Michael A. Derby

High-Avidity CTL Exploit Two Complementary Mechanisms to Provide Better Protection Against Viral Infection Than Low-Avidity CTL

Previously, we observed that high-avidity CTL are much more effective in vivo than low-avidity CTL in elimination of infected cells, but the mechanisms behind their superior activity remained unclear. In this study, we identify two complementary mechanisms: 1) high-avidity CTL lyse infected cells earlier in the course of a viral infection by recognizing lower Ag densities than those distinguished by low-avidity CTL and 2) they initiate lysis of target cells more rapidly at any given Ag density. Alternative mechanisms were excluded, including: 1) the possibility that low-avidity CTL might control virus given more time (virus levels remained as high at 6 days following transfer as at 3 days) and 2) that differences in efficacy might be correlated with homing ability. Furthermore, adoptive transfer of high- and low-avidity CTL into SCID mice demonstrated that transfer of a 10-fold greater amount of low-avidity CTL could only partially compensate for their decreased ability to eliminate infected cells. Thus, we conclude that high-avidity CTL exploit two complementary mechanisms that combine to prevent the spread of virus within the animal: earlier recognition of infected cells when little viral protein has been made and more rapid lysis of infected cells.

Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections

Summary: We used several approaches to develop enhanced vaccines for chronic viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), I) Selected epitopes were used to avoid potentially harmful immune responses. 2) Linkage between helper and cytotoxic T‐lymphocyte (CTL) epitopes was found to be important, 3) We developed an “epitope enhancement” approach modifying the sequences of epitopes to make more potent vaccines, including examples for HIV and HCV epitopes presented by murine class II and human class I major histocompatibility complex (MHC) molecules, 4) CTL avidity was found to be important for clearing viral infections in vivo, and the mechanism was examined. High‐avidity CTLs, however, were found to undergo apoptosis when confronted with high‐density antigen, through a mechanism involving tumor necrosis factor (TNF), TNF‐RII, and a permissive state induced through the T‐cell receptor. 5) We employed cytokines in the adjuvant to steer immune responses toward desired phenotypes, and showed synergy between cytokines, 6) Intrarectal immunization with pep‐tide vaccine induced mucosal and systemic CTL, Local mucosal CTL were found to be critical for resistance to mucosal viral transmission and this resistance was enhanced with mucosally delivered interleukin‐12, 7) We used an asymmetry in induction of mucosal and systemic immune responses to circumvent pre‐existing vaccinia immunity for use of recombinant vaccinia vaccines.

An abrupt and concordant initiation of apoptosis: antigen-dependent death of CD8+ CTL

The ability of CD8+ cytotoxic T lymphocytes (CTL) to clear viral infections may be limited when high avidity CTL encounter supra‐optimal antigen density on antigen‐presenting cells (APC) and undergo antigen‐dependent apoptosis of CTL (ADAC). Previously, we have shown ADAC in CD8+ populations to be Fas independent, TNF‐α receptor 2 (TNFR2) mediated, caspase dependent, and accompanied by a decrease in Bcl‐2. We now employ flow cytometry to follow ADAC within individual CD8+ cells to demonstrate that the intense TCR signal induced in high avidity CTL by supra‐optimal antigen density results 8 – 16 h later in a caspase‐independent TNFR2 down‐modulation that is directly related to the stimulating APC antigen density and concludes in a rapid onset of apoptosis by 18 – 24 h. Individual CTL undergoing apoptosis exhibit a dramatic and concurrent: (1) positive staining with Annexin V and propidium iodide; (2) transformation to a smaller cell size characteristic of apoptosis; and (3) a nearly complete loss of Bcl‐2, c‐IAP1, and TRAF2. We conclude that the antigen‐dependent apoptosis of CD8+ CTL occurs when a tandem TCR / TNFR2 signalinitiates an abrupt and concordant onset of multiple apoptotic events.

Competitive Inhibition In Vivo and Skewing of the T Cell Repertoire of Antigen-Specific CTL Priming by an Anti-Peptide-MHC Monoclonal Antibody

We have recently described a mAb, KP15, directed against the MHC-I/peptide molecular complex consisting of H-2Dd and a decamer peptide corresponding to residues 311–320 of the HIV IIIB envelope glycoprotein gp160. When administered at the time of primary immunization with a vaccinia virus vector encoding gp160, the mAb blocks the subsequent appearance of CD8+ CTL with specificity for the immunodominant Ag, P18-I10, presented by H-2Dd. This inhibition is specific for this particular peptide Ag; another H-2Dd-restricted gp160 encoded epitope from a different HIV strain is not affected, and an H-2Ld-restricted epitope encoded by the viral vector is also not affected. Using functional assays and specific immunofluorescent staining with multivalent, labeled H-2Dd/P18-I10 complexes (tetramers), we have enumerated the effects of blocking of priming on the subsequent appearance, avidity, and TCR Vβ usage of Ag-specific CTL. Ab blocking skews the proportion of high avidity cells emerging from immunization. Surprisingly, Vβ7-bearing Ag-specific TCR are predominantly inhibited, while TCR of several other families studied are not affected. The ability of a specific MHC/peptide mAb to inhibit and divert the CD8+ T cell response holds implications for vaccine design and approaches to modulate the immune response in autoimmunity.