Article Prior Puma Infection Modifies Early Immune Responses and Attenuates Feline Immunodeficiency Infection in

Wendy S. Sprague 1,2,*, Ryan M. Troyer 1,3, Xin Zheng 1, Britta A. Wood 1,4, Martha Macmillian 1, Scott Carver 1,5 ID and Susan VandeWoude 1

1 Department of Molecular Biology, Immunology and Pathology, College of Veterinary Medicine and Biological Sciences, Colorado State University, Fort Collins, CO 80523, USA; [email protected] (R.M.T.); [email protected] (X.Z.); [email protected] (B.A.W.); [email protected] (M.M.); [email protected] (S.C.); [email protected] (S.V.) 2 Sprague Medical and Scientific Communications, LLC, Fort Collins, CO 80528, USA 3 Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A5C1, Canada 4 The Pirbright Institute, Pirbright, Surrey GU24 0NF, UK 5 School of Natural Sciences, University of Tasmania, Hobart, Tasmania 7001, Australia * Correspondence: [email protected]; Tel.: +1-970-223-0333

 Received: 22 March 2018; Accepted: 12 April 2018; Published: 20 April 2018 

Abstract: We previously showed that cats that were infected with non-pathogenic Puma lentivirus (PLV) and then infected with pathogenic feline immunodeficiency virus (FIV) (co-infection with the host adapted/pathogenic virus) had delayed FIV proviral and RNA viral loads in blood, with viral set-points that were lower than cats infected solely with FIV. This difference was associated with global CD4+ T cell preservation, greater interferon gamma (IFN-γ) mRNA expression, and no cytotoxic T lymphocyte responses in co-infected cats relative to cats with a single FIV infection. In this study, we reinforced previous observations that prior exposure to an apathogenic lentivirus infection can diminish the effects of acute infection with a second, more virulent, viral exposure. In addition, we investigated whether the viral load differences that were observed between PLV/FIV and FIV infected cats were associated with different immunocyte phenotypes and cytokines. We found that the immune landscape at the time of FIV infection influences the infection outcome. The novel findings in this study advance our knowledge about early immune correlates and documents an immune state that is associated with PLV/FIV co-infection that has positive outcomes for lentiviral diseases.

Keywords: feline immunodeficiency virus (FIV); puma lentivirus (PLV); innate immunology; CD8; FAS (death receptor; CD95)

1. Introduction Feline immunodeficiency virus (FIV) infection, which is a pathogenic lentivirus of domestic cats, causes fatal immune dysfunction that is characterized by progressive depletion of CD4+ T lymphocytes that is similar to HIV infection of humans [1,2]. More than two dozen felid species have demonstrated seroreactivity against FIV antigens, and FIV that was isolated from puma (Puma concolor) has been characterized genetically as Puma lentivirus (PLV) infection in domestic cats causes a nonpathogenic disease that results in initially high viral titers, but marked diminution early in infection [3–6]. Previously, we found that cats that were infected with non-pathogenic PLV and then infected with pathogenic FIV (co-infection with the host adapted/pathogenic virus) had delayed FIV proviral and RNA viral load detection in blood, with an overall viral set-point decrease compared to cats

Viruses 2018, 10, 210; doi:10.3390/v10040210 www.mdpi.com/journal/viruses Viruses 2018, 10, 210 2 of 15 infected solely with FIV. This difference was associated with global CD4+ T cell preservation and greater interferon gamma (IFN-γ) mRNA expression, but not cytotoxic T lymphocyte responses in co-infected cats relative to cats with single FIV infection [7,8]. Co-infected cats also had accelerated anti-FIV capsid antibody development soon after FIV infection when compared to cats with single PLV or single FIV infection [9], and multivariate analysis implicates immediate anti-PLV immune responses involving CD8+ T cells, CD25+ cells, IL-4, IFN-γ, and the death receptor (FAS) as correlates of attenuated disease in co-infected cats [10]. Furthermore, co-infection results in a severe bottleneck restricting virulent FIV infection in the face of prior PLV infection, as evidenced by genomic analysis, suggesting a redistribution of viral infection and viral fitness [11,12]. Taken together, we hypothesize that the differences in available cell targets and early immune activation parameters are plausible explanations for these observations. Therefore, the current study aimed to compare lymphocyte and cytokine responses in blood, bone marrow, and lymphoid tissues of cats that were acutely infected with a pathogenic FIV isolate vs. those acutely that were infected with a nonpathogenic PLV isolate, followed by co-infection with pathogenic FIV.

2. Materials and Methods

2.1. Viral Stocks Stocks of FIV-C36 and PLV-1695 were prepared, as described previously [7]. Briefly, stocks of FIV-C virus were propagated by co-culture of retropharyngeal cells from an FIV-C positive and 7.2 domestic cat MYA-1 cells with a final titer of 10 tissue culture infectious dose 50 (TCID50)/mL. Stocks of PLV-1695 were similarly propagated by the co-culture of PLV-infected puma PBMC with 4.7 domestic cat MYA-1 cells, resulting in a titer of 10 TCID50/mL.

2.2. Animals Twenty-four specific-pathogen-free (SPF) cats were used from a breeding colony at Colorado State University (CSU). Animals were randomized by litter and gender and they were housed in groups of six per pen in isolation rooms of an AAALAC-international accredited animal facility. All of the procedures were approved by the CSU Institutional Animal Care and Use Committee prior to initiation (approval number: 01-246A-08, 1 March 2008).

