Zeling Cai

Dual Function of Drosophila Cells as APCs for Naive CD8+ T Cells: Implications for Tumor Immunotherapy

With unseparated mouse spleen cells as responders, Drosophila cells expressing MHC class I (L(d)) molecules alone lead to peptide-specific responses of CD8+ cells in the absence of exogenous cytokines. Under these conditions, DNA released from dying cells stimulates the B cells in spleen to up-regulate costimulatory molecules; these activated B cells then provide bystander costimulation for CD8+ cells responding to class I-peptide complexes on the Drosophila APCs. By stimulating B cells and presenting antigen to T cells, Drosophila cells thus serve two different functions in promoting primary responses of CD8+ cells in vitro. With this system, we show that Ld-transfected Drosophila cells are able to induce autologous spleen cells to respond to a tumor-specific peptide in vitro and, after transfer, cause tumor rejection in vivo.

Early Antigen-Specific Response by Naive CD8 T Cells Is Not Altered with Aging

Both a dramatic decline in CD8 responses and a switch to memory T cell predominance occur with aging. The extent to which the loss of responsiveness is the consequence of the accumulation of more differentiated vs intrinsically defective T cells (or both) has been unclear. Using similar conditions of Ag stimulation, we have examined the responses generated by CD8+ cells isolated from aged TCR transgenic mice. We found that the naive transgene+ CD8+ cells from aged 2C mice expressed activation markers, produced IL-2, proliferated, and differentiated into cytotoxic T cells as efficiently as their young counterparts. The extent of responsiveness and the level of the responses were comparable in both age groups regardless of the stimulatory conditions used, i.e., partial costimulation/adhesion molecule expression on APCs, or presentation of lower affinity peptide or diminished peptide concentrations. By day 4 after Ag stimulation, no significant age-related differences were observed in the number of effector cells generated nor in the levels of secreted IL-2 or IFN-γ. Upon restimulation of effector cells, IL-2 secretion and to a lesser extent TNF-α expression, but not IFN-γ secretion, were diminished with age. These findings suggest that age-associated alterations in naive CD8 cell function are not found after primary stimulation, but may become apparent upon restimulation.

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor‐ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T‐cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T‐cell receptors (TCR). whereas Signal 2 is induced by the interactions of co‐stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T‐cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)‐l can provide the second signal for CD8 T‐cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM‐1 on the same APC. Thus, the data suggest that at least two qualitatively different co‐stimulation signals are required for full activation of CD8 T cells under physiological conditions.

Regulation of Transporter Associated with Antigen Processing by Phosphorylation

The ATP-binding cassette transporter associated with antigen processing (TAP) is required for transport of antigenic peptides, generated by proteasome complexes in the cytoplasm, into the lumen of the endoplasmic reticulum where assembly with major histocompatibility complex class I molecules takes place. The TAP transporter is a heterodimer of TAP1 and TAP2. Here we show that both TAP1 and TAP2 are phosphorylated under physiological conditions. Phosphorylation induces formation of high molecular weight TAP complexes that contain TAP1, TAP2, tapasin, and class I heterodimers. In addition, a 43-kDa phosphoprotein, which appears to be a kinase, is contained in the phosphorylated TAP-containing complexes. Phosphorylated TAP complexes are able to bind peptides and ATP, however, they are not capable of transporting peptides. After de-phosphorylation, TAP complexes regain the ability to transport peptides. Interestingly, phosphorylation levels of TAP complexes induced by viral infection inversely correlates with a significant reduction in TAP-dependent peptide transport activity. Enhanced TAP phosphorylation appears to be one of several strategies that viruses have exploited to better escape from host immune surveillance. These results demonstrate that major histocompatibility complex class I antigen processing and presentation is modulated by reversible TAP phosphorylation, and implicate the importance of TAP phosphorylation in the regulation of cytotoxic immune response.

Constructing Artificial Antigen-Presenting Cells from Drosophila Cells

Stimulation of unprimed T cells is controlled by professional antigen-presenting cells (APC) such as dendritic cells (DC).1–3 The strong APC function of DC is presumed to reflect that these cells express a high density of major histocompatibility complex (MHC) molecules and a variety of costimulatory molecules. In this respect, activation of naive T cells by APC is thought to require two distinct signals: Signal t reflects T cell receptor (TCR) contact with specific peptides bound to MHC molecules, and Signal 2 is the consequence of other molecules on T cells, e.g. CD28, interacting with costimulatory molecules, e.g. B7 (B7-1, B7-2), on APC. Since many different accessory molecules on APC can express costimulatory function for T cells under defined conditions, which particular accessory molecules are essential for stimulating naive T cells is unclear. We have addressed this issue by constructing artificial APC from a Drosophila cell line by gene transfection.

THE THYMUS AND T CELL DEATH

Through the combined effects of positive and negative selection, T cell differentiation in the thymus generates a repertoire of mature T cells that is tailored to tolerate self antigens but mount strong responses to foreign antigens1,2. Thymic selection is associated with extensive cell death, and only a very small proportion of thymocytes are selected for survival and export to the extrathymic environment. This article provides an overview of thymic selection and the fate of thymocytes.