In Vitro Stimulation of Sensitized Lymphocytes by Herpes Simplex Virus and Vaccinia Virus

Abstract

Splenic lymphocytes from rabbits immunized with herpes simplex virus (HSV) were incubated in vitro with ultraviolet light-inactivated HSV, and the degree of lymphocyte transformation was determined by measurement of the incorporation of [3Hjthymidine into acid-insoluble material. Lymphocytes from immunized rabbits were stimulated as much as 30-fold, whereas lymphocytes from control rabbits were unaffected. Lymphocyte sensitization occurred within 3 days after immunization; sensitized lymphocytes could still be detected 120 days after immunization. The antigenicity of the ultraviolet light-inactivated crude virus pool was found to reside primarily in the virion. Infectious virus, in comparison with inactivated virus, produced less lymphocyte stimulation. Studies on the interaction of the humoral and cellular immune responses showed that incubation of anti-HSV antibody with HSV antigens did not reduce the capacity of the viral antigens to stimulate sensitized lymphocytes. Other experiments revealed that lymphocytes from both the spleen and peripheral blood of animals immunized with vaccinia virus could be stimulated by vaccinia, but not by HSV. Conversely, lymphocytes from animals immunized with HSV could not be stimulated by vaccinia. The transformation of sensitized lymphocytes by viral antigens appears to be a simple, highly specific, and quantitative in vitro technique for the study of the cellular immune response to viral infections.Cell-mediated immunity is thought to be an important factor in the defense against certain viral infections (1), but its precise role has been difficult to assess because of the lack of a simple in vitro assay. A number of in vitro systems, however, have been developed and are widely used for the study of the cellular immune response to nonviral antigens (2, 3). Recently, studies from several laboratories suggest that lymphocytes from specifically immunized animals will respond to viral antigens in vitro, as measured by blastogenesis, migration inhibitory factor production, and cytotoxicity (4-11). There is, however, little if any quantitative information on the preparation and nature of the viral antigens, the time of appearance and magnitude of the immune response, and the specificity of the reaction. The present report on virusinduced lymphocyte stimulation describes a simple and quantitative in vitro system for the study of the cellular immune response to viral antigens, and provides some information on the factors and conditions involved in viruslymphocyte interaction. were grown and assayed in primary rabbit kidney cells (12, 13). The stock pools of crude HSV and vaccinia contained 7.5 X 107 plaque-forming units (PFU)/ml and 4.0 X 106 PFU/ml, respectively. Partially purified virus was prepared by centrifuging the stock virus pools at 78,500 X g for 60 min in a Beckman model L ultracentrifuge. The supernatant fluid was used as the source of soluble viral antigens; the viral pellet was suspended in phosphate-buffered saline (PBS, 0.02 M, pH 7.2), centrifuged at 78,50...

Cellular Immune Response to Human Cytomegalovirus

“…The use of adjuvant did not signifi cantly increase background incorporation of [3H]-thymidine or compromise specificity. These two problems were noted in studies with HSV [14], Virus antigen stimulation of rabbit lymph ocytes previously has been studied using spleen cells or peripheral blood cells after removal of erythrocytes and/or plasma [6,15]. We found that unwashed whole blood cultures exhibited comparable or greater reactivity than frac tionated cells.…”

“…Other workers have shown that lympho cyte transformation is a useful method for monitoring cellular immunity to herpesviruses in animals and man [5,6]. As a prelude to studying the latter, it was considered desirable to characterize the stimulating antigen and evaluate its specificity in an experimental animal model.…”

Disseminated Herpes simplex in Untreated Multiple Myeloma

“…Cutaneous delayed hypersensitivity has been demonstrated after intradermal administration of the killed virus [24], HSV antigens stimulate splenic lymphocytes of rabbits previously immunized with HSV [25], W ilton el al. [26] were able to correlate susceptibility to recurrent HSV infections with impaired lymphocyte cytotoxicity to infected cells and impaired macro phage migration inhibition factor production in the presence of HSV anti gens.…”

Insulin Inhibits Cellular and Herpes Simplex Virus DNA Synthesis in Transformed Lymphoblastoid Cell Lines

“…The effect of insulin on DNA synthesis in lymphoblastoid cells that originated from Burkitt's lymphoma is the subject of the present study. Since herpes simplex virus (HSV) can infect human lymphoblastoid cells originally transformed by Epstein-Barr virus (EBV) [12][13][14][15][16], the effect of insulin on the synthesis of viral DNA in these cells was also studied.…”