2.3. Study Design The cats were divided and housed in the following four groups (n = 6 per group): (1) cats receiving only PLV-1965 (PLV), (2) cats receiving PLV-1695 followed by FIV-C36 one month later (CO), (3) cats receiving only FIV-C36 (FIV), and (4) cats receiving sham inoculations of media (SHAM). Blood samples were obtained by venipuncture of the cephalic vein on conscious animals at −5, −2, 0, 1, 2, 3, and 4 weeks (post-FIV inoculation; FIV PI) (Figure1). Bone marrow samples were collected from the humerus following ketamine/acepromazine/butorphanol anesthesia at −2 and 2 weeks FIV PI (Figure1). At −4 weeks FIV PI, 12 26-week-old cats were inoculated intravenously (IV) with 1 mL of PLV, as previously described [7], while the remaining 12 cats received 1 mL of culture supernatant from un-infected MYA-1 cells IV. Four weeks later (week 0), six of the PLV-inoculated animals and six of the SHAM controls received 1 mL of FIV stock IV that had been diluted 1:80 in a 0.9% NaCl solution. The remaining 12 animals received 1 mL of culture supernatant from un-infected MYA-1 cells IV. The study termination was eight weeks post-PLV inoculation and four weeks post-FIV challenge. Animals were humanely euthanized, and bone marrow, thymus, and mesenteric and prescapular lymph nodes were collected at necropsy (see Figure1 below). Viruses 2018, 10, 210 3 of 15 Viruses 2018, 10, x FOR PEER REVIEW 3 of 15

Figure 1. Study timeline. Figure 1. Study timeline. 2.4. Physical Examinations 2.4 Physical Examinations Animals were monitored daily for clinical signs of illness, as well as general health throughout the study.Animals Physical were examinations, monitored daily including for clinical weight signs and of temperature illness, as well measurements, as general health were performed throughout atthe each study. blood Physical collection. examinations, including weight and temperature measurements, were performed at each blood collection. 2.5. Cell Isolation 2.5 Cell Isolation Cells were isolated and purified from peripheral blood, bone marrow, and tissues throughout the studyCells for were flow isolated cytometry and analysis. purified Peripheral from peripheral blood mononuclear blood, bone cellsmarrow, (PBMC) and and tissues bone throughout marrow cellsthe were study purified for flow on cytomet a Histopaquery analysis. 1.077 Peripheral (Sigma, blood St. Louis, mononuclear MO, USA) cells gradient, (PBMC) according and bone marrow to the manufacturer’scells were purified instructions. on a Histopaque Tissue cells 1.077 were (Sigma, purified St. using Louis, a 100 MO,µm USA) cell strainer. gradient, according to the manufacturer’s instructions. Tissue cells were purified using a 100 μm cell strainer. 2.6. Hematology 2.6 Hematology Total white and red blood cell counts were measured using a Coulter Z1 (Coulter, Miami, FL, USA). OneTotal hundred-cell white and red differential blood cell counts counts were were performed measured using us aing microscope a Coulter (Olympus Z1 (Coulter, BX40 Miami, clinical FL, microscope,USA). One Center hundred Valley,‐cell PA, differential USA). counts were performed using a microscope (Olympus BX40 clinical microscope, Center Valley, PA, USA). 2.7. Flow Cytometry 2.7Percentages Flow Cytometry of PBMC and tissue cells positive for each subset examined were determined by flow cytometryPercentages using monoclonal of PBMC orand polyclonal tissue cells antibodies positive (Table for each1). Markers subset examined were selected were to determined identify the by significantflow cytometry subsets using of lymphocytes, monoclonal including or polyclonal T cells antibodies in various states(Table of 1). activation Markers and were maturation, selected to andidentify B cells the (Table significant2). Antibodies subsets were of lymphocytes, conjugated toincluding fluorochromes T cells in using various Zenon states kits, of according activation to and 5 6 manufacturer’smaturation, and instructions B cells (Table (Invitrogen, 2). Antibodies Carlsbad, were CA, USA).conjugated 2 × 10 to tofluorochromes 1 × 10 PBMCs using were Zenon blocked kits, usingaccording goat serum to manufacturer’s (MP Biomedicals, instructions Solon, OH, (Invitrogen, USA) at a Carlsbad, 1:10 dilution CA, and USA). were 2 × incubated 105 to 1 × for 10 306 PBMCs min ◦ ◦ atwere 4 C. Afterblocked washing, using thegoat cells serum were (MP incubated Biomedicals, for 30 min Solon, at 4 COH, with USA) the primary at a 1:10 antibody dilution at varyingand were dilutionsincubated (Table for1 ).30 Cells min wereat 4 °C. then After washed washing, three timesthe cells in flow were buffer incubated (phosphate for 30 buffered min at 4 saline °C with + 5% the fetalprimary bovine antibody serum) and at varying were resuspended dilutions (Table in 200 µ1).L ofCells a buffer were with then 1% washed paraformaldehyde three times in for flo fixation.w buffer Samples(phosphate were buffered analyzed saline on a DAKO + 5% fetal Cyan bovine ADP serum) (Beckton-Dickinson, and were resuspended Brea, CA, USA).in 200 μ GatesL of a were buffer set with to eliminate1% paraformaldehyde small particles, neutrophils,for fixation. andSamples eosinophils were analyzed using forward on a andDAKO side scatter.Cyan ADP A total (Beckton of at ‐ leastDickinson, 10,000 cells Brea, were CA, counted, USA). Gates and the were percentage set to eliminate of cells small that were particles, stained neutrophils, with each and antibody eosinophils was determined.using forward Gates and were side set scatter. based onA total the isotype of at least controls 10,000 (Table cells1 )were when counted, used at theand same the percentage dilution as of thecells antibody, that were such stained that 1% with or fewer each cells antibody were positive.was determined. Gates were set based on the isotype controlsList mode (Table files 1) when were analyzedused at the using same FlowJo dilution (Tree as the Star antibody, Inc., San such Carlos, that CA, 1% USA). or fewer For cells PBMC, were absolutepositive. cell counts were determined by multiplying the percent of gated cells expressing each subset by theList total mode white fi bloodles were cell analyzed count minus using the FlowJo absolute (Tree neutrophil, Star Inc., eosinophil,San Carlos, andCA, basophilUSA). For counts, PBMC, asabsolute determined cell bycounts blood were smear determined differential by counts.multiplying the percent of gated cells expressing each subset by the total white blood cell count minus the absolute neutrophil, eosinophil, and basophil counts, as determined by blood smear differential counts.

