Previous studies (1-9) have suggested that peripheral human T cells can be divided into two mutually exclusive functional subsets by the two monoclonal antibodies, OKT8 and OKT4. The OKT8+ subset does not provide helper activity but contains cells capable of suppressing B cell differentiation . Importantly, the suppression observed with OKT8+ cells requires the presence of radiosensitive OKT4 + cells . Potent helper activity is found in a radiosensitive OKT4' subset . Irradiated OKT4+ cells also induced B cell differentiation, but only at high ratios of T cells to B cells . In our previous studies (7), we also found that the addition of graded numbers of radiosensitive OKT4 + cells to B cells eventually decreased the net helper activity observed . These experiments raised the possibility that precursors of suppressor cells may be contained within the OKT4 + population .The current study was undertaken to further investigate the functional heterogeneity within the OKT4+ population. In particular, we asked whether OKT4 + cells could be induced to differentiate into immunoregulatory cells capable of suppressing B cell differentiation . In the experiments reported here, we observed that although in vitro pokeweed mitogen (PWM) -activated' OKT4+ cells can function as radioresistant helper cells, these activated OKT4+ cells could also exert potent feedback suppression . This suppression mediated by activated OKT4+ cells required the presence of radiosensitive cells contained within the resting OKT4 + population. These data emphasize the potential role of interactions of T cell subsets contained exclusively within the OKT4 + population in the immunoregulation of B cell differentiation . Volume 154 August 1981 459-467 Materials and MethodsLymphocyte Preparation and Isolation ofHuman T and B Cells . Fresh peripheral blood lymphocytes were isolated from consenting healthy human volunteers by Ficoll-Hypaque density gradient centrifugation . Highly enriched population of T and B cells were then isolated by methods previously described in detail (10) . In brief, human mononuclear cells were washed in minimum essential medium (Grand Island Biological Co ., Grand Island, N . Y .) containing 5% fetal calf serum (FCS ; Microbiological Associates, Bethesda, Md .) and then separated into surface Ig* Supported in part by grants AI-14969 and AI-11524 from the National Institutes of Health, and by The Robert Wood Johnson, Jr . Charitable Trust and The Arthritis Foundation .'Abbreviations used in this paper: C, complement ; E+ , E rosette positive ; FCS, fetal calf serum ; PFC, plaqueforming cells ; PWM, pokeweed mitogen. J . Exp . MED .

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Human T cell hybrids were generated by fusing lectin-activated normal and leukemic human T cells with an aminopterin-sensitive human T cell line. This mutant cell line, designated CEM-T15, was derived from the human T cell line CEM after chemical mutagenesis with ethane methylsulfonate and subsequent culture in medium containing 6-thioguanine. After polyethylene glycol-induced fusion, the cells were cultured in hypoxanthine-aminopterin-thymidine selective medium. More than 5 wk after fusion, evidence for successful hybridization was obtained by three independent criteria: (a) The majority of the cultures contained cells expressing the OKT3 surface antigen: this antigen is expressed on normal T cells but not on CEM-T15 cells. (b) Most of the cultures contained polyploid cells. (c) Some of the cultures provided helper activity in the generation of antibody-forming cells. This functional activity is absent from the CEM-T15 parental cell line. Evidence for functional stability of the hybrids greater than 20 wk after fusion was provided by several clones that not only continue growing exponentially but also maintain expression of OKT3 surface antigen and high levels of helper function. These T cell hybrids constructed using antigen-specific human T cells should be of considerable importance in further studies of the immunobiology of human T cells.

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