Phenotypic changes in CD8+ peripheral blood lymphocytes in cats infected with feline immunodeficiency virus

It is well documented that several cell surface molecules of T lymphocytes are altered by immune activation. We previously reported that feline immunodeficiency virus (FIV) infection induces a reduction in CD8beta chain expression of peripheral blood lymphocytes (PBLs) in cats. In this study, we performed three-color flow-cytometric analysis for activation-associated cell surface molecules (CD2, CD11a, CD45RA-like and major histocompatibility complex antigen class II (MHC II)) and light scatters (cellular size and complexity) to examine whether phenotypic changes also occurred in CD4(+) PBLs in addition to CD8(+) PBLs, of five FIV-infected cats and one uninfected cat. It was shown that (i) CD8alpha(+) PBLs, but not CD4(+) PBLs, had a distinct subpopulation with increased CD11a expression accompanying a reduced CD8beta chain and increased intracellular granules (ii) CD8alpha(+) PBLs, but not CD4(+) PBLs, expressed CD45RA-like antigen with diverse expression levels and (iii) MHC II expression was greater in CD8alpha(+) PBLs than CD4(+) PBLs, and the CD8beta chain reduction was correlated with the MHC II decrease within CD8alpha(+) PBLs. These results suggest that FIV infection induces phenotypically heterogeneous subpopulations in CD8(+) PBLs, including activated phenotypes, rather than in CD4(+) PBLs.     

Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus.

Within 6 months of infection with the Petaluma isolate of feline immunodeficiency virus, specific-pathogen-free domestic cats exhibited a decrease in the percentage and number of circulating CD4+ lymphocytes and in the CD4+/CD8+ T-cell ratio, along with a marginally significant depression of pokeweed mitogen-induced lymphocyte proliferation in vitro. There was no loss of responsiveness to concanavalin A during this stage, and the cats were capable of mounting a satisfactory antibody response to a T-dependent, synthetic polypeptide immunogen. The pokeweed mitogen response deficit became clearly demonstrable by 11 to 12 months postinfection. A decline in the lymphocyte proliferative response to concanavalin A and a diminished ability to mount an in vivo antibody response to the T-dependent immunogen evolved by 25 to 44 months postinfection. Virus infection did not affect the ability of cats to mount an antibody response to a T-independent synthetic polypeptide immunogen. These data indicate that feline immunodeficiency virus produces a slowly progressive deterioration of T-cell function but does not affect the ability of B cells to recognize and respond to a T-independent antigenic stimulus.     

Immunologic abnormalities in pathogen-free cats experimentally infected with feline immunodeficiency virus.

Blood mononuclear cells from 47 cats experimentally infected with feline immunodeficiency virus (FIV) were examined by using monoclonal antibodies directed against feline CD4 and CD8 homologs, a pan-T-cell antigen, and cell surface immunoglobulin. Significant inversion of the CD4+/CD8+ T-cell ratio was observed only in cats that were infected for 18 months or more. This inversion was associated with a decrease in the absolute numbers of CD4+ T cells and a concomitant increase in CD8+ cells. However, the total numbers of circulating T and B cells were not significantly reduced. Cats infected with FIV for 24 to 28 months also had significantly elevated levels of serum immunoglobulin G (IgG), but normal levels of IgA and IgM. The long-term decline in CD4+ T cells and hypergammaglobulinemia observed in FIV-infected cats resemble the abnormalities occurring in humans after human immunodeficiency virus infection.     

Decline in CD4+ cell numbers in cats with naturally acquired feline immunodeficiency virus infection.

T-cell subsets were studied by fluorescence-activated cell sorter analysis in 57 feline immunodeficiency virus (FIV)-seropositive cats with naturally acquired FIV infection to see whether CD4(+)-CD8+ alterations were comparable to those observed in human immunodeficiency virus-infected patients. CD4+ values were decreased and CD8+ values were increased. The CD4+/CD8+ ratio was reduced to 1.6, compared with 3.3 in 33 FIV-seronegative control cats. Variance analysis of data showed a significant influence of FIV seropositivity, sex, and spaying of female cats on CD4+ values. CD8+ values were significantly influenced by FIV seropositivity, age, and breed. These findings indicate a similarity between FIV and human immunodeficiency virus infections, as far as alterations of T-cell subsets are concerned.     

