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Tuesday, 30 October 2007

Sick Cats


Loss of naïve (CD45RA+) CD4+ lymphocytes during pediatric infection with feline immunodeficiency virus
By Abigail D. Carreñoa
and Ayalew Mergiab, Janelle Novakb, Nazareth Gengozianc, d and Calvin M. Johnsona, ,
aDepartment of Pathobiology, Auburn University, Auburn, AL, United States
bDepartment of Infectious Diseases and Pathology, University of Florida, Gainesville, FL, United States
cUniversity of Tennessee Graduate School of Medicine, Knoxville, TN, United States
dThompson Cancer Survival Center, Knoxville, TN, United States
Received 10 July 2007; revised 14 August 2007; accepted 5 September 2007. Available online 11 September 2007.

Abstract
Feline immunodeficiency virus (FIV) infection of cats is an animal model for the pathogenesis of CD4+ lymphopenia and thymus dysfunction in HIV-infected humans. Recently, a monoclonal antibody (755) was reported to recognize the feline homologue to CD45RA, allowing the enumeration of naïve T cells in cats. We tested the hypothesis that pediatric FIV infection would be associated with a selective loss of naïve CD4+ lymphocytes by inoculating newborn cats with a pathogenic clone of FIV (JSY3) or a related clone with an inactive ORF-A gene (JSY3-ΔORFA), and compared the data to age-matched uninfected control cats. Both FIV inocula were associated with a reduction in the CD4–CD8 ratio (p = 0.01), which was attributable to a disproportionate loss of naïve CD4+ cells (p = 0.01) vs. naïve CD8+ cells. Therefore, the reduced CD4:CD8 ratio in FIV-infected juvenile cats is associated with a selective depletion of naïve CD4+ cells from the blood.
Keywords: FIV; HIV; Naïve CD4 lymphocytes; CD45RA

1. Introduction
Feline immunodeficiency virus (FIV) infection of cats is associated with a depletion of CD4 T lymphocytes and an increase in CD8 T lymphocytes from the peripheral blood, resulting in a decreased CD4–CD8 ratio. The pathogenesis of these changes is similar to that of HIV infection, and FIV has become a well established model for HIV immunopathogenesis (Kolenda-Roberts et al., 2007).

The course of FIV infection can be characterized by a gradual transition through three general phases defined by changes in clinical signs, level of viremia, antiviral immunity, and the ratio of helper-T (CD4+) lymphocytes to T-cytotoxic/suppressor (CD8+) lymphocytes (CD4:CD8) in peripheral blood.

The acute stage of infection typically occurs within the first 3 months of virus exposure. Clinical signs during this period include transient fever, neutropenia, and generalized lymphadenopathy (Yamamoto et al., 1988). The dynamic events of the acute stage precede a relatively stable, clinically asymptomatic stage of infection that characterizes most lentivirus infections of animals. In nature, the median age of asymptomatic cats (4 years) is much less than the median age for FIV-infected cats with AIDS-like disease (10 years), suggesting that this asymptomatic stage lasts for several years (Shelton et al., 1989). The third phase of FIV infection is the classical form of feline AIDS often marked by the emergence of multiple degenerative, neoplastic, and secondary infectious diseases, including chronic periodontal disease, weight loss, upper respiratory tract disease, abscesses, neurologic disease, and lymphoma. While there is considerable overlap in clinical findings between this and the previous category, manifestations may become multiple or more severe. This stage is best characterized in naturally infected animals, but has also been reproduced in an experimental setting ([Diehl et al., 1995] and [English et al., 1994]).