Viruses 2018, 10, 210 4 of 15

Table 1. Antibodies used for flow cytometry.

Source Cell Receptor Species Against Isotype Dilution Antibody Clone Southern Biotech CD4 Anti-feline Mouse IgG1 1:100 Clone 3-4F4 Southern Biotech CD8 Anti-feline Mouse IgG1 1:100 Clone fCD8 Serotec CD134 Anti-feline Mouse IgG1 1:10 Clone 7D6 Southern Biotech B220 Anti-mouse Rat IgG2a 1:100 Clone RA3-6B2 R&D Systems FAS Anti-feline Mouse IgG1 1:500 Clone 431014 BD Pharmingen CD21 Anti-human Mouse IgG1 1:5 Clone B-ly4 Calvin Johnson CD45RA Anti-feline Mouse IgG1 1:7 Auburn University BD Pharmingen MHC II (HLA-DR, DP, DQ) Anti-human Mouse IgG1 1:10 Clone TÜ39 Wayne Thompkins CD25 Anti-feline North Carolina Mouse IgG2a 1:20 State University Bio-Rad CD14 Anti-human Mouse IgG1 1:20 Clone TÜK4 FAS: death receptor (CD95); MHC: major histocompatibility complex; HLA-DR: Human Leukocyte Antigen-antigen D Related; DP: DP chain; DQ: DQ chain.

Table 2. Cell subsets examined in the different tissue compartments and the proposed function of each phenotype.

Cell Subsets Examined Tissue Compartments Examined Proposed Function CD4+ Blood, Thymus, MLN, PLN T cells, helper of cytotoxic CD8+ Blood, Thymus, MLN, PLN Cytotoxic T cells, NKT cells CD14+ Blood, Thymus, MLN, PLN Monocytes, Macrophages, myeloid DC CD4+ CD134+ Blood, Thymus, MLN, PLN Activated CD4+ T cells CD8+ FAS+ Blood Activated CD8+ T cells CD4+ MHC II+ Blood, Thymus, MLN, PLN Activated CD4+ T cells CD4+ CD45RA+ Blood, Thymus Naïve CD4+ T cells CD8+ CD45RA+ Blood, Thymus Naïve CD8+ T cells CD4+ CD8+ Thymus Double-positive T cells CD4− CD8− Thymus Double-negative T cells CD4+ CD25+ Blood Activated or Regulatory CD4+ T cells B220+ CD21+ Blood B cells MLN = mesenteric lymph node, PLN = peripheral lymph node.

2.8. Genomic DNA Extraction DNA was extracted from tissues using a bead-based disruption/homogenization system and the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA). Briefly, 40 mg of tissue were placed in Lysing Matrix A tubes (M.P. Biomedicals, Irvine, CA, USA) before adding 450 µL tissue lysis buffer ATL (QIAGEN) and 50 µL proteinase K (QIAGEN). Tissues were homogenized by high-speed bead disruption in the FastPrep®-24 instrument (M.P. Biomedicals) for 40 s at a speed setting of 6.0. The resulting homogenate was centrifuged at 14,000 × g for 10 min, and the supernatant was transferred Viruses 2018, 10, 210 5 of 15 to a new microcentrifuge tube. DNA was extracted as per the manufacturer’s instructions. DNA was ◦ eluted with 100 µLH2O and stored at −20 C until use. DNA was extracted from 1 million PBMCs using the Qiamp blood mini DNA kit (QIAGEN). ◦ DNA from each sample was eluted with 50–100 µL of H2O and stored at −20 C until use.

2.9. RNA Extraction & cDNA Synthesis RNA was extracted from tissues using the FastRNA pro-green kit (M.P. Biomedicals, Irvine, CA, USA) with FastPrep®-24 homogenizer (M.P. Biomedicals), following the manufacturer’s protocol. Briefly, 100 mg of tissue was homogenized in RNApro™ Solution and Lysing Matrix D using the FastPrep®-24 instrument for 40 s at a setting of 6.0. RNA was then purified according to the ◦ manufacturer’s instructions, resuspended in 100 µL RNase-free H2O and stored at −80 C. Cellular RNA was then converted to cDNA using random primers and Superscript II (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was extracted from 2 million bone marrow cells were resuspended in 0.5mL Trizol (Sigma, St. Louis, MO, USA) at 4 million cells/mL. RNA was purified by phenol:chloroform extraction and ethanol precipitation. Viral RNA was extracted from plasma using the QIAamp viral RNA kit (QIAGEN, Valencia, CA, USA) spin protocol.

