Marie-Luise Blue

Evidence for specific association between class I major histocompatibility antigens and the CD8 molecules of human suppressor/cytotoxic cells

Human T lymphocytes, metabolically labeled with 35S-cysteine and 35S-methionine, were reacted with the homobifunctional cross-linking reagent, dithiobis (succinimidyl propionate) (DSP). When detergent lysates from these cells were immunoprecipitated with a monoclonal antibody reactive with the CD8 antigen, a radiolabeled protein of approximately 44 kd was coprecipitated with the CD8 molecule. Immunoprecipitates from detergent lysates prepared without prior chemical cross-linking contained only the 33 kd CD8 molecule. Similar results were obtained when T lymphocytes or a cytotoxic T cell clone (T4T8Cl) were radiolabeled with 32P-orthophosphoric acid. The 44 kd CD8-associated protein was identified as the heavy chain of the class I major histocompatibility antigen by depletion in preclearing experiments with anti-class I MHC antibody and by peptide mapping. Further analyses indicated that the CD8-class I MHC association is due, in part at least, to disulfide bonding, which may be susceptible to cleavage during processing of cell lysates.

Crosslinking CD3 with CD2 using Sepharose-immobilized antibodies enhances T lymphocyte proliferation☆

T lymphocyte proliferation can be triggered through interactions with either CD3:Ti, the target of antigen-specific activation, or CD2, the target of an antigen-independent activation pathway. Sepharose-immobilized antibody reactive with CD3 was used to aggregate the T cell receptor complex resulting in T lymphocyte activation. When CD3 was simultaneously crosslinked with CD2 using Sepharose beads coupled to antibodies directed at both determinants, T cell proliferation was markedly enhanced (stimulation index = 8- to 11-fold). A smaller enhancement was induced when CD3 was crosslinked with several other functionally relevant T cell surface molecules. The relative mitogenic potency of the accessory molecules tested was CD2 greater than CD4 greater than CD8 greater than 2H4. Little or no increased proliferation resulted from crosslinking CD3 with class I or class II major histocompatibility antigens. The added proliferation induced by CD3: CD2 crosslinking did not occur in the presence of soluble antibodies directed against CD2. Human thymocytes, the majority of which express both CD3 and CD2, were similarly activated by Sepharose-immobilized antibodies. Our results suggest that specific interactions between T cell surface molecules may play a role in the regulation of lymphocyte activation.

Secondary immune amplification following live poliovirus immunization in humans

Eight subjects inoculated orally with live attenuated poliovirus were investigated to study the effects of live virus infection on human T-cell responses. Proliferation to poliovirus and unrelated recall antigens were measured serially over a 3-week period. Five of eight subjects inoculated demonstrated a clear anamnestic response to poliovirus, but three did not. Only the five subjects demonstrating an anamnestic response to poliovirus were found to have augmented secondary immune responses to two unrelated recall antigens (tetanus toxoid and reovirus) and in the autologous mixed lymphocyte response (AMLR). No consistent changes were found in circulating T-cell surface activation antigens whether or not the subjects responded to poliovirus. These studies suggest that an asymptomatic poliovirus infection associated with immunization in humans can induce nonspecific secondary immune amplification as measured by in vitro T-cell proliferative response. This amplification pathway is a potential mechanism for immune responses against antigens other than those of the infecting virus.

Human thymocyte maturation in vitro: A flow cytometric analysis☆☆☆

Using an in vitro culture system, light scatter analyses, and two-color flow cytometry, we provide evidence that the interleukin-2 (IL-2) and transferrin receptors can be induced within 48 hr on nonproliferating immature thymocytes. The thymocytes (greater than 35%) that expressed the transferrin and IL-2 receptors demonstrated nuclear activation as measured by log 90 degrees light scatter analysis. Increases in antigen-receptor-associated T3-antigen expression followed transferrin and IL-2-receptor induction and occurred on maximally activated T4+T8+ thymocytes on Day 3 of culture. Maximal T3 expression did not occur until Days 5-7 and paralleled loss of T4, T8 coexpression, suggesting an association between a mature T3-Ti antigen receptor complex and a mature T4, T8 phenotype.

