Figure 1.
HSV-specific ASC residing long-term in the female genital tract, lumbosacral ganglia and adjacent spinal cord are predominantly plasma cells.
Hartley guinea pigs were infected ivag with HSV-2 strain MS. Lymphocytes from the spleen, bone marrow, vagina/cervix, and lumbosacral ganglia and adjacent spinal cord of individual animals were harvested between days 49–70 after infection and stimulated with medium or LPS/CpG and HSV-specific ASC quantified by ELISPOT. Each data point represents results from an individual animal. Results shown include tissues from three experiments for spleen and bone marrow and two experiments for vagina/cervix and spinal cord/ganglia. HSV-specific ASCs from tissues of uninfected animals were always less than five ASC/tissue. (* P<0.05; *** P<0.0001, Student's t test).
Figure 2.
Location of HSV-specific, tissue-resident ASCs in guinea pigs infected previously with HSV-2.
Hartley guinea pigs were infected ivag with HSV-2 strain MS and lymphocytes isolated from the indicated tissues on days 48–57 post infection. HSV-specific ASCs were quantified by ELISPOT on HSV-2 glycoprotein-coated plates. Each data point represents results from an individual animal. (* P<0.05; ** P<0.01, Student's t test).
Figure 3.
Isotype and IgG subclass of HSV-specific antibodies produced by ASCs isolated from lymphoid tissue and non-lymphoid sites of HSV-2 infection.
Hartley guinea pigs were infected ivag with HSV-2 strain MS, serum was collected and lymphocytes were isolated from the indicated tissues on days 48–57 post infection. Supernatants from ASC cultures were collected on day three after ASC isolation. The immunoglobulin isotype and IgG subclass of HSV-specific antibodies from serum and ASC supernatant were determined by ELISA. Each data point represents ASC supernatant from an individual animal. (* P<0.05; ***P<0.0001, Student's t test).
Figure 4.
IgG antibody from tissue-resident ASC isolated from HSV-2-infected guinea pigs is reactive with HSV-2 glycoproteins and neutralizes HSV-2.
Hartley guinea pigs were infected ivag with HSV-2 strain MS, serum was collected and lymphocytes were isolated from the indicated tissues between days 48–57 post infection. Supernatants from ASC cultures were collected on day three after isolation and the endpoint titer of antibodies reactive with full-length recombinant HSV-2 gD (rgD2), truncated rgD2, or truncated recombinant HSV-2 rgG (rgG2) was determined by ELISA. (*P<0.05; ** P<0.01; *** P<0.001 compared to HSV-2 gG2 titer). Virus neutralization was detected by incubating a constant virus titer in serial dilutions of ASC supernatant to determine a 50% neutralizing titer (E) or by incubating serial dilutions of HSV-2 virions in undiluted ASC supernatant (F). Results shown are from a representative experiment of three performed.
Figure 5.
Detection and quantification of HSV-specific IFN-γ SCs in spleen, genital tract and neural tissues of HSV-2 infected guinea pigs.
Lymphocytes from the indicated tissues of HSV-2-infected Strain 13 guinea pigs (n = 5) were harvested on day 7 post infection. A) IFN-γ ELISPOT of lymphocytes harvested from sensory ganglia/spinal cord and genital tract. B) Total number and frequency of HSV-specific IFN-γ SC in the spleen, genital tract, sensory ganglia/spinal cord of HSV-2 infected guinea pigs. Each data point represents results from an individual animal. *** P<0.0001, Chi square.
Figure 6.
Tissue distribution of HSV-2 specific memory T cells following genital HSV-2 infection of guinea pigs.
Hartley guinea pigs were infected ivag with HSV-2 strain MS and lymphocytes isolated from the indicated tissues on days 99-150 post infection. HSV-specific, IFN-γ SCs were detected and quantified by IFN-γ ELISPOT. The frequency of HSV-specific, IFN-γ SC per 106 cells is given in A,C,E,G and the total number of HSV-specific, IFN-γ SC is given in B,D,F,H for each tissue. Each data point represents results from an individual animal. (* P<0.05; ** P<0.01, Student's t test; *** P<0.0001, Chi square).
Figure 7.
Presence of CD4+ and CD8+ HSV-specific memory T cells in genital tracts and neural tissue of HSV-2 infected guinea pigs.
Hartley guinea pigs were infected ivag with HSV-2 strain MS and lymphocytes isolated from the indicated tissues on days 99–150 post infection. Enriched populations of CD4+ and CD8+ T cells were obtained using a magnetic-based kit. HSV-specific, IFN-γ SCs were detected and quantified by IFN-γ ELISPOT. Each data point represents results from an individual animal. (* P<0.05 Student's t test).