Frequent perinatal transmission of feline immunodeficiency virus by chronically infected cats.

Vertical transmission of feline immunodeficiency virus (FIV) was studied in cats infected with either of two FIV clinical isolates (FIV-B-2542 or FIV-AB-2771) prior to breeding and conception. Queens infected 4 to 30 months (mean = 14 months) prior to conception transmitted FIV to 59 of 83 (71%) kittens; 50.6% were virus positive on the day of birth. To examine potential routes of FIV transmission from mother to offspring, kittens were delivered via either vaginal or cesarean birth and nursed by either their virus-infected natural mothers or uninfected surrogate mothers. Comparison of FIV infection rates at birth with those at 6 months of age in kittens delivered by cesarean and surrogate raised demonstrated that late in utero transmission occurred in approximately 20% of kittens. Comparison of kittens nursed by FIV mothers with those by uninfected surrogate mothers demonstrated a 13.5% increase in infection rate of kittens exposed to milk-borne virus. Isolation of virus from 40% of maternal vaginal wash samples and the slightly greater infection rate in vaginally versus cesarean-delivered surrogate-nursed kittens suggested that intrapartum transmission may occur. In addition, we found that low maternal CD4 count (<200 cells per microl), longer duration of maternal infection (>15 months), and maternal symptoms of clinical immunodeficiency correlated with increased rates of mother-to-kitten FIV transmission, paralleling observations in human immunodeficiency virus-infected women. We conclude that FIV infection provides a model in which to explore aspects of human immunodeficiency virus vertical transmission and intervention difficult to address in human patients.     

Changes in soluble factor-mediated CD8+ cell-derived antiviral activity in cynomolgus macaques infected with simian immunodeficiency virus SIVmac251: relationship to biological markers of progression

Cross-sectional studies have shown that the capacity of CD8+ cells from human immunodeficiency virus (HIV)-infected patients and simian immunodeficiency virus (SIV) SIVmac-infected macaques to suppress the replication of human and simian immunodeficiency viruses in vitro depends on the clinical stage of disease, but little is known about changes in this antiviral activity over time in individual HIV-infected patients or SIV-infected macaques. We assessed changes in the soluble factor-mediated noncytolytic antiviral activity of CD8+ cells over time in eight cynomolgus macaques infected with SIVmac251 to determine the pathophysiological role of this activity. CD8+ cell-associated antiviral activity increased rapidly in the first week after viral inoculation and remained detectable during the early phase of infection. The net increase in antiviral activity of CD8+ cells was correlated with plasma viral load throughout the 15 months of follow-up. CD8+ cells gradually lost their antiviral activity over time and acquired virus replication-enhancing capacity. Levels of antiviral activity correlated with CD4+ T-cell counts after viral set point. Concentrations of beta-chemokines and interleukin-16 in CD8+ cell supernatants were not correlated with this antiviral activity, and alpha-defensins were not detected. The soluble factor-mediated antiviral activity of CD8+ cells was neither cytolytic nor restricted to major histocompatibility complex. This longitudinal study strongly suggests that the increase in noncytolytic antiviral activity from baseline and the maintenance of this increase over time in cynomolgus macaques depend on both viral replication and CD4+ T cells.     

Prevalence of feline immunodeficiency virus and feline leukemia virus infections in random-source cats.

Retroviral serologic profiles were generated for 506 random-source cats (Felis catus) that were received by our facility during a twenty-month period. Feline leukemia virus antigens were detected in plasma samples from 26 (5.1%) of the cats. Antibodies to feline immunodeficiency virus were present in 24 (4.7%) of the samples tested. A single cat (0.2%) was positive for both viruses. Neither gender nor vendor correlation with retroviral seropositivity could be demonstrated.     

