Chemokines and HIV: The First Close Encounter

The first “close encounter” between the fields of chemokines and HIV occurred in the late spring of 1995, when my group at NCI’s Laboratory of Tumor Cell Biology (LTCB) received from Harvard Microchemistry and Proteomics the amino acid sequences of three peptide fragments from an HPLC-purified fraction that we had submitted a few weeks earlier. The sequences showed a perfect match with the human chemokine RANTES/CCL5. The fraction had been isolated from the culture supernatant of an immortalized Tcell clone (FC36.22) producing the elusive “CD8-derived HIV-suppressive factor” whose existence had been postulated by Jay Levy’s group at UCSF since 1986 (1), but whose identity had remained enigmatic for nearly a decade. Two years earlier, when Fiorenza Cocchi, at that time a post-doctoral fellow from Milan, Italy, had approached me with the proposal to embark on the quest for the “Levy factor,” my first reaction had been anything but enthusiastic. Not that I had any doubt about the significance of identifying an endogenous factor that was believed to help HIV-infected individuals remain asymptomatic by suppressing the virus in a non-cytolytic fashion (2). On several occasions, we had heard Levy illustrate his model, and Bob Gallo, our inspiring lab chief at the LTCB, had often remarked on the importance of resolving this longstanding riddle. However, all attempts to identify the factor until then had failed in the midst of confusion among different designations, experimental models, and mechanistic hypotheses (3), and skepticism about the very existence of such factor was on the rise. Furthermore, my laboratory’s focus was on pathogenesis, with very limited expertise in protein chemistry. And so, we concurred to give it a try, but I made it clear that we would soon drop the project unless we could come up with a robust and reproducible experimental system to justify a long-term commitment. The original phenomenological observation on the non-cytolytic HIVsuppressive activity of CD8 T cells was made by Chris Walker and Jay Levy in the mid-80s while they were attempting to increase the rate of HIV-1 isolation from asymptomatic seropositive individuals. They found that removal of CD8 T cells from the cultures greatly enhanced their odds of success; when autologous CD8 T cells were added back to the cultures, virus replication was again suppressed but the number of CD4 T cells remained constant, thus ruling out a classic “cytotoxic T-lymphocyte” effect (1). In subsequent years, the same group went on to show that the activity was not bound to MHC restriction and was at least in part mediated by a soluble factor – initially nicknamed “CAF”– that was diffusible through a semipermeable membrane (4). Moreover, they established a correlation between the levels of CAF production and the asymptomatic state of HIV-1 infection (2), corroborating the clinical significance of this unconventional CD8 T-cell activity. Despite intensive efforts, however, no progress toward the identification of the factor was made over the following years. One of the key challenges was the extremely low level of factor that could be rescued from primary CD8 T-lymphocyte cultures, further complicated by marked donor– donor variability. Yet, Levy insisted that the activity was an exclusive product of primary CD8 T cells and a specific attribute of HIV-seropositive individuals (4), which posed major challenges for production scale-up. We reasoned that the first critical step to tackle this project was to devise a highyield and reproducible cellular source for the factor, and we began testing primary and immortalized CD8 T cells from both seropositive and seronegative donors under diverse conditions of activation and culture. The LTCB was an ideal site in this respect because a major focus for over two decades had been the optimization of Tcell growth conditions, culminating in the discovery of “T-cell growth factor,” subsequently named interleukin-2, by Doris Morgan, Frank Ruscetti, and Gallo in 1976 (5), and the isolation of the first human retrovirus, HTLV-1, which can immortalize both CD4 and CD8 T cells ex vivo, by Bernie Poiesz and Gallo in 1980 (6). Thus, besides testing primary CD8 T cells, we derived CD8 T-cell lines immortalized with HTLV-1 or its little brother, HTLV-2, and dug deep into the freezers in search for every vintage CD8 Tcell line that we could test. Among the many cells that we screened was 67-I, an HTLV-I-immortalized clone obtained a few years earlier by Anita DeRossi at the LTCB (7), which eventually turned out to be the key to our success. Derived from the peripheral blood of a healthy blood donor, 67-I retained many features of primary CD8 T cells, but unlike the latter it provided a stable and scalable source of soluble factors and was adaptable to grow under serum-free culture conditions, which would eventually simplify purification of the factor. In parallel to developing an efficient “factor factory,” a second critical need was to establish a highly standardized readout system for the quantitative determination of antiviral activity. Again, it was essential to overcome the inconsistencies of primary cells and, even worse,