Preparation of Stable Target Cells for Anti‐Herpes Simplex Virus Cytotoxic T Lymphocytes

Abstract

Using an avirulent strain of herpes simplex virus (HSV), SKa, and a methylcholanthrene induced sarcoma cell line, Meth A cells, we have developed a reliable target cell system for detection of cell-mediated cytotoxicity directed against HSV-infected cells. SKa-infection in Meth A produced no progeny virus but induced HSV-specific surface antigens as revealed by radioimmunoassay using 125I-labeled HSV antibody. Spontaneous release of 51Cr from the SKa-infected Meth A cells was no more than that from uninfected control cells but a strong spontaneous 51Cr release was produced in Meth A cells infected with KOS, a virulent strain which produced a progeny virus in Meth A and was lytic for the cells. When used as a target, SKa-infected Meth A cells could detect HSV-specific cytotoxicity by spleen and lymph node lymphocytes of mice immunized with SKa and KOS. This system also detected effector cytotoxic lymphocytes stimulated in vitro by mixed cultures of immune spleen cells and KOS-infected Meth A cells. Thus, the system should be valuable in studies of cell-mediated cytotoxicity directed against HSV-infected cells.

Protection of Mice from Fatal Herpes Simplex Virus Type 1 Infection by Adoptive Transfer of Cloned Virus-specific and H-2-restricted Cytotoxic T Lymphocytes

“…Extensive evidence has accumulated which suggests that cell-mediated immune mechanism(s) are critical in recovery and protection against HSV infections (Notkins, 1974). It has become evident that HSV-specific cytotoxic T lymphocytes (CTL), which are restricted by the major histocompatibility complex (H-2 in mouse and HLA in man), although difficult to demonstrate during primary infection (often weak response), can be generated to high levels when primed lymphocytes are secondarily stimulated in vitro, both in murine (Pfizenmaier et al, 1977;Sethi & Brandis, 1977;Lawman et al, 1980;Nash et al, 1980;Kastrukoffet al, 1981 ;Sonoda et al, 1981) and human models. The present study demonstrates that cloned cultures of in vitro generated HSV-specific and H-2-restricted CTL which are expanded in the presence of T cell growth factor (TCGF) when transferred intraperitoneally (i.p.)…”

Preparation of Membrane Fraction from Herpes Simplex Virus‐Infected Cells which Induce Cytotoxic T Lymphocytes

“…Avirulent SKa-HSV was used for priming mice and preparing stable target cells for anti-HSV CTL assay. KOS-HSV, virulent for mice, was used for secondary immunization and for preparation of a large quantity of HSV antigen, as described previously (22). Both strains were grown and titrated in Vero cells.…”

“…Meth A cells were infected with KOS-HSV and used as the immunogen for anti-HSV CTL. SKa-HSV infected Meth A cells were used for the CTL target as described previously (22). Vero and Meth A cells were cultured in I placed on 50% sucrose layer centrifuged at 100,000 xg for I hr at 4 C Banding material at the interface adjusted to 50% sucrose, placed at the bottom of a discontinuous sucrose gradient, centrifuged at 100,000 xg for 2 hr at 4 C Fraction I plasma membrane 2} membranes con-3 taining impurities diluted with PBS centrifuged at 100,000 X g for I hr Eagle's minimum essential medium (MEM) supplemented with 2 mM glutamine, 100 units of penicillin per ml, 100 fJg of streptomycin per ml and 5% calf serum.…”

“…Female BALBjc mice, 6-10 weeks old, were infected with 10 6 PFU oflive SKa-HSV by intraperitoneal injection and boosted by a second injection of 10 7 PFU of live KOS-HSV after an interval of 2-4 weeks. One to 2 weeks after the second injection, the mice were bled and their spleen cells were isolated as described previously (22).…”

“…The standard method of testing for anti-HSV cytotoxicity described previously (22) was used. In brief, 10 4 51Cr-Iabeled SKa-infected Meth A cells and graded numbers of the cultured spleen cells to be tested were incubated for 4 hr in a microtray with 0.2 ml of RPMI-1640 supplemented with 5% calf serum in triplicate, and O.l-ml samples of the supernatant medium were taken from each well.…”