Longitudinal analysis of feline leukemia virus-specific cytotoxic T lymphocytes: correlation with recovery from infection

Abstract

Feline leukemia virus (FeLV) is a common naturally occurring gammaretrovirus of domestic cats that is associated with degenerative diseases of the hematopoietic system, immunodeficiency, and neoplasia. Although the majority of cats exposed to FeLV develop a transient infection and recover, a proportion of cats become persistently viremic and many subsequently develop fatal diseases. To define the dominant host immune effector mechanisms responsible for the outcome of infection, we studied the longitudinal changes in FeLV-specific cytotoxic T lymphocytes (CTLs) in a group of naïve cats following oronasal exposure to FeLV. Using (51)Cr release assays to measure ex vivo virus-specific cytotoxicity, the emerging virus-specific CTL response was correlated with modulations in viral burden as assessed by detection of infectious virus, FeLV p27 capsid antigen, and proviral DNA in the blood. High levels of circulating FeLV-specific effector CTLs appeared before virus neutralizing antibodies in cats that recovered from exposure to FeLV. In contrast, persistent viremia was associated with a silencing of virus-specific humoral and cell-mediated host immune effector mechanisms. A single transfer of between 2 x 10(7) and 1 x 10(8) autologous, antigen-activated lymphoblasts was associated with a downmodulation in viral burden in vivo. The results suggest an important role for FeLV-specific CTLs in retroviral immunity and demonstrate the potential to modulate disease outcome by the adoptive transfer of antigen-specific T cells in vivo.

FIG. 1.

FeLV provirus loads in PBMC. FeLV provirus load was determined by quantitative Taqman PCR. DNA was extracted from PBMC at regular intervals prior to and following FeLV exposure. The sensitivity of this technique was determined to be 1 copy per reaction, which typically contained 2.5 × 10⁵ cells. Solid lines, recovered cats; dotted lines, persistently viremic cats. The arrowhead indicates the time of adoptive transfer.

FIG. 2.

Detection of virus neutralizing antibodies. Virus neutralizing antibodies (VNA) were assayed in the peripheral plasma preexposure and at 3-week intervals following exposure to FeLV by focus reduction of FeLV-A/Glasgow-1 on QN10S cells. Solid lines, recovered cats; dotted lines, persistently viremic cats. The arrowhead indicates the time of adoptive transfer. Note that the values for persistently viremic cats are uniformly negative, so that individual cats are not discernible in the figure.

FIG. 3.

Elicitation of virus-specific CTLs following FeLV exposure. Mononuclear cells were prepared from the peripheral blood of recovered cats (a; n = 5) and persistently viremic cats (b; n = 4) preexposure and at regular intervals following intranasal and oral exposure to FeLV. FeLV-specific effector CTL activity was measured directly ex vivo on autologous and allogeneic skin fibroblast targets labeled with ⁵¹Cr and infected with recombinant vaccinia viruses expressing either FeLV Gag/Pro (solid bars), Env (hatched bars), or wild-type vaccinia virus as a negative control (consistently below 5% and not shown). Alternatively, targets were infected with FeLV-A/Glasgow-1 in vitro (shaded bars). The release of ⁵¹Cr into the culture supernatant was detected after 4 h of incubation at 37°C. Results represent the mean values ± SEM from triplicate cultures at an effector-to-target cell ratio of 25:1, from which the values for recognition of allogeneic targets have been subtracted.

FIG. 4.

Adoptively transferred T cells migrate to the lymph nodes. FeLV-specific effector CTL activity was compared in the peripheral lymph nodes and blood of three cats 6 weeks after the infusion of autologous antigen-activated lymphocytes (cats 6, 8, and 9), and in one persistently viremic control cat that did not receive an infusion (cat 10). FeLV-specific CTLs were measured directly ex vivo on autologous (solid lines and symbols) and allogeneic (dashed lines and open symbols) skin fibroblast targets labeled with ⁵¹Cr and infected with recombinant vaccinia viruses expressing either FeLV Gag/Pro (▪), Env (•), or wild-type vaccinia virus as a negative control (✖). Alternatively, targets were infected with FeLV-A/Glasgow-1 in vitro (▴). The release of ⁵¹Cr into the culture supernatant was detected after 4 h of incubation at 37°C. Results represent the mean values from triplicate cultures.