FIV infection is characterized by consistent changes within the CD8 T cell population, including an expansion of activated memory CD8 lymphocytes (CD44hi, CD8betalow, CD62Llow), which progressively accumulate within the lungs and peripheral lymph nodes during the course of adult infection and mediate non-cytotoxic cell-mediated suppression of FIV replication ex vivo (Gebhard et al., 1999). During pediatric FIV infection, these cells first occupy the thymus and blood, where strong CD8 cell-mediated antiviral activity is linked to reduce virus load in lymphoid tissues (Crawford et al., 2001). In contrast, the phenotypic and functional heterogeneity of CD4 T cells has not been well characterized to date. This is particularly important in the case of pediatric FIV infection, as CD4 T cells greatly outnumber CD8 cells in the blood and lymphoid tissues of neonates before CD8 T cells emerge within 3 weeks of age ([Bortnick et al., 1999] and [Sellon et al., 1994]). One approach to the characterization of subpopulations of CD4 T cells is the enumeration of cells that express markers of the naïve or memory phenotypes. CD45RA is one of the four isoforms of the leukocyte common antigen, and is the predominant form expressed on the surface of naïve T cells. Gengozian et al. described a mAb (755) recognizing the feline CD45RA isoform based on three lines of evidence: avidity to a membrane protein with a molecular weight of >200 kDa, enhanced suppressor function of CD8 cells following addition of mAb 755 to a culture system stimulating IgG function, and predictable declines in the percentage of CD4 cells reactive with the antibody in aging kittens (Gengozian et al., 2005). Using the mAb, we identified CD45RA on feline leukocytes and report a decline in naïve CD4 T cells during the acute stage of pediatric FIV infection. Cell populations with low/negative expression of CD45RA were designated as memory cells (Gengozian et al., 2005).

2. Materials and methods
Six newborn cats from specific pathogen free (SPF) queens were assigned to protocols approved by the Institutional Animal Care and Use Committees at the University of Florida or Auburn University. Blood was drawn from cats for pre-inoculation analysis within 24 h of birth. After random assignment to groups, 1-day-old kittens (n = 4) were injected intraperitoneally with 200 μl of cell culture supernatant containing virions from either the wild type clone JSY3 (n = 1) (Yang et al., 1996) or the ORF-A mutant JSY3ΔORF-A/2 (n = 3) (Norway et al., 2001) at a dose of approximately 104 TCID50. Two cats were administered a sham inoculum consisting of 200 μl sterile saline solution. Blood was collected at biweekly intervals and processed for flow cytometry. Briefly, the blood was centrifuged at 200 × g for 5 min at room temperature and the serum was withdrawn and frozen for later analysis. Fetal bovine serum (FBS) (Gibco, Carlsbad, CA) was added to the cellular fraction in an equal amount to the withdrawn serum and resuspended for a complete blood count (CBC) and the remainder was used for flow cytometric analysis. CBCs were analyzed by a Heska CBC-Diff Veterinary Hematology System (Heska, Inc., Loveland, CO) when the blood samples were less than 200 μl, and samples over 200 μl were analyzed with an Advia 120 (Siemens Diagnostics, Inc., Tarrytown, NY).

One hundred to 500 μl of EDTA-treated blood was used for whole blood lysis. Fourteen millilitres of room temperature 1× lysis solution was made from 10× lysis solution (1.7 M ammonium chloride, 0.1 M potassium bicarbonate and 1.1 mM tetrasodium EDTA) by the addition of sterile phosphate buffered saline solution (PBS; 1.1 M potassium phosphate, 0.2 M anhydrous monobasic sodium phosphate and 3.1 M sodium chloride, pH 7.4). At least 100 μl of whole blood was added and mixed by inversion. The solution was incubated at room temperature for 3 min and was centrifuged at 300 × g for 6 min. The supernatant was decanted and 10 ml of flow buffer (1× PBS, 2% FBS and 0.1% sodium azide) was added to the cells. The solution was mixed gently and spun again under the same conditions. The supernatant was decanted and 1 ml of flow buffer was added to the cells. The cells were gently mixed and filtered through a 35 μm cell strainer cap into a 5 ml round bottom tube.

Monoclonal mouse anti-feline CD4 antibody (clone vpg34, Serotec, Inc., Raleigh, NC), conjugated to fluorescein isothiocyanate (FITC) was prepared at an optimal concentration based on a preliminary study. The monoclonal mouse anti-feline CD8 conjugated to R-phycoerythrin (RPE) (clone vpg9, Serotec, Inc., Raleigh, NC) was used at the manufacturer's recommended dilution. A mouse anti-feline CD4 antibody conjugated to RPE (clone #3-4F4) (Southern Biotech, Birmingham, AL) was used at a 1:400 dilution of the manufacturer's preparation. The monoclonal mouse anti-feline CD45RA (mAb 755) (Gengozian et al., 2005) antibody was conjugated to Alexa 488 using the Zenon® Mouse IgG1 Labeling Kit (Molecular Probes, Invitrogen, Inc., Carlsbad, CA) according to the manufacturer's instructions. A mouse IgG monoclonal antibody of irrelevant specificity conjugated to fluorescein isothiocyanate (FITC) was used at similar concentration (Serotec, Inc., Raleigh, NC) as a negative control. Tubes were placed on ice throughout the procedure, and were incubated in the dark.

