No abstract

Cytotoxic T lymphocytes (CTL) generated in C57BL/6 (H-2b) mice in response to infection with the serologically distinct herpes simplex virus type 1 (HSV-t) or type 2 (HSV-2) were cross-reactive against target cells infected with either serotype. However, HSV-2-infected cells were shown to be much less susceptible to CTL-mediated lysis, and analysis through the use of HSV-1 x HSV-2 intertypic recombinants mapped the reduced susceptibility to a region contained within 0.82 to 1.00 map units of the HSV-2 genome. The study reported here was undertaken to determine the possible reasons for the reduced susceptibility of HSV-2infected cells to lysis by CTL. Competition for the specific lysis of labeled HSV-1-infected cells by either HSV-1or HSV-2-infected, unlabeled inhibitor cells and frequency analysis of the CTL precursor able to recognize HSV-1and HSV-2-infected cells suggested that the reduced susceptibility of HSV-2-infected cells to lysis could be explained, at least in part, by reduced levels of target cell recognition. A determination of the surface expression of the critical elements involved in target cell recognition by CTL following infection with HSV-1 or HSV-2 revealed that all the major HSV-specific glycoprotein species were expressed. Infection with both HSV-1 and HSV-2 caused a reduction in the expression of the class I H-2 antigens. However, this reduction was much greater following infection with HSV-2. This suggested that one important factor contributing to reduced lysis of HSV-2-infected cells may be the altered or reduced expression of the class I H-2 self-antigens.

Cytotoxic T lymphocytes (CTL) were generated in C57BL/6 mice with herpes simplex virus type 1 (HSV-1) (strains KOS, 17, HFEM, and mP) and HSV-2 (strains 186, G, and GP6). Effector lymphocytes were tested for cytotoxicity against syngeneic HSV-1-and HSV-2-infected cells in a 5-h 51Cr release assay. HSV-1 strain HFEM was found to induce CTL efficiently only when 100-fold more virus was used as compared with HSV-1 strains KOS, 17, and mP. All HSV-1 and HSV-2 strains induced cross-reactive populations of CTL. CTL generated by HSV-1 KOS and HSV-2 186 also demonstrated cross-reactivity in an earswelling model for delayed-type hypersensitivity. Lymphocytes generated by all HSV-2 strains were highly efficient at lysing HSV-1-infected target cells. However, HSV-2-infected target cells were found to be less susceptible to lysis by either HSV-1 or HSV-2 CTL than were HSV-1-infected target cells. The lowered susceptibility of HSV-2-infected cells was not due to an inefficient infection of BL/ 6 WT-3 cells as measured by standard growth assays and infectious center assays. Varying the multiplicity of infection or the time of infection did not increase the susceptibility of HSV-2-infected target cells to lysis by CTL. Increasing the effector-to-target-cell ratio resulted in an increased lysis of both HSV-1and HSV-2-infected target cells by CTL, but the level of HSV-2-infected target cell lysis still did not approach the level of HSV-1-infected target cell lysis. HSV-2-infected cells were as efficient as HSV-1-infected cells in the cold cell competition assay employed in reducing the lysis of 51Cr-labeled, HSV-1-infected target cells. In addition, HSV-2-infected cells were susceptible to lysis by HSV-immune serum and complement.

The objective of this study was to identify and test a convenient means for long-term storage of lymphocytes taken from clinically characterized patients without losing B- or T-cell function. Accordingly, peripheral blood lymphocytes were frozen and stored, and portions of each sample were subsequently assayed for T-cell blastogenic response and B-cell Jerne plaquing at various time intervals after freezing. A comparison of the cell counts of fresh and frozen cultures indicated that cell were recovered after freezing. Furthermore, these cells showed no significant differences in (i) cell viability; (ii) blastogenic response to antigens of Actinomyces maeslandii, Bacteroides melaninogenicus, Fusobacterium nucleatum, and tetanus toxoid; (iii) blastogenic response to phytohemagglutinin and pokeweed mitogen; and (iv) polyclonal B-cell response to pokeweed mitogen as measured by the direct Jerne plaque assay. The retained blastogenic and plaquing responses seen in frozen cultures indicated the maintenance of both T-cell and B-cell function, respectively. This is the first reported demonstration of Jerne plaquing of normal human lymphocytes after freezing. It appears that freezing techniques provide a means for repeating and extending both T- and B-cell assays using frozen stored portions of the same cell samples.

A function(s) involved in the altered susceptibility of herpes simplex virus type 2 (HSV-2)-infected cells to specific lysis by cytotoxic T lymphocytes was mapped in the S component of HSV-2 DNA by using HSV-1 x HSV-2 intertypic recombinants (RH1G44, RS1G25, RSOBG10, A7D, and C4D) and HSV-1 MP. Target cells infected with RSOBG10, A7D, and C4D exhibited reduced levels of cytolysis, as did HSV-2-infected cells, whereas RH1G44 and RS1G25 recombinant-infected and HSV-1 MP-infected cells showed levels of lysis equal to that of HSV-1 KOS-infected cells. The intertypic recombinants R50BG10, RS1G25, RH1G44,