Activation requirements of circulating antigen-specific human CD8+ memory T cells probed with insect cell-based artificial antigen-presenting cells

Abstract

We sought to define the molecular setup of an antigen‐presenting cell that elicits antigen‐specific T cell responses in vitro using insect cells that were infected with recombinant baculoviruses. Expression of single‐chain HLA was complemented step‐by‐step with costimulatory molecules, including CD54 and CD80, by co‐infection with the relevant viruses. Role of CD8 was assessed by introducing hybrid class I molecules where the alpha‐3 domain of the HLA heavy chain molecule was replaced by its murine Kb counterpart. Circulating T cells that respond to the EBV‐derived HLA‐A2‐restricted peptide GLGCTLVAML were previously shown to bear hallmarks of memory cells. We found that the HLA+peptide complex alone displayed on the surface of insect cells was sufficient toelicit IFN‐γ secretion from these freshly isolated CD8+ T cells in ELISpot assays. Binding of CD8 was absolutely required, but coexpression of costimulatory molecules resulted only in minimal increase in the number of spots. Tumor antigen‐specific CTL clones also reacted in a strictly antigen‐specific manner, but required CD54 for quantitative responses. The amount of IFN‐γ produced by the individual reactive T cells was evaluated as spot size, and was also influenced by the costimulatory molecules: CD54 increased also the response magnitude of cultured CTL lines, while CD80 enhanced cytokine release from freshly isolated CD8+ T cells. Understanding the stimulatory requirements of functionally competent effector/memory T cells and their exact enumeration will be helpful for increasing the efficacy of vaccines.

Artificial Antigen-Presenting Cells Transduced with Telomerase Efficiently Expand Epitope-Specific, Human Leukocyte Antigen–Restricted Cytotoxic T Cells

“…Telomerase activity of each sample was detected by the extension of oligonucleotide, which serves as the substrate for the telomerase, and followed by [g- 32 For the radioactive PCR reaction, the extract of each sample (2 AL; corresponding to the adjusted protein concentrations) was combined with the 48 AL reaction mixture containing a [g-32 P]ATP-labeled TS primer and 2 units Taq DNA polymerase. After a 30-minute telomerase extension reaction at room temperature, samples were subjected to PCR for 27 cycles followed by electrophoresis on 12% PAGE.…”

“…Ideally, one would want a ready-to-use APC applicable to any patient, which permits selective expansion of potent T cells specific for any tumor antigen. Artificial APCs (AAPCs) have thus been developed to generate clinically relevant quantities of CTLs for the purpose of adoptive therapy (30)(31)(32)(33)(34)(35)(36). AAPCs have been engineered to optimize the stimulation and expansion of CTLs by presenting antigen in the context of a specific HLA allele along with a variety of costimulatory molecules, including B7.1 (CD80), intercellular adhesion molecule-1 (ICAM-1; CD54), LFA-3 (CD58), and 4-1BB ligand (36).…”

Development of the PANVAC™-VF vaccine for pancreatic cancer

“…Following T-cell receptor engagement, CD28 appears at the T cell-APC interface and interacts with B7.1 and B7.2 to provide T-cell signaling resulting in proliferation and cytokine expression by the T cell. Under the influence of additional costimulation through tumor necrosis factor (TNF)-like molecules and local cytokines, the T cell enters into an activated state leading to expansion of effector T cells and induction of memory responses [41]. This activation, however, may be short-lived due to the eventual accumulation of CTL antigen (CTLA)-4 at the T-cell surface, which outcompetes CD28 for B7.1 and B7.2 binding [42].…”

Prime Boost Vaccination Strategies: CD8 T Cell Numbers, Protection, and Th1 Bias

“…(30). The area of each ELISPOT spot was measured and was taken to represent a measure of the amount of IFN-␥ secreted by each TYQ-specific CD8 T cell (12). Unlike conventional bulk IFN-␥ assays, which measure secretion from a population of cells, this ELISPOT-based method allows IFN-␥ secretion profiles to be measured for individual Agspecific CD8 T cells.…”

“…The DNA and MVA vaccines both encode the murine polyepitope or polytope immunogen, which contains the following CD8 T cell epitopes: TYQRTRALV (TYQ) from the influenza nucleoprotein, RPQASGVYM (RPQ) from the lymphocytic choriomeningitis virus nucleoprotein, and SYIPSAEKI (SYI) from the Plasmodium berghei circumsporozoite protein (10). Epitope-specific CD8 T cell numbers were enumerated using IFN-␥ ELISPOT technology, and the size of the IFN-␥ spots produced by individual T cells was taken as a measure of the amount of IFN-␥ secreted by each T cell (12). An influenza protection model was used to investigate the effect of different vaccination strategies on the capacity of a given number of vaccine-induced CD8 T cells to mediate protection.…”