Association of plasma viral RNA load with prognosis in cats naturally infected with feline immunodeficiency virus

We measured the quantity of plasma feline immunodeficiency virus (FIV) RNA using a real-time sequence detecting system. Plasma viral RNA load was shown to correlate with the clinical stage, survival time, and disease progression in naturally FIV-infected cats. The present study indicates that the plasma viral RNA load can be used as a clinical marker representing the impairment of the immune system and predicting the clinical outcome in FIV-infected cats.     

Mature dendritic cells infected with canarypox virus elicit strong anti-human immunodeficiency virus CD8+ and CD4+ T-cell responses from chronically infected individuals

Recombinant canarypox virus vectors containing human immunodeficiency virus type 1 (HIV-1) sequences are promising vaccine candidates, as they replicate poorly in human cells. However, when delivered intramuscularly the vaccines have induced inconsistent and in some cases transient antigen-specific cytotoxic T-cell (CTL) responses in seronegative volunteers. An attractive way to enhance these responses would be to target canarypox virus to professional antigen-presenting cells such as dendritic cells (DCs). We studied (i) the interaction between canarypox virus and DCs and (ii) the T-cell responses induced by DCs infected with canarypox virus vectors containing HIV-1 genes. Mature and not immature DCs resisted the cytopathic effects of canarypox virus and elicited strong effector CD8+ T-cell responses from chronically infected HIV+ individuals, e.g., cytolysis, and secretion of gamma interferon (IFN-gamma) and beta-chemokines. Furthermore, canarypox virus-infected DCs were >30-fold more efficient than monocytes and induced responses that were comparable to those induced by vaccinia virus vectors or peptides. Addition of exogenous cytokines was not necessary to elicit CD8+ effector cells, although the presence of CD4+ T cells was required for their expansion and maintenance. Most strikingly, canarypox virus-infected DCs were directly able to stimulate HIV-specific, IFN-gamma-secreting CD4 helper responses from bulk as well as purified CD4+ T cells. Therefore, these results suggest that targeting canarypox virus vectors to mature DCs could potentially elicit both anti-HIV CD8+ and CD4+ helper responses in vivo.     

Lentivirus-specific cytotoxic T-lymphocyte responses are rapidly lost in thymectomized cats infected with feline immunodeficiency virus

To what extent the thymus is needed to preserve the virus-specific cytotoxic T-lymphocyte (CTL) response of lentivirus-infected adults is unclear. Presented here is the first definitive study using thymectomized (ThX) animals to directly evaluate the contribution of thymic function to lentivirus-specific CTL response and the control of lentivirus infections. ThX and mock-ThX cats were inoculated with feline immunodeficiency virus (FIV) and monitored for their FIV-specific CTL responses. Early in infection, both FIV-ThX and FIV-mock-ThX cats produced similar CTL responses, but surprisingly, after 20 weeks, the FIV-ThX cats showed a statistically significant loss of FIV-specific CTL activity, while FIV-infected cats with intact thymuses continued to maintain FIV-specific CTL. The loss of CTL did not affect plasma virus load, which remained elevated for both groups. These results emphasize the importance of thymic integrity in maintaining immunity to lentiviruses, but also bring into question the notion that virus load is regulated predominantly by the virus-specific CTL response.     

T cell subpopulations mediating inhibition of feline immunodeficiency virus replication in mucosally infected cats

Feline immunodeficiency virus (FIV) infection induces an increase in two subpopulations (CD8alpha(+)beta(low) and CD8alpha(+)beta(-)) within CD8(+) peripheral blood lymphocytes (PBLs) of cats. It is known that depletion of CD8(+) cells often results in augmentation of FIV proliferation in PBL culture, similarly to the case of human immunodeficiency virus. In this study, we attempted to define PBL subpopulations mediating antiviral activity in five cats intravaginally infected with a molecularly cloned FIV isolate. Several subpopulations (CD8alpha(+)beta(+), CD8alpha(+)beta(-), and CD4(+) cells) were shown to participate in inhibition of the FIV replication, at least in part, in a major histocompatibility complex-unrestricted manner. Moreover, the subpopulations showing anti-FIV activity were different among the individual cats.     

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.     