2.10. Quantitation of Proviral Load, mRNA Viral Load, & Cytokine Transcripts by Real-Time qPCR Briefly, 5 µL of tissue extracted DNA was quantitated by comparison to standard curves that were generated using plasmids containing the FIV-C gag or the PLV pol. The number of cell equivalents for each DNA sample was determined, as described by Terwee et al. [7]. FIV and PLV mRNA was quantified in tissues by qPCR using the previously described FIV-C gag and the PLV pol assays [13,14]. While these assays target two different lentiviral genes, the abundance of gag and pol-containing mRNAs during feline lentiviral infection are similar, suggesting that these assays are reasonable for comparing viral mRNA levels of FIV and PLV. [15] To allow for the accurate comparison between samples, viral mRNA expression was normalized to mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample using the 2−∆Ct method, in which ∆Ct is the cycle at which the threshold is reached for GAPDH that was subtracted from the cycle threshold for viral mRNA. Conversion of ∆Ct to 2−∆Ct produced a value that indicates the fold abundance of viral mRNA relative to that of GAPDH mRNA. Cytokine mRNA expression was quantitated by qPCR, as previously described [7]. Expression of IL-10, IL12p40, and IFNγ mRNA was quantitated relative to GAPDH mRNA expression using this the 2−∆Ct method. All of the qPCR assays in the study were performed in triplicate, and qPCR efficiencies were within the accepted range of 90–110%.

2.11. Statistical Analyses To evaluate the effects of infection, time, and the interaction on the response variables, we utilized repeated measures ANOVA after the log transformation of the data. To achieve this, we split the experiment into a PLV group (at week 0), a CO group, and a SHAM group (week 0 to week 4) and analyzed these independently. Analyses were undertaken using the program R (http://www.r-project. org/). Individual time point differences between the FIV and CO groups in blood and tissues were evaluated using the Student’s t-test. Linear regression was used to predict the effect of previous PLV infection on decreased FIV proviral and viral loads in the CO group. T-tests and linear regression analyses were analyzed with PRISM software (GraphPad Software, LaJolla, CA, USA). For all of the analyses, a P < 0.05 was considered to be statistically significant. Viruses 20182018,, 1010,, 210x FOR PEER REVIEW 6 of 15

3. Results 3. Results

3.1. Prior PLV InfectionInfection ResultsResults in in Lower Lower FIV FIV Proviral Proviral Loads Loads in in PBMC, PBMC, Bone Bone Marrow, Marrow, and and LNs, LNs, and and Decreased PlasmaDecreased Viremia Plasma Viremia FIV proviralproviral loadsloads were were significantly significantly lower lower at at three three and and four four weeks, weeks, and and FIV FIV plasma plasma viremia viremia was significantlywas significantly lower lower at two at andtwo fourand weeksfour weeks FIV PI FIV in PI CO in versus CO versus FIV cats FIV (Figure cats (F2igureA,B; 2A,B;p-values p‐values < 0.05). < FIV0.05). proviral FIV proviral loads loads were trendingwere trending lower lower in the in bone the bone marrow marrow of the of CO the versus CO versus FIV catsFIV atcats two at andtwo fourand four weeks weeks FIV PIFIV (P PI= 0.084(P = 0.084 and Pand= 0.196, P = 0.196, respectively; respectively; Figure Figure2C). Decreased 2C). Decreased proviral proviral loads wereloads alsowere noted also noted in the in mesenteric the mesenteric and prescapular and prescap lymphular lymph nodes nodes four weeksfour weeks FIV PIFIV in PI CO in versus CO versus FIV catsFIV (Figurecats (Figure2D, E). 2D, Viral E). Viral load differencesload differences were notwere found not found in the in thymus the thymus (Supplementary (Supplementary Figure Figure S1). In S1). all ofIn theall of tissues, the tissues, the range the range of viral of loads viral loads was narrower was narrower in CO in than CO FIV than cats. FIV cats.

Figure 2. Feline immunodeficiencyimmunodeficiency virus (FIV) proviral loads ( A) and FIV plasma viremia (B) were statistically decreased decreased at at various various times times post post FIV FIV infection infection (FIV (FIV PI) PI) in inCO CO versus versus FIV FIV cats. cats. At 3 At(P 3= (0.016)P = 0.016) and 4 and (P = 4 0.049) (P = 0.049) weeks weeks FIV PI FIV for PI FIV for proviral FIV proviral loads, loads, and at and 2 (P at = 0.0 2 (P48)= 0.048)and 4 ( andP = 0.011) 4 (P = weeks 0.011) weeksFIV PI FIVfor plasma PI for plasma viremia. viremia. However, However, no statistical no statistical differences differences were werefound found by group by group and time and time(P = (0.190).P = 0.190). Bone Bone marrow marrow proviral proviral loads loads (C) ( Cwere) were not not statistically statistically different different by by group group and and time time ( (PP == 0.182), 0.182), but a trending difference was noted betweenbetween CO and FIV cats at 2 weeks FIV PI ((P == 0.084) and no difference was found at 4 weeksweeks FIVFIV PIPI ((PP == 0.196). Proviral loads in mesenteric (P = 0.0367) (D) and prescapular (P = 0.0152) ( E) lymph nodes were lower in the CO as com comparedpared with FIV cats at 4 weeks FIV PI. Bars indicate statistical differences ((PP <0.05)<0.05) between groups.