Dual fluorescence of the following combinations of antibodies was used: anti-feline CD4-RPE vs. anti-feline CD45RA-Alexa 488; anti-feline CD8-RPE vs. anti-feline CD45RA-Alexa 488; and anti-feline CD4-FITC vs. anti-feline CD8-RPE. After antibodies were added to 5 ml polystyrene tubes, 2 × 105 cells were added in 100 μl and incubated on ice in the dark for 30 min. The cells were washed twice with flow buffer at 1000 rpm for 5 min at 4 °C. Cells were then washed twice and resuspended in isotonic 0.5% paraformaldehyde. Samples were analyzed either with a DakoCytomation MoFlo flow cytometer and Summit software (DakoCytomation, Inc., Fort Collins, CO) or with a FACScan flow cytometer and Consort-32 computer system and LYSYS-II software (Becton Dickinson, Inc.).

Naïve cells were expressed as the percentage of CD4 or CD8 lymphocytes that co-labeled with CD45RA, or as absolute numbers calculated as the product of the percentage and the absolute lymphocyte count. All statistical analyses were performed with the Statistica 7.0 software package (StatSoft, Inc., Tulsa, OK). Numbers of naïve CD4 and CD8 cells were subjected to log10 transformation. All data were analyzed by mean plots ± 95% confidence intervals in repeated measures ANOVA, and comparisons of means were performed by the unequal N honest significant difference (HSD) test. Differences at p = 0.05 in the HSD were considered significant.


3. Results and discussion
Despite the physiologic decline in CD4:CD8 ratio associated with neonatal development (Bortnick et al., 1999), we found that the CD4:CD8 ratio in all FIV-infected cats was less than the ratio of uninfected cats between 4 and 12 weeks of age (p = 0.010; data not shown). Both wild-type FIV (generated from clone JSY3) and FIV with a defective ORF-A gene (JSY3ΔORF-A/2) were capable of inducing similar changes over the 12-week course of study, as reported ([Norway et al., 2001] and [Novak et al., 2007]), therefore the data from three JSY3-infected and one ORF-A/2-infected cats were pooled for this study. The decline in CD4:CD8 ratio was associated with a significant reduction in the absolute number of naïve CD4 T cells (p = 0.014; Fig. 1B), resembling the preferential decline in absolute numbers of naïve CD4 T cells in HIV-infected adults ([Roederer et al., 1995] and [Spina et al., 1997]). Similar to data from HIV-infected human neonates (Sleasman et al., 1996), the percentages of naïve CD4 and CD8 lymphocytes in FIV-infected cats did not differ significantly from uninfected cats in proportions of naïve CD4 and CD8 lymphocytes when compared with memory cells (Fig. 1A and B). In the current study, the loss of naïve CD4+ cells without a decline in their percentages was attributable to a significant decline in total leukocyte counts in the infected cats (p = 0.001; data not shown) and a trend toward a reduced percentage of naïve CD4+ cells. The absolute numbers and percentages of naïve CD8 lymphocytes were not significantly different between infected and uninfected cats, although there was a trend for naïve CD8 cell percentages to be lower in infected cats (Fig. 1B and D). In all cases, a scatterplot of the data was best fit by a polynomial curve as depicted in Fig. 1. Roederer et al. (1995) reported a decline in naïve CD8 lymphocytes and an expansion in memory CD8 lymphocytes in HIV-infected adults, resulting in an overall increase in total CD8 lymphocytes. This phenomenon has also been reported in FIV infection, and is attributable to a marked expansion of activated memory CD8 lymphocytes that suppress FIV replication ex vivo (Gebhard et al., 1999).
Fig. 1. Quantification of naïve CD4 T cells and naïve CD8 T cells expressed as percentages and absolute numbers over the course of juvenile FIV infection. Percent naïve CD4 and naïve CD8 T cells are expressed as a percentage of total CD4 and CD8 cells, respectively. Solid curves indicate uninfected cats; dotted curves indicate infected cats. Scatterplot of the data is represented as a polynomial curve over time. (A) Naïve CD4 T cell percentages over a 12-week time period of FIV infection vs. sham infection. (B) Naïve CD4 T cell numbers over a 12-week time period of FIV infection vs. sham infection. (C) Naïve CD8 T cell percentages over a 12-week time period of FIV infection vs. sham infection. (D) Naïve CD8 T cell numbers over a 12-week time period of FIV infection vs. sham infection.