Suppression of human immunodeficiency virus replication by CD8+ cells from infected and uninfected chimpanzees.

Over a 4-year period, infectious human immunodeficiency virus type 1 (HIV-1) has been recovered from cultured peripheral blood mononuclear cells (PBMC) of virus-infected animals only intermittently and at relatively low titers. In examining the possible mechanism for this observation, CD4+ cells or CD8+ cells were removed by panning from the PMBC before culture. A dramatic increase in frequency of HIV-1 recovery as well as in the level of virus replication was observed in the CD4+ cell-enriched or CD8+ cell-depleted cultures of PBMC from 3/3 infected animals. Moreover, addition of purified CD8+ effector cells from all 6 HIV-infected and 5/10 uninfected animals to an equal number of HIV-1 acutely infected purified CD4+ target cells resulted in 75-100% suppression of virus production. CD8+ cells from 3 additional uninfected animals caused delayed replication kinetics and moderate to low suppression of peak virus production. These findings contrast with the previously recognized absence of this HIV-1-suppressing activity of CD8+ cells from seronegative humans. This CD8+ cell-mediated suppression of viral replication could help explain the natural resistance of chimpanzees to HIV-induced disease.     

Simian immunodeficiency virus infection of CD8+ lymphocytes in vivo.

To determine the lymphoid target cells of simian immunodeficiency virus (SIV) in vivo, peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) were positively selected (>97% purity) for surface expression of CD4, CD8, or CD20 and then analyzed for SIV provirus using semiquantitative DNA amplification. We found provirus in CD4+ and CD8+ lymphocytes but none in CD20+ lymphocytes. During acute SIV infection (< or = 214 days postinoculation), the percentage of PBL and LNL CD4+ cells containing proviral DNA ranged from 0.2 to 20% and from 0.2 to 2%, respectively. Proviral burden in the CD8+ population of either PBL or LNL ranged from 0.01 to 0.2%. Virus isolation by cocultivation was positive for both CD4+ and CD8+ purified populations. No difference in proviral burden was observed between PBL and LNL subsets during acute SIV infection. Up to 19.4% of positively selected CD8+ cells also expressed CD4, and thus the provirus may reside within a dual-positive population. This dual-positive population may represent activated lymphocytes that are particularly susceptible to infection and may provide an opportunity for virus entry into the CD8+ CD4- lymphocytes in vivo.     

Simian immunodeficiency virus-specific CD8+ lymphocyte response in acutely infected rhesus monkeys.

To assess the possible role of cytotoxic T lymphocytes (CTLs) in containing the spread of human immunodeficiency virus in acutely infected individuals, the temporal evolution of the virus-specific CD8+ lymphocyte response was defined in simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys. A brief period of SIVmac plasma antigenemia was seen 9 to 16 days following intravenous infection with SIVmac, ending as the absolute number of CD8+ peripheral blood lymphocytes (PBLs) increased. In a prospective assessment of the ability of CD8+ lymphocytes of these monkeys to suppress SIVmac replication in autologous PBLs, inhibitory activity was detected as early as 4 days, with a more pronounced effect 12 to 16 days following infection. SIVmac Gag- and Nef-specific CD8+ effector cell activities were demonstrable in PBLs of animals by 2 weeks following virus inoculation. In fact, SIVmac-specific CTL precursors were documented in the PBLs of rhesus monkeys 4 to 6 days after SIVmac infection. These studies indicate that AIDS virus-specific CD8+ CTLs are present in PBLs within days of infection and may play an important role in containing the early spread of virus.     

Passive antibody protection of cats against feline immunodeficiency virus infection.

All six cats passively immunized with sera from either feline immunodeficiency virus (FIV)-vaccinated cats or cats infected with FIV (Petaluma strain) were protected from homologous FIV infection at a challenge dose that infected all six control cats. Passive immunization with sera from cats vaccinated with uninfected allogeneic T cells used to grow the vaccine virus did not protect either of two cats against the same FIV challenge. These results suggest that antiviral humoral immunity, perhaps in synergy with anticellular antibodies, may be responsible for previously reported vaccine protection.