+ 3.2. Global and Naïve CD4 + TT Cells Cells are are Preserved Preserved by by Co Co-Infection‐Infection Compared Compared to FIV Infection Infection

+ Global CD4+ cellscells were were increased increased in the CO when compared to the FIV cats at 2 and 4 weeks FIV + + + PI (P < 0.05, Figure3 3A).A). CD4 CD4+ subset preservation waswas documenteddocumented inin thethe naïve naïve CD4 CD4+CD45RACD45RA+ cell compartment at 2 weeks FIV PI in the CO verses FIV cats, but not to the same extent as the global + + + CD4+ cellscells (Figure 33B,B, P = 0.001).CD4+CD45RACD45RA+ cellscells in in the the CO CO cats cats decreased decreased at four weeks; and therefore, no longerlonger differed betweenbetween thethe groupsgroups ((PP == 0.332).

Viruses 2018, 10, 210 7 of 15 Viruses 2018, 10, x FOR PEER REVIEW 7 of 15

Figure 3. CD4CD4+ +cellscells were were preserved preserved in inthe the blood blood of CO of CO as compared as compared with with FIV FIVcats catsat 2 atweeks 2 weeks (P = (0.0492),P = 0.0492), and approached and approached preservation preservation at 4 weeks at 4 weeks (P = 0.0612) (P = 0.0612) FIV PI. FIV At 2 PI. and At 4 2 weeks and 4 FIV weeks PI, control FIV PI, + controlSHAM SHAMcats had cats higher had higherCD4+ cells CD4 thancells FIV than cats FIV (P cats = 0.002 (P = and 0.002 P and < 0.0001,P < 0.0001, respectively), respectively), and CO and cats CO + + cats(2 weeks, (2 weeks, P = 0.0090;P = 0.0090; 4 weeks, 4 weeks, P = 0.0034)P = 0.0034) (A). Naïve (A). Naïve CD4+ CD4CD45RACD45RA+ cells werecells also were preserved also preserved in the inCO the cats CO compared cats compared with FIV with cats FIV at 2 cats weeks at 2 ( weeksP = 0.0012), (P = 0.0012),but not at but 4 weeks not at 4(P weeks = 0.332) (P FIV= 0.332) PI. At FIV 2 and PI. + + At4 weeks 2 and FIV 4 weeks PI, SHAM FIV PI, CD4 SHAM+CD45RA CD4 +CD45RA cells werecells greater were than greater FIV cats than (2 FIV weeks, cats (2 P weeks,= 0.0069;P 4= weeks, 0.0069; 4P weeks,= 0.0012),P = yet 0.0012), the SHAM yet the was SHAM only was different only different than the than CO thecats CO at 4 cats weeks at 4 weeksFIV‐PI FIV-PI (P = 0.0062) (P = 0.0062) CD4+ + CD4cells (cellsB). Bars (B). Barsindicate indicate statistical statistical differences differences between between the the indicated indicated groups. groups. Significant Significant differences + + + were notednoted amongamong the the three three groups groups over over time time (CD4 (CD4+cells, cells,P P= = 0.0004; 0.0004; CD4 CD4+CD45RACD45RA+ cells, P = 0.0012).

3.3. Prior PLV Infection Resulted in Greater CD8 + Cell,Cell, CD8 CD8++FASFAS++ cell,cell, and and large large granular granular lymphocytes lymphocytes Numbers During Early FIV Infection Compared With SHAM AnimalsAnimals + Global CD8 + cellscells (Figure (Figure 4A)4A) were were higher higher in in the the CO CO cats cats as as compared compared with with the the FIV FIV cats cats at at 0, 0,1, + 1,and and 2 weeks 2 weeks FIV FIV PI PI(p‐ (valuesp-values <0.05); <0.05); however, however, by by4 weeks 4 weeks PI all PI of all the of thegroups groups had had similar similar CD8 CD8+ cell + cellnumbers. numbers. CD8+ CD8 cells incells the inCO the and CO SHAM and SHAM cats were cats similar were at similar all time at allpoints time (0–4 points weeks), (0–4 whereas weeks), whereasthe FIV cats the had FIV decreased cats had decreased levels compared levels compared with SHAM with cats SHAM at 0, 1, cats and at 2 0, weeks 1, and FIV 2 weeks PI (p < FIV 0.05). PI + + (CD8p < 0.05).+FAS+ CD8cell numbersFAS cell were numbers greater were in the greater CO than in the FIV CO cats than at 0, FIV 1, catsand at2 weeks 0, 1, and FIV 2 PI weeks (p‐values FIV PI < + (0.05),p-values and < this 0.05), difference and this was difference more pronounced was more pronounced than that seen than in that the seenglobal in CD8 the global+ cell population CD8 cell + + + + population(Figure 4B). (FigureBy four4 weeksB). By fourFIV PI, weeks CD8 FIV+FAS PI,+ cells CD8 in theFAS FIVcells cats in increased. the FIV cats CD8 increased.+FAS+ cells CD8 in theFAS CO cellscats were in the higher CO cats than were the higherSHAM than cats at the all SHAM of the catstime at points all of (0–4 the timeweeks, points P <0.05), (0–4 and weeks, the PFIV<0.05), cats andhad higher the FIV levels cats hadas compared higher levels with asSHAM compared cats at with 2 and SHAM 4 weeks cats FIV at PI 2 ( andP <0.05). 4 weeks As previously FIV PI (P <0.05). noted + + As[13], previously large granular noted [lymphocyte13], large granular (LGL)numbers lymphocyte mirrored (LGL)numbers the CD8+ mirroredFAS+ cell the populations, CD8 FAS andcell populations,significant differences and significant were differencestrending at wereone week trending (P = at 0.074) one week and were (P = 0.074)significant and were at two significant weeks FIV at twoPI between weeks FIVthe PICO between and FIV the cats CO (P and = 0.002; FIV catsFigure (P =4C). 0.002; LGLs Figure were4C). higher LGLs in were the CO higher versus in the SHAM CO versuscats at SHAM2, 3 and cats 4 FIV at 2,PI 3 (P and <0.05). 4 FIV However, PI (P <0.05). no significance However, no was significance found in wasLGL found numbers in LGL between numbers the betweenFIV and the the SHAM FIV and cats the at SHAM any time cats point. at any time point.