An analysis of dot plots generated by flow cytometry of dual antibody labeling with combinations of CD4 and CD45RA in 8-week-old control cats demonstrated similar proportions of memory and naïve CD4+ cells, while naïve CD8+ cells clearly predominated over memory CD8+ cells at the same age (Fig. 2). This contrasted with FIV-infected cats, in which memory cells outnumbered naïve cells in both cases, and in which there was minimal overlap in CD45RA expression between naïve and memory CD4+ subpopulations (Fig. 3).
Fig. 2. Flow cytometric analysis of dual antibody labeling with anti-CD4-PE and anti-CD45RA (755)-Alexa 488 (left plot) and dual antibody labeling with anti-CD8-PE and anti-CD45RA (755)-Alexa 488 (right plot) in a control animal (H4-2) at 8 weeks of age.
Fig. 3. Flow cytometric analysis of dual antibody labeling with anti-CD4-PE and anti-CD45RA (755)-Alexa 488 (left plot) and dual antibody labeling with anti-CD8-PE and anti-CD45RA (755)-Alexa 488 (right plot) in an FIV-infected cat (K3-1) at 8 weeks of age.

Four isoforms of the CD45 (leukocyte common antigen) family of highly glycosolated transmembrane proteins have been identified in humans: CD45RA, CD45RB, CD45RC, and CD45RO (Serra-Pages et al., 1995). The relative molecular weights of the corresponding antigens range from 180 kDa for CD45RO to 200–220 kDa for CD45RA (Thomas, 1989). The CD45 cytoplasmic domain has been identified as a protein tyrosine kinase, which is involved in signaling events in B and T cell activation ([Altin and Sloan, 1997] and [Dahlke et al., 2004]). For T cells the pattern of CD45 expression varies with the stage of development and activation by antigenic stimulation. Initial studies showed naïve T cells (CD4 or CD8) to express CD45RA, and memory cells to express CD45RO ([Clement et al., 1988], [Rudd et al., 1987] and [Sanders et al., 1988]). However, cells can convert phenotypically from CD45RA to CD45RO following 14 days in vitro activation by mitogens ([Geginat et al., 2001] and [Poppema et al., 1996]), and there is cyclic expression of these surface markers in vitro (Warren and Skipsey, 1991).

Mechanisms other than direct killing of infected CD4 T cells may contribute to CD4 T cell depletion, including activation-induced cell death (Grossman et al., 2002). Activation of resting T cells by HIV can occur through several mechanisms, including direct activation in the context of MHC II presentation by antigen presenting cells (Weissman et al., 1996), indirect stimulation through pro-apoptotic inflammatory cytokines (Matsuyama et al., 1991), or exposure to activating viral proteins (e.g., gp120) ([Capobianchi, 1996] and [Delobel et al., 2006]). Proliferation may precede apoptosis or killing by virus-specific CTL ([Grossman et al., 2002] and [Hellerstein et al., 1999]).

l-Selectin (CD62L) expression has been used as a marker of either naïve/memory or activated (effector) CD8 cells ([Hamann et al., 1997] and [Zimmerman et al., 1996]). In addition, reduced expression of the CD8 β chain defines activated CD8 cells (Schmitz et al., 1998). The persistently high levels of activated CD8 cells is a profile associated with HIV and FIV infection; typically, healthy uninfected individuals have very few circulating activated T cells, except during active immune responses (Picker et al., 1993). Gebhard et al. (1999) reported that FIV infection is characterized by a loss of naïve CD8 cells and an increase in activated CD8 expressing CD62L, the adhesion marker CD44, and the integrins CD49d and CD18. Within this activated CD8 population are cells with the capacity to potently suppress FIV replication in vitro through a contact-independent mechanism, implying that the loss of naïve CD8 lymphocytes is driven by an antiviral immune response (Gebhard et al., 1999). In normal human neonates, the majority of CD4 T cells express CD45RA, and a progressive age related transition occurs so that by late infancy, most CD4 T cells express CD45RO ([Bradley et al., 1989], [De Paoli et al., 1988] and [Sleasman et al., 1996]). In adults, CD45RA-expressing T cells are a long-lived, nearly static population of virgin T cells, while immunologic memory is maintained by the more numerous and proliferative CD45RO-expressing T cells (Michie et al., 1992).

