investigate the mechanisms and genetic restrictions that govern the interactions between TsF3 and its target cells in the plaque-forming cell (PFC) response. The results show that the target of TsF3 is a splenic adherent cell. Suppression will occur only if the restriction specificity of the TsF3 matches the H-2 genotype of the adherent population. Once this TsF3-adherent cell interaction has occurred, suppression of NP-specific B cells can occur across an H-2 barrier. The data also demonstrate that Igh-linked gene products do not appear to play a part in the TsF3-mediated suppression of in vitro PFC responses, which contrasts with the requirements for regulation of T cell-mediated contact sensitivity responses.     

Characterization of a muscle-associated antigen from Wuchereria bancrofti.

A recombinant clone, WbN1, isolated from a genomic expression library of Wuchereria bancrofti and showing restricted specificity at the DNA level (Southern and PCR analyses) for Wuchereria bancrofti and Brugia malayi has been previously described. Sequence analysis of WbN1 indicated that it had notable similarity to myosin. Further characterization using in situ hybridization has localized the mRNA in the muscle of the adult parasite and in the microfilariae. Rabbit polyclonal antiserum, raised against the recombinant WbN1 fused to the maltose-binding protein, recognized a 200-kDa polypeptide in immunoblots containing B. malayi antigen extracts. The same antibody also recognized myosin extracted from Brugia pahangi, Onchocerca volvulus, and Caenorhabditis elegans. Localization using the rabbit antiserum revealed the presence of the antigen in the adult muscle tissue and in the microfilariae; the same antibody inhibited the binding of a monoclonal antibody 28.2 (directed toward MHC B of C. elegans myosin) to the recombinant WbN1 antigen and also to purified C. elegans myosin. Based on homology data, structural location, competitive ELISA, and immunoblot we conclude that WbN1 is related to myosin or a similar myofibrillar protein.     

Cloned suppressor T cells derived from mice tolerized with conjugates of antigen and monomethoxypolyethylene glycol. Relationship between monoclonal T suppressor factor and the T cell receptor

Cloned Ts cells specific for the Ag, human monoclonal (myeloma) IgG, were derived from spleen cells of mice that had been immunosuppressed by treatment with a tolerogenic conjugate of HIgG and monomethoxypolyethylene glycol. The cloned Ts cells (clone 23.32) suppressed in vitro antibody responses in an Ag-specific and MHC-restricted manner. By FMF with appropriate antibody reagents, these cells were shown to be Thy-1+, CD4-, CD5-, and CD8+ and to express CD3 and the alpha beta-TCR. These results are consistent with the view that Ts cells use Ag recognition structures similar to those reported for Th cells and CTL. A soluble factor (TsF) extracted from the cloned Ts cells also suppressed in vitro antibody responses in an Ag-specific and H-2Kd-restricted manner, i.e., restricted to MHC class I molecules. The suppressive activity of this TsF could be abrogated by addition of mAb H28-710 that reacts with a determinant on the alpha-chain of TCR. Moreover, the TsF bound to and could be recovered from an immunosorbent consisting of the anti-alpha-TCR mAb H28-710 coupled to Sepharose 4B. In contrast, the TsF was not bound by immunosorbents consisting of mAb to the beta-chain of TCR (H57-597) or to V beta 8 (F23.1). It was, therefore, concluded that the TsF of clone 23.32 is serologically related to the alpha-chain of the TCR; however, it is not identical to TCR, because it lacks the determinants expressed on the TCR beta-chain that are recognized by the two anti-beta mAbs used in this study.     

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.