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FigureFigure 4. 4. CD8CD8+ +cellscells are are higher higher in in the the blood blood of of CO CO compared compared with with FIV FIV cats cats at at 0, 0, 1 1 and and 2 2 weeks weeks FIV FIV-PI‐PI ((PP == 0.0015, 0.0015, PP == 0.045, 0.045, PP == 0.011, 0.011, respectively). respectively). CD8 CD8+ +cellscells in in CO CO cats cats are are similar similar to to SHAM SHAM cats cats at at all all timetime points points (P (P >> 0.05), 0.05), whereas whereas FIV FIV cats cats had had decreased decreased levels levels compared compared with with SHAM SHAM cats cats at at0, 0,1 and 1 and 2 weeks2 weeks FIV FIV PI ( PIP = ( P0.005,= 0.005, 0.0136, 0.0136, and 0.003, and 0.003, respectively) respectively) (A). CD8 (A).+FAS CD8++ cellsFAS +incells the CO in the cats CO were cats higher were thanhigher SHAM than cats SHAM at all cats of the at all time of points the time (0 pointsweek, P (0 = week,0.00098;P =1 week, 0.00098; P = 1 0.0198; week, P2 weeks,= 0.0198; P = 2 0.0292; weeks, 4P weeks,= 0.0292; P = 4 0.00135), weeks, P =and 0.00135), higher and in FIV higher as compared in FIV as compared with SHAM with cats SHAM at 2 and cats 4 at weeks 2 and 4FIV weeks PI (P FIV = 0.02577,PI (P = 0.02577,0.00088, respectively) 0.00088, respectively) (B). Large ( Bgranular). Large lymphocyte granular lymphocyte (LGL) numbers (LGL) mirrored numbers the mirrored CD8+FAS the+ cellCD8 populations,+FAS+ cell populations, and differences and differenceswere trending were at trending one week at one (P = week 0.074) (P and= 0.074) were and significant were significant at two weeksat two ( weeksP = 0.002) (P = FIV 0.002) PI FIVbetween PI between CO and CO FIV and cats. FIV Significant cats. Significant differences differences in LGLs in LGLs were were trending trending at oneat one week week (P = ( P0.074)= 0.074) and andwere were significant significant at two at twoweeks weeks FIV PI FIV in PI between in between the CO the and CO FI andV cats FIV ( catsP = 0.002).(P = 0.002). LGLs LGLswere higher were higher in the in CO the than CO SHAM than SHAM cats at cats 2, 3, at and 2, 3, 4 and FIV 4 PI FIV (P PI= 0.0076, (P = 0.0076, 0.0059, 0.0059, and 0.0044,and 0.0044, respectively). respectively). However, However, no significant no significant difference difference was found was in found LGL numbers in LGL numbers between between the FIV andthe sham FIV and inoculations sham inoculations of media (SHAM) of media cats (SHAM) at any cats time at point any ( timeC). Bars point ind (Cicate). Bars statistical indicate differences statistical betweendifferences the betweenindicated the groups. indicated Significant groups. differences Significant were differences also noted were among also noted the three among groups the threeover + + + timegroups (CD8 over+ cells, time P (CD8 = 0.0005;cells, CD8P +=FAS 0.0005;+ cells, CD8 P = FAS0.0004;cells, LGLs,P = P 0.0004; = 0.003). LGLs, P = 0.003).

3.4.3.4. Cytokine Cytokine Differences Differences in in PBMC PBMC Between Between Co Co-Infected‐Infected and and FIV FIV-Infected‐Infected Cats Cats FIV FIV PI PI PBMCPBMC IFN IFN-‐γ γexpressionexpression was was significantly significantly higher higher in in CO CO than than FIV FIV cats cats at 0, at 2, 0, and 2, and 3 weeks 3 weeks FIV FIV PI (PIp‐values (p-values < 0.05; < 0.05; Figure Figure 5A),5 despiteA), despite no overall no overall differences differences among among the groups the groups over time. over IL time.‐10 mRNA IL-10 expressionmRNA expression was significantly was significantly different different among amongall groups all groupsover time, over and time, significantly and significantly higher higherIL‐10 expressionIL-10 expression was appreciated was appreciated at 0, 2, at and 0, 2, 3 andweeks 3 weeks (p‐values (p-values < 0.05) < in 0.05) CO in as CO compared as compared with withFIV cats FIV (Figurecats (Figure 5B). 5NoB). Nodifferences differences were were seen seen among among groups groups for for IL IL-12‐12 mRNA mRNA expression expression (Supplementary (Supplementary FigureFigure S2). S2).