Characterization of the T3+T4+T8+ thymocyte intermediate in vitro

Sensitive two-color fluorescence staining and cell-sorting techniques were used to isolate a T4+T8+ thymocyte subpopulation with high T3 density from human thymocyte cultures. Previously, this population was shown to give rise to both T4+T8- and T8+T4- thymocytes. In the present study, this T3+T4+T8 population was shown to be functionally as well as phenotypically distinct from either T8+T4- cells or T4+T8- cells present in the same culture. The T3+T4+T8+ cell had intermediate cytotoxic capacities relative to T8+T4- and T4+T8- thymocyte fractions. The proliferative capacity of the T4+T8+ population although less than that of the T8+T4- subset exceeded the proliferative response of the T4+T8- population. The time of appearance of large numbers of T3+T4+T8+ cells in culture as well as functional properties exhibited by T3+T4+T8+ cells are consistent with the notion that the T3+T4+T8+ cell represents an activated intermediate in thymocyte differentiation. The T3+T4+T8+ thymocyte may be an important intermediate in in vivo as well as in in vitro thymic differentiation. Moreover, the analysis of its functional properties may contribute to an understanding of functional responses exhibited by the most mature (T3+) population isolated from human thymus.

Lectin Activation Induces T4, T8 Coexpression on Peripheral Blood T Cells

Recent studies from this and other laboratories have indicated that the T4 and T8 glycoproteins preferentially interact with class II and class I MHC molecules, respectively (1–3). Although the precise mechanism of interaction between T4, T8, and MHC antigens is not known, it has been proposed that T4 and T8 molecules bind to nonpolymorphic regions of MHC antigens (2,4). In this report, using the sensitive technique of two-color fluorescence flow cytometry, we demonstrate the existence of small numbers of T4+T8+ cells in freshly isolated T cell populations and that lectins can induce the coexpression of T4 and T8 on a fraction of peripheral blood T cells. Our results suggest that T4, T8 coexpression occurs on an activated blast-like peripheral blood T cell. Since lymphocyte cell surface phenotype and function appear to be closely linked, the T4+T8+ cell may prove to have an important role in either regulation or differentiation.

Human Thymocyte Subpopulations: Maturational Stages Defined by the Expression of the T3-T Cell Receptor Complex

Using sensitive fluorescence flow-cytometric techniques, human thymocyte subpopulations were fractionated according to their surface expression of the T3-T cell receptor (T3-Ti) complex. Two major subpopulations, one expressing low (immature subpopulation) and one high T3 antigen surface density (mature fraction) were characterized in detail with respect to surface antigen expression, right-angle scatter and proliferative capacity. Thymocyte subpopulations were activated through the T11 molecule (alternate pathway) and compared with regard to interleukin-2 (IL-2) receptor expression, changes in right-angle scatter and 3H-thymidine incorporation. We report that both populations could be activated through the T11 pathway to undergo nuclear activation and express IL-2 receptors. Moreover, in the absence of accessory cells, only the most mature population, expressing high T3 density, could be induced to proliferate, whereas immature cortical thymocytes required accessory cells for proliferation. These findings suggest that the cellular microenvironment may have a critical role in regulating the activation of immature cortical thymocytes.

T Cell Ontogeny: Acquisition of a Functional Program

Three discrete stages of thymocyte activation and maturation have been defined in an in vitro thymocyte culture system. The major population of immature cortical thymocytes (T3LT4+ T8+ T6+), which were previously considered to be unresponsive dead end cells, could be fully activated through the T11 molecule provided accessory cells were present. These results suggest that the T3LT4+T8+T6+ cell is a viable intermediate in thymic maturation. Furthermore, the development of functionally competent subsets was also regulated by accessory cells and could be blocked by the presence of anti-class II antibodies. Ia+ adherent cells promoted the development of the T4+T8– inducer subset, whereas cytotoxic T8+T4– cells predominated in the absence of accessory cells. In addition to cytotoxic and inducer T cell subsets, T cell receptor complex (T3–Ti)-positive cells bearing the NKH1 antigen arose at a later time in thymocyte culture. These NKH1+ cells exhibited NK activity and their development could be suppressed by adherent cells. We believe that this thymocyte culture system has permitted the isolation and identification of some of the events involved in the acquisition of a mature T cell phenotype and functional program. The identification and isolation of thymic subsets and cell to cell interactions provide new insights into the role of the major histocompatibility complex and accessory cell function during thymic maturation.