Analysis of cytotoxic T lymphocyte precursor frequencies against herpes simplex virus

A limiting dilution microculture system is described in which the frequencies of cytotoxic T lymphocyte precursors reactive against herpes simplex virus type 1 were determined. Analysis by Poisson statistics demonstrated that the estimated frequency of HSV-1 reactive cells in the spleens of normal mice was less than 1/250,000. In contrast, spleen cells from HSV-1-immune mice stimulated in vitro with HSV-1-infected irradiated splenocytes resulted in CTL-p frequencies between 1/6519 and 1/15632. The same responder population of spienocytes when heterologously stimulated with HSV-2-infected stimulators yielded much lower frequencies of anti-HSV-1 CTL-p, 1/22045- 1/49196.

To analyze the cross-reactivity between HSV-1 and HSV-2, mice were infected in vivo with HSV-2, and the resulting immune splenocytes were then heterologously or homologously stimulated in vitro. When the effectors were assayed against HSV-1-infected target celis, CTL-p frequencies of 1/13664-1 /18476 and 1/12784-1/16644 were obtained for homologously and heterologously stimulated splenocytes, respectively.

Split culture analysis comparing cytotoxicity against syngeneic and allogeneic virus-infected targets provided evidence for specificity, H-2 restriction and the T cell nature of the CTL-p. It was determined that the putative CTL-p derived cytolytic effector progeny were elimi nated by treatment with anti-Lyt 2.1 plus complement, thus indicating that the effector progeny were Lyt 1^-2⁺ cells.

These experiments demonstrate the feasibility of using the limiting dilution analysis as a sensitive and quantitative means to measure the ceil mediated immune response to HSV-1 antigens.