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Figure 5. Peripheral blood mononuclear cell (PBMC) interferon gamma (IFN‐γ) expression was Figure 5. Peripheral blood mononuclear cell (PBMC) interferon gamma (IFN-γ) expression was higher higher in the CO as compared to FIV cats at 0, 2 and 3 weeks FIV PI (p‐values <0.05) (A), while no in the COdifferences as compared were found to FIV among cats the at 0,groups 2 and over 3 weeks time (P FIV = 0.2269). PI (p -valuesPBMC IL <0.05)A‐10 mRN (A), expression while no was differences were foundappreciated among at the 0, 2, groups and 3 weeks over timeFIV PI (P in= the 0.2269). CO when PBMC compared IL-10 with mRNA FIV expressioncats (P = 0.003), was 2 appreciated(P = at 0, 2, and0.044),Figure 3 weeks and5. Peripheral 3 (P FIV = 0.005) PI blood in Significant the mononuclear CO when differences comparedcell (PBMC) were noted withinterferon for FIV IL cats‐gamma10 expression (P =(IFN 0.003),‐γ among) expression 2 (P the= 0.044),three was and 3 (P = 0.005)groupshigher Significant overin the time CO differences( Pas = compared 0.0001) (B were ).to Bars FIV noted indicatscate at for 0, statistical 2 IL-10 and 3 expression weeksdifferences FIV PIbetween among (p‐values the the <0.05)CO three and (A groupsFIV), while cats. over no time (P = 0.0001)differences (B). Bars were indicate found among statistical the groups differences over time between (P = 0.2269). the PBMC CO and ILA‐10 FIV mRN cats. expression was 3.5. Tissueappreciated Lymphocyte at 0, 2, Phenotypes and 3 weeks Differ FIV BetweenPI in the CoCO‐Infected when compared and FIV‐ Infectedwith FIV Cats cats (P = 0.003), 2 (P = 0.044), and 3 (P = 0.005) Significant differences were noted for IL‐10 expression among the three 3.5. Tissue3.5.1. Lymphocyte Thymus Phenotypes Differ Between Co-Infected and FIV-Infected Cats groups over time (P = 0.0001) (B). Bars indicate statistical differences between the CO and FIV cats. + + 3.5.1. ThymusAt 4 weeks FIV PI, Thymic CD4 CD45RA cell percentages among live cells were trending lower in3.5. the Tissue CO when Lymphocyte compared Phenotypes to FIV Differcats (P Between = 0.059, Co Figure‐Infected 6A), and and FIV both‐Infected the FIV Cats and CO cats (p‐values At< 4 0.05) weeks had FIVhigher PI, CD4 Thymic+CD45RA CD4+ cell+ CD45RApercentages+ cellthan percentagesSHAM cats. CD4 among+CD8+ double live cells positive were cell trending 3.5.1. Thymus lower inpercentages the CO when in the CO compared cats were toalso FIV trending cats (higherP = 0.059, as compared Figure with6A), the and FIV both cats (P the = 0.071, FIV Figure and CO cats 6B), and both the FIV (P = 0.004) and+ the CO (+P = 0.0165) cats had lower CD4+CD8+ double positive (p-values

Figure 6. Thymic CD4+CD45RA+ cell percentages were trending lower in the CO compared to FIV cats (P = 0.059), and both the FIV (P <0.0001) and CO cats (P = 0.0014) had higher CD4+CD45RA+ live cell percentages than the SHAM cats at 4 weeks FIV PI (A). CD4+CD8+ double positive cell percentages in the CO cats were trending higher when compared with those in the FIV cats (P = 0.071), and both the FIV (P = 0.004) and CO (P = 0.0165) cats had lower CD4+CD8+ double positive cells compared with Figure 6.FigureThymic 6. Thymic CD4+ CD4CD45RA+CD45RA+ cell+ cell percentages percentages were trending trending lower lower in the in CO the compared CO compared to FIV cats to FIV cats SHAM cats (B). Bars indicate statistical differences between the indicated groups. + + (P = 0.059),(P = and0.059), both and theboth FIV the (FIVP <0.0001) (P <0.0001) and and CO CO cats cats ((PP = 0.0014) 0.0014) had had higher higher CD4 CD4CD45RA+CD45RA live cell+ live cell percentages than the SHAM cats at 4 weeks FIV PI (A). CD4+CD8+ double positive cell percentages in percentages than the SHAM cats at 4 weeks FIV PI (A). CD4+CD8+ double positive cell percentages in 3.5.2. thePrescapular CO cats were Lymph trending Node higher when compared with those in the FIV cats (P = 0.071), and both the the CO cats were trending higher when compared with those+ + in the FIV cats (P = 0.071), and both the AtFIV 4 (weeksP = 0.004) FIV and PI, CO and (P similar= 0.0165) to cats observations had lower CD4in theCD8 peripheral double positive blood, cellsCD4 compared+ cell percentages with + + FIVwere (P = SHAMsignificantly 0.004) cats and (B ). COpreserved Bars (P indicate= 0.0165) in prescapularstatistical cats had differences lymph lower betweennodes CD4 CD8(PLNs) the indicateddouble in CO groups. when positive compared cells compared to FIV cats with SHAM(P = 0.0475, cats (B ).Figure Bars indicate7A). While statistical the CD4 differences+ cell percentages between in the the CO indicated cats were groups. not different from the SHAM3.5.2. Prescapular cats (P > 0.05), Lymph the FIV Node cats were different (P < 0.05). While CD8+ cell percentages did not vary 3.5.2. PrescapularamongAt groups 4 weeks Lymph (data FIV not NodePI, shown),and similar the CD4/CD8to observations ratio differencesin the peripheral were significantly blood, CD4 +lower cell percentages in the FIV whenwere significantlycompared with preserved CO cats in (P prescapular = 0.020, Figure lymph 7B), nodes presumably (PLNs) duein CO to whenthe differences compared in to the FIV CD4 cats+ + Atcells( 4P weeks= between0.0475, FIV Figure the PI, groups. 7A). and While similar the to CD4 observations+ cell percentages in the in the peripheral CO cats were blood, not different CD4 cell from percentages the were significantly SHAM cats ( preservedP > 0.05), the in FIV prescapular cats were different lymph (P nodes< 0.05). While (PLNs) CD8 in+ COcell percentages when compared did not vary to FIV cats (P = 0.0475,among Figure groups7A). (data While not shown), the CD4 the +CD4/CD8cell percentages ratio differences in the were CO significantly cats were not lower different in the FIV from the SHAM catswhen (P compared> 0.05), thewith FIV CO cats ( wereP = 0.020, different Figure ( 7B),P < presumably 0.05). While due CD8 to the+ cell differences percentages in the didCD4 not+ vary among groupscells between (data the not groups. shown), the CD4/CD8 ratio differences were significantly lower in the FIV when compared with CO cats (P = 0.020, Figure7B), presumably due to the differences in the CD4 + cells between the groups. Viruses 2018, 10, 210 10 of 15 Viruses 2018, 10, x FOR PEER REVIEW 10 of 15