Another pathogenetic mechanism that has been defined during FIV infection is mediated by the activation of CD4+CD25+ T regulatory T cells (T-reg). Iwashiro et al. reported T-reg cell activation in vivo under chronic retroviral stimulation (Iwashiro et al., 2001). T-reg cells are defined by their inability to produce IL-2 and are anergic to antigenic or mitogenic stimulation ([Jonuleit et al., 2001] and [Maloy and Powrie, 2001]). Vahlenkamp et al. found, using these criteria, that cells from both the lymph node and PMBCs of FIV+ cats possess the characteristics of T-reg cells (Vahlenkamp et al., 2004). Furthermore, they found that freshly isolated, unstimulated CD4+CD25+ T cells from FIV+ cats inhibited the proliferative response of autologous Con A-stimulated CD4+CD25− T cells when compared with T-reg cells from FIV− cats, implicating T cell anergy as a component of FIV immunosuppression (Vahlenkamp et al., 2004).

During the course of HIV infection, the absolute reduction in CD4 T cells is attributable to a loss of both CD4+ CD45RA+ cells and CD4+ CD45RO+ cells ([Hengel et al., 1999], [Roederer et al., 1995] and [Spina et al., 1997]). Preferential HIV infection of the memory CD4 cells may predispose them to lytic depletion ([Saavedra-Lozano et al., 2004] and [Sleasman et al., 1996]). Naïve T cells may also serve as viral reservoirs during some cases of HIV infection, albeit at a lower frequency of infection than memory T cells ([Blaak et al., 2000] and [Ostrowski et al., 1999]).

One plausible explanation for the loss of naïve CD4 T cells is through virus-mediated dysfunction of the thymus, a model that could incorporate both intrathymic and extrathymic pathways ([Brenchley et al., 2004], [Delobel et al., 2006] and [McBreen et al., 2001]). The findings of Delobel et al. implicate apoptosis of naïve CD4 T cells in the periphery through persistent T cell activation and gp120-CXCR4 interactions, rather than through a coordinated loss within the thymus (Delobel et al., 2006). In contrast, Ye et al. (2004) have reviewed evidence to suggest that the thymus may serve as a centralized site for thymocyte destruction, particularly during in utero infection (Ye et al., 2004). Very few infected naïve CD4 and CD8 T cells can be identified in the blood of HIV-infected children (Sleasman et al., 1996), despite the fact that progenitor CD4+CD8+ thymocytes are highly susceptible to HIV infection in vivo (Bonyhadi et al., 1993). Such findings would seem to suggest an important role for the thymus under the conditions of neonatal infection by virus strains with tropism for naïve T cells. We and others have implicated thymus dysfunction as a potential mechanism for centralized T cell depletion in the FIV model (reviewed by Kolenda-Roberts et al., 2007) based on the temporal association between productive and destructive infection of the thymus and a global decline in T cells from the peripheral blood of cats infected in utero with FIV. The ability to directly identify naïve CD4 lymphocytes in blood and lymphoid tissues, including lymph nodes, thymus, and the gastrointestinal tract will extend the utility of the FIV model for the investigation of mechanisms responsible for the depletion of naïve T cells in HIV/AIDS patients.

Acknowledgements
This work was supported by NIH R29HD33983 (CMJ), NIH R01AI42563 (AM), Auburn University Biogrant Program, and the Grace Kemper Research Fund. The authors thank Cynda Crawford, Holly Kolenda-Roberts, Peter Nadeau, Robert Norway, George Papadi, and Chengming Wang for their contributions.

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(Abigail Carreño is a frequent contributor to Junto).

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