We investigated the mechanisms by which two human T-T cell hybridoma-derived suppressor factors (SFs) (designated 160 and 169) (Platsoucas et al., Hybridoma 1987;6:589; Kunicka et al., Hybridoma 1989;8:127) inhibit the proliferative response to mitogens by human peripheral blood mononuclear cells (PBMCs). Interleukin 2 (IL-2) production by human PBMCs cultured with concanavalin A or OKT3 monoclonal antibody for 12 or 36 hr in the presence of 160 or 169 SF was found to be inhibited >80% when compared to control PBMC cultures stimulated with mitogen in the absence of SFs. This suppression of IL-2 production was not due to the SFs interfering with IL-2-induced proliferation of the IL-2-dependent murine cell clone used to determine the levels of IL-2. The proliferative responses of SF-treated PBMCs could not be restored by addition of exogenous recombinant human IL-2 (rIL-2) (1-100 U/ml). Furthermore, inhibition of the proliferative responses by the SFs could not be reversed by addition of exogenous rIL-1, rIL-2, or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on concanavalin A-activated cultures at 12- or 36-hr time points was not affected by treatment with the SFs. Both the 160 and 169 hybridoma-derived SFs were found to cause the accumulation of an mRNA of 2.8 kb that hybridized with an IL-2-specific oligonucleotide probe. This 2.8-kb transcript was in addition to the expected 1.0-kb, transiently expressed IL-2 message, and it could be superinduced in the presence of cycloheximide. These results suggest that these SFs may be influencing RNA splicing pathways. These SFs appear to be useful molecules for probing the regulatory controls of lymphocyte proliferation and may constitute important physiological regulators of the immune response. In addition, they may have clinical activity for the treatment of patients that received transplants, patients with autoimmune diseases, and others.

We have previously identified a suppressor factor (SF), designated 160 constitutively produced by human T-T-cell hybridomas generated by fusing Con A-activated human peripheral blood lymphocytes from a normal donor with cells of the Jurkat tumor T-cell line (Hybridoma 8:127-151, 1989). The 160 SF inhibited in vitro proliferative responses to polyclonal activators and allogeneic cells, and immunoglobulin synthesis and secretion of human and mouse lymphocytes. We investigated whether the hybridoma-derived 160 SF and transforming growth factor-beta (TGF-beta) are distinct molecules. TGF-beta has been shown to inhibit a number of lymphocyte responses. In agreement with our previous findings, the 160 SF abrogated the proliferative responses of human peripheral blood mononuclear cells (PBMC) to mitogens and allogeneic cells in mixed lymphocyte culture. In contrast, TGF-beta, added to the PBMC cultures at the same time with the mitogen or the stimulating allogeneic cells, had no effect on the proliferative response. Acid treatment of the 160 SF completely abolished the 160 SF activity. In contrast, this treatment results in activation of the latent TGF-beta form to the active form, and acidification does not affect the function of existing active TGF-beta. A polyclonal anti-TGF-beta antibody did not detect TGF-beta by Western blotting in concentrated (10x) 160 SF preparations. In addition, the 160 SF did not induce the anchorage-independent growth of NRK fibroblasts in the presence of EGF.TGF-beta at concentrations as low as 1 ng/ml, in the presence of EGF, induced the anchorage-independent growth of the anchorage-dependent indicator NRK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

With the objective of developing human T-T cell hybrids producing B-cell growth factor, we fused concanavalin A-activated T lymphocytes with cells of the Jurkat T cell line. The hybrids were selected on the basis of their ability to form colonies in soft agar, whereas the parent Jurkat T cell line did not. T-T cell hybrids were HLA-typed, screened by functional tests, and recloned by limiting dilution. In addition to obtaining B-cell growth factor-producing hybrids, we also obtained certain other T-T cell hybrids (as determined by HLA-typing) producing suppressor factors inhibiting proliferative responses and antibody production by human lymphocytes. Subsequently, a suppressor factor with similar inhibitory properties was identified in supernatants of the Jurkat T cell line. However, the Jurkat factor exhibited different biochemical and functional properties than the hybridoma-derived suppressor factors. Using two-parameter cell cycle analysis and the metachromatic fluorochrome acridine orange, we found that the hybridoma-derived 160 and 169 suppressor factors arrested phytohemagglutinin-induced proliferative of peripheral blood mononuclear cells in the G0/G1 phase of the cell cycle, whereas the Jurkat suppressor factor arrested proliferation in the S phase. Incubation of peripheral blood mononuclear cells with the 160, 169, or Jurkat suppressor factors for 24 hr at 37 degrees C, followed by washing, did not alter their cell cycle progression (or RNA content) in response to stimulation with phytohemagglutinin. The hybridoma-derived 160 and 169 suppressor factors and the Jurkat factor inhibited the growth but not the viability of cells from the following human tumor cell lines: A673 sarcoma cell line, SK-LC-6 and SK-LC-14 lung cell lines, SB, Raji, and Daudi lymphoblastoid cell lines, and FARR malignant melanoma cell line. In contrast, it did not affect the growth of murine L1210 cells and FS-4 normal human diploid fibroblasts. The hybridoma-derived 160 suppressor factor was selected to investigate its effect on cell-mediated cytotoxicity. The 160 suppressor factor did not inhibit natural killer cytotoxicity or its augmentation by interferon alpha or interleukin 2 or the generation of lymphokine-activated killer cells. However, this factor partially inhibited the generation of specific T cell-mediated cytotoxicity.

The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2′deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5′phosphate (FaraAMP), and 9-β-d-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by PBMC in the PWM-induced system. These results demonstrate that purine nucleoside analogs significantly inhibited a number of functions of human lymphocytes. Although selectivity for T lymphocyte subpopulations and B cells was not observed, a differential effect of Tub and FaraAMP on LAK cytotoxicity versus NK cytotoxicity and specific T cell cytotoxicity was found.

Human T-T cell hybrids are developed by fusing activated T lymphocytes exhibiting a desired immunological function or producing soluble factors with a human tumor T cell line with the objective to immortalize the T cell properties of interest. Mutagenized human tumor T cell lines, deficient for the enzyme hypoxanthine-guanine phosphoribosyl transferase have been used for the development of T-T cell hybrids. Unfused tumor cells are removed by using appropriate selection media. Certain of these media contain components (such as thymidine) that inhibit the growth of the hybrids. A different method involves the use of tumor T cell lines chemically treated, before the fusion, with irreversible biochemical inhibitors. This treatment eliminated any unfused cells of the T cell line. Recently, a method has been developed for the generation of human T-T cell hybrids without the use of mutagenized or chemically treated tumor T cell lines. Hybrids are selected on the basis of their ability to form colonies in soft agar, and their hybrid nature is confirmed by HLA typing and functional tests. The human lymphoblastoid cell lines used did not form colonies in agar. Hybrids developed by this method exhibit excellent growth characteristics and increased stability. A large number of human T-T cell hybrids producing growth, differentiation or immunoregulatory factors have been developed. Certain hybrids exhibiting immunological functions requiring direct cell-cell contact have been developed also. The advantages of using T-T cell hybrids over other methods for immortalizing T cell functions or lymphokine production are summarized. Also, the obstacles in developing T-T cell hybrids are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

We have recently developed a new method (Hybridoma 6:589, 1987) for the generation of human T-T cell hybrids. This method is based on a new selection procedure that involves cloning the hybrids in soft agar, screening by HLA-typing or appropriate functional tests and recloning by limiting dilution. T-T cell hybrids were separated from the parent line on the basis of their ability to form colonies in soft agar, whereas the parent lymphoblastoid T cell lines did not. HAT medium was not used in our selection procedure. Using this method, we have succeeded in developing human T-T cell hybrids (as determined by HLA-typing) constitutively producing B cell growth factor (BCGF) (Hybridoma 6:589, 1987) or suppressor factors. These hybrids were obtained by fusing MLC or Con A T cell blasts with cells from the Molt 4 or Jurkat lymphoblastoid T cell lines. T-T cell hybridomas, derived by fusing Con A-stimulated lymphocytes with cells from the Jurkat T cell line, produced suppressor factors inhibiting: (1) proliferative response in vitro of human peripheral blood mononuclear leukocytes to mitogens and to allogeneic cells in mixed lymphocyte culture; and (2) immunoglobulin synthesis and secretion by mononuclear leukocytes in the PWM-induced differentiation system in vitro. A suppressor factor with these inhibitory properties was also identified in supernatants of the Jurkat T cell line. These suppressor factors were ammonium sulphate precipitable, pH 2 labile, non-dialyzable and they were inactivated by treatment at 56 degrees C for 30 minutes. They exhibited a molecular weight in the range of 50,000-70,000, as determined by gel filtration, and were not gamma or alpha interferon or lymphotoxin/TNF. They did not lyse human lymphoblastoid tumor cell lines nor did they affect the viability and cell numbers of human mononuclear cells even after prolonged incubation (88 hr). They appeared to be cytostatic rather than cytotoxic molecules. The Jurkat suppressor factor is different from those produced by the hybrids on the basis of: (a) different isoelectric points; and (b) the ability of the Jurkat factor to arrest proliferation to PHA of human mononuclear cells in the S phase, whereas the 160 and 169 factors arrest proliferation at the G1 phase of the cell cycle. Certain of these suppressor factors (produced by the hybrids 153, 160, 170, and the Jurkat T cell line) also inhibited proliferative responses of mouse lymphocytes in vitro. In contrast, suppressor factors produced by the 169 and 77 hybrids did not inhibit any murine responses.