Viruses 2018, 10, x FOR PEER REVIEW 10 of 15

FigureFigure 7. Prescapular 7. Prescapular lymph lymph node node CD4 CD4++ livelive cell cell percentages percentages ((A (() PA =) 0.0475)P = 0.0475) and CD4/CD8 and CD4/CD8 ratios ((B ratios) P = 0.02) were higher in CO when compared to FIV cats at 4 weeks FIV PI. CD4+ live cell percentages ((B) P = 0.02) were higher in CO when compared to FIV cats at 4 weeks FIV PI. CD4+ live cell percentages and CD4/CD8 ratios were also higher in the SHAM compared to FIV cats (CD4+: P = 0.008; CD4/CD8 and CD4/CD8 ratios were also higher in the SHAM compared to FIV cats (CD4+: P = 0.008; CD4/CD8 Figureratio: P 7. = Prescapular0.0011), but lymphnot between node CD4the SHAM+ live cell and percentages the CO cats. ((A ) P = 0.0475) and CD4/CD8 ratios ((B) ratio:PP == 0.02) 0.0011), were buthigher not in between CO when the compared SHAM to and FIV the cats CO at cats.4 weeks FIV PI. CD4+ live cell percentages 3.6. PLVand CD4/CD8Infection Altersratios werethe Immune also higher Landscape in the SHAM at the Timecompared of FIV to Inoculation FIV cats (CD4 +: P = 0.008; CD4/CD8 3.6. PLV Infectionratio: P = 0.0011), Alters thebut not Immune between Landscape the SHAM at and the the Time CO ofcats. FIV Inoculation To understand the possible reasons for attenuation of FIV proviral and the viral loads in CO To3.6.versus understand PLV FIV Infection cats, theweAlters evaluated possible the Immune reasonsblood Landscape cell for and attenuationat cytokinethe Time ofdifferences FIV of FIV Inoculation proviral between and the groups the viral four loads weeks in CO versusafter FIV PLV cats, inoculation we evaluated (at week blood 0 FIV cell ).PI andAfter cytokine four weeks differences of PLV infection, between the cats groups in the four PLV weeks group after To understand the possible reasons for attenuation of FIV proviral and the viral loads in CO PLV inoculationhad decreased (at B220 week+CD21 0 FIV+ B PI). cells After (Figure four 8A, weeks P = 0.026) of PLV and infection, CD4+MHC the II+ cats (Figure in the 8B, PLV P = 0.0287) group had versus FIV cats, we evaluated blood cell and cytokine differences between the groups four weeks cells, and increased+ + CD8+FAS+ (Figure 8C, P < 0.0001) and CD4+CD134+ + cells+ as compared with SHAM decreasedafter PLV B220 inoculationCD21 B (at cells week (Figure 0 FIV ).8PI A,AfterP = 0.026)four weeks and of CD4 PLVMHC infection, II (Figurethe cats 8inB, theP PLV= 0.0287) group cells, cats (Figure 8D,+ P = +0.0206). IL‐10 (Figure 8E, P = 0.0001) +expression+ was also increased in PBMC at and increased CD8 FAS+ (Figure+ 8C, P < 0.0001) and CD4 CD134 +cells as+ compared with SHAM cats hadthis timedecreased point. B220 CD21 B cells (Figure 8A, P = 0.026) and CD4 MHC II (Figure 8B, P = 0.0287) (Figurecells,8D, andP =increased 0.0206). CD8 IL-10+FAS (Figure+ (Figure8E, 8C,P P= < 0.0001)0.0001) and expression CD4+CD134 was+ cells also as increased compared inwith PBMC SHAM at this time point.cats (Figure 8D, P = 0.0206). IL‐10 (Figure 8E, P = 0.0001) expression was also increased