Linda Rogozinski

Relationship between Human T Cell Functional Heterogeneity and Human T Cell Surface Molecules

Our knowledge of human T cell differentiation and function has expotentially increased during recent years. With this growth in knowledge there has been an increase in our appreciation of the complexity of the T-T interactions which initiate and control immune responses. A great deal remains to be learned concerning the mechanisms of these complex cellular interactions. In particular, it will be important to precisely understand the clear heterogeneity of functions within isolated subsets of OKT4+ and OKT8+ T cells. Perhaps, as importantly, it will be necessary to define more clearly the functions of the T4 and T8 molecules as well as the precise function of the other defined glycoproteins on the T cell surface. The evidence is clearly emerging that many of those molecules are not solely markers of unique functional subsets and are intimately involved in the functions of T cells.

Interactions among human T cell subsets

TB and TT interactions involved in the regulation of human B cell differentiation were studied in vitro. The strategy employed here involved the isolation of OKT4+ and OKT8+ subsets and subsequent quantitative assessment of their effects on pokeweed mitogen (PWM) driven B cell differentiation as measured by the reverse hemolytic plaque assay. Thus, graded numbers of either untreated or irradiated OKT4+ and OKT8+ cell subsets were added to autologous B cells and after 6 days, cultures were assayed for plaque forming cells (PFC) activity. We found that the helper activity which is exclusively contained within the OKT4+ population is radiosensitive. Radioresistant OKT4+ helper cells could also be demonstrated but only at high T/B ratios. In contrast, the OKT8+ population is depleted of helper function and contains radiosensitive cells important in the suppression of B cell differentiation. However, the suppression observed with radiosensitive OKT8+ cells requires the presence of radiosensitive OKT4+ cells. Cooperative interactions between these subsets were further analyzed by investigating the immunoregulatory function of OKT4+ and OKT8+ cells in the induction of helper factor production in response to alloantigens. Evidence was obtained that the production of mixed lymphocyte culture (MLC) derived helper factor is dependent on OKT4+ cells but not OKT8+ cells. Furthermore, the alloantigen induced helper factor production and/or release was found to be suppressed by the radiosensitive OKT8+ subset. Finally, additional studies demonstrated that alloreactive as well as trinitrophenyl (TNP) altered-self reactive cytotoxic T lymphocytes (CTL) precursors are contained within the OKT8+ T cell subset. However, the killer cell activity was amplified by the presence of OKT4+ cells. This amplifying effect is mediated by factors released by OKT4+ cells after specific interaction with either soluble or alloantigens. These data, taken together, underscore the importance of functional TT interactions in the immunoregulation of both T and B cell differentiation.

Human helper-T-cell function does not require T4 antigen expression

The relationship between immunoregulatory T-cell function and the expression of T-cell subset-specific differentiation antigens was examined using a phenotypically anomalous human T-cell line (TCL), termed H-1. H-1 cells were found to express T11, extremely high levels of T3, but no T4 nor T8 antigen. Despite their lack of T4 antigen expression, H-1 cells could be activated by coculture with pokeweed mitogen (PWM), anti-T3 antibody, or autologous B cells to provide potent help for B-cell differentiation into plaque-forming cells (PFC). In contrast, H-1 cells did not suppress the PFC response triggered by PWM-activated T4+ cells. These results demonstrate that the expression of the T-cell subclass-specific differentiation antigen, T4, is not required for a T cell to become activated and to implement the program for helper function. In addition, enhanced expression of T3 on the T4-, T8-, H-1 cell surface may reflect a compensatory upregulation of the T3/Ti receptor complex on T cells which are deficient in these nonpolymorphic associative recognition structures.

Construction of Human T-Cell Hybrids with Helper Function

Human T-cell hybrids with helper activity were obtained after fusion of phytohemagglutinin-activated normal human T cells with a 6-thioguanine-resistant, aminopterin-sensitive human T-cell line. This mutant line, designated CEM-T15, was derived from the human T-cell line CEM after mutagenesis with ethyl methanesulfonate. The polyethylene glycol induced fusion and the selection in hypoxanthine- aminopeterin -thymidine medium were performed by modification of standard somatic cell hybridization techniques. After fusion, the strategy for selecting hybrids consisted in screening growing cultures for the presence of cells expressing the OKT3 cell surface differentiation antigen. OKT3 was chosen because it is present in 85-95% of normal human T cells but absent from CEM-T15 cells. Thus, OKT3+ cells growing 5-7 weeks after fusion most likely represented hybrids between normal T cells (OKT3+) and continuously growing CEM-T15 cells (OKT3-). Several of the hybrids were tested for their capacity to promote pokeweed mitogen-induced antibody production by B cells. These experiments demonstrated that many of the hybrids had helper activity. Periodical testing of these uncloned hybrids for helper activity revealed functional instability, with most of the hybrids losing helper activity after 20 weeks of continuous culture. However, early and repeated cloning of the same hybrids resulted in a series of hybrid clones with helper activity still present more than 8 months after fusion. In more recent fusions, we have demonstrated that human helper hybrids producing helper factor(s) can also be obtained. These and similar hybrids with different functions will be of considerable importance in further studies of the immunobiology of human T lymphocytes.