The structure-function relationship of several recombinant human alpha interferons (IFN-alpha) (IFN-alpha 1, IFN-alpha 2, IFN-alpha 4, IFN-alpha 7, IFN-alpha 2/alpha 1 and IFN-delta 4 alpha 1) was investigated with respect to their ability to augment natural killer (NK) cytotoxicity of human peripheral blood mononuclear cells (PBMC) against hemopoietic tumor cell lines. Although all these IFNs significantly augmented NK cytotoxicity against the K562, Daudi and U937 targets, significant quantitave differences were observed in their ability to augment NK. INF-alpha 4, IFN-alpha 2 and IFN-alpha 2/alpha 1 were able to augment NK at low concentrations (less than 0.1 ng/ml), whereas IFN-alpha 7, IFN-alpha 1 and IFN-delta 4 alpha 1 required significantly higher concentrations (3 ng/ml or higher). The cumulative rank order of INFs on the basis of NK augmenting ability was found to be: IFN-alpha 4 approximately IFN-alpha 2 approximately IFN-alpha 2/alpha 1 greater than IFN-alpha 7 greater than IFN-alpha 1 approximately IFN-delta 4 alpha 1. To determine synergism or potentiation in the ability of IFNs to augment NK cytotoxicity, we investigated the effect of simultaneous, sequential and reversed order of treatment of human PBMC by these IFNs. Such potentiation or synergism was not observed. In addition, all these IFNs were able to augment NK cytotoxicity against targets from malignant melanoma cell lines. IFN-alpha 7 augmented regularly and reproducibly NK cytotoxicity in 15 of 19 normal donors examined (79%). This augmentation was blocked by an anti-IFN-alpha antibody. Concentrations of IFN-alpha 7 as low as 0.06 ng/ml were able significantly to augment NK cytotoxicity of PBMC after incubation for one hour at 37 degrees C. In contrast to these findings, IFN-alpha J, an interferon similar to IFN-alpha 7, has been report to be incapable of augmenting NK cytotoxicity and also of interfering with augmentation of NK by other IFNs. Sequential treatment of PBMC first with IFN-alpha 7 and then with other interferons did not prevent the augmentation of NK. Similarly, simultaneous treatment with IFN-alpha 7 and other interferons did not prevent augmentation of NK. In both treatments IFN-alpha J has been reported to prevent augmentation of NK. IFN alpha J and IFN-alpha 7 differ only by one amino acid, at position 107, where a lysine in IFN-alpha J has been replaced by a glutamic acid in the IFN-alpha 7.(ABSTRACT TRUNCATED AT 250 WORDS)

We compared the effects of natural and recombinant (r) alpha (IFN-α) and gamma (IFN-γ) interferons on the proliferative responses of human peripheral blood mononuclear cells to mitogens and allogeneic cells in mixed lymphocyte culture (MLC) and on the generation of specific T-cell-mediated cytotoxicity. In 14 of 19 donors, natural IFN-γ and rIFN-γ had no significant effect on the proliferative responses to mitogens or allogeneic cells in MLC, even at very high IFN-γ concentrations (10,000 U/ml). In the remaining 5 donors, a statistically significant (p <0.001) enhancement by 49 ± 8% of the proliferative responses was observed. In contrast, natural IFN-α and rIFN-α2 significantly inhibited (p <0.001) proliferative responses to mitogens and to allogeneic cells, even at concentrations as low as 10 U/ml, in agreement with previous reports. Although natural and recombinant IFN-α significantly inhibited these proliferative responses, they did not affect interleukin-2 (IL-2) production in these cultures, suggesting that they inhibit proliferation by a mechanism that does not involve inhibition of IL-2 production. rlFN-γ did not affect the generation of specific cytotoxicity in MLC, although it was significantly enhanced by natural IFN-α and rIFN-α2. Additionally, we compared the ability of human rIFN-α subtypes to inhibit proliferative responses to allogeneic cells in MLC. rIFN-α2, rIFN-α4, and rIFNα7 displayed the most potent inhibitory activity of allogeneic responses and were active at concentrations as low as 0.3–0.6 ng/ml. The rIFN-α2/α1 hybrid molecule was also capable of significantly inhibiting proliferative responses in MLC at concentrations of 0.6 ng/ml or higher. rIFN-α1 and rIFN-δ4α1 inhibited at considerably higher concentrations (30 ng/ml or higher).

We compared the effects of natural and recombinant (r) alpha (IFN-alpha) and gamma (IFN-gamma) interferons on the proliferative responses of human peripheral blood mononuclear cells to mitogens and allogeneic cells in mixed lymphocyte culture (MLC) and on the generation of specific T-cell-mediated cytotoxicity. In 14 of 19 donors, natural IFN-gamma and rIFN-gamma had no significant effect on the proliferative responses to mitogens or allogeneic cells in MLC, even at very high IFN-gamma concentrations (10,000 U/ml). In the remaining 5 donors, a statistically significant (p less than 0.001) enhancement by 49 +/- 8% of the proliferative responses was observed. In contrast, natural IFN-alpha and rIFN-alpha 2 significantly inhibited (p less than 0.001) proliferative responses to mitogens and to allogeneic cells, even at concentrations as low as 10 U/ml, in agreement with previous reports. Although natural and recombinant IFN-alpha significantly inhibited these proliferative responses, they did not affect interleukin-2 (IL-2) production in these cultures, suggesting that they inhibit proliferation by a mechanism that does not involve inhibition of IL-2 production. rIFN-gamma did not affect the generation of specific cytotoxicity in MLC, although it was significantly enhanced by natural IFN-alpha and rIFN-alpha 2. Additionally, we compared the ability of human rIFN-alpha subtypes to inhibit proliferative responses to allogeneic cells in MLC. rIFN-alpha 2, rIFN-alpha 4, and rIFN alpha 7 displayed the most potent inhibitory activity of allogeneic responses and were active at concentrations as low as 0.3-0.6 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

We developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the gamma chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, we identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125I-labeled denatured lysates of T3+ WT31- lymphocytes expanded in culture from a SCID patient. These polypeptide chains were not disulfide linked and were not present on human peripheral blood lymphocytes from normal donors cultured for 5 days with phytohemagglutinin or for 2 weeks with rIL-2 and polyclonal activators or on cells of the Jurkat lymphoblastoid human T-cell line. Chemical crosslinking of 125I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These results were confirmed by sequential immunoprecipitation with anti-Leu-4 mAb followed by 9D7 anti-P13K mAb. The 9D7 anti-P13K mAb immunoprecipitated two polypeptide chains of 43 and 64 kDa from denatured lysates of lymphocytes from a patient with severe common variable immunodeficiency (CVI) that were expanded in culture with rIL-2 and Con A. Thus, this second TCR may be composed of two polypeptide chains (gamma gamma'), both of which appear to be the product of the gamma-chain gene. These experiments were done with polyclonal cell populations. Cloned T3+ WT31- cell populations are required to determine whether this TCR contains two gamma polypeptide chains. In contrast, only one polypeptide chain of 56 kDa was immunoprecipitated by the 9D7 anti-P13K mAb from peripheral blood lymphocytes from a patient with mild CVI expanded in culture with rIL-2 and polyclonal activators. Using the same 9D7 anti-P13K mAb and immunoblotting analysis, we identified a 35 kDa gamma-chain polypeptide under reducing conditions expressed on purified L3T4- Lyt2- BALB/c mouse thymocytes. This gamma-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.

We developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the gamma chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, we identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125I-labeled denatured lysates of T3+ WT31- lymphocytes expanded in culture from a SCID patient. These polypeptide chains were not disulfide linked and were not present on human peripheral blood lymphocytes from normal donors cultured for 5 days with phytohemagglutinin or for 2 weeks with rIL-2 and polyclonal activators or on cells of the Jurkat lymphoblastoid human T-cell line. Chemical crosslinking of 125I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These results were confirmed by sequential immunoprecipitation with anti-Leu-4 mAb followed by 9D7 anti-P13K mAb. The 9D7 anti-P13K mAb immunoprecipitated two polypeptide chains of 43 and 64 kDa from denatured lysates of lymphocytes from a patient with severe common variable immunodeficiency (CVI) that were expanded in culture with rIL-2 and Con A. Thus, this second TCR may be composed of two polypeptide chains (gamma gamma'), both of which appear to be the product of the gamma-chain gene. These experiments were done with polyclonal cell populations. Cloned T3+ WT31- cell populations are required to determine whether this TCR contains two gamma polypeptide chains. In contrast, only one polypeptide chain of 56 kDa was immunoprecipitated by the 9D7 anti-P13K mAb from peripheral blood lymphocytes from a patient with mild CVI expanded in culture with rIL-2 and polyclonal activators. Using the same 9D7 anti-P13K mAb and immunoblotting analysis, we identified a 35 kDa gamma-chain polypeptide under reducing conditions expressed on purified L3T4- Lyt2- BALB/c mouse thymocytes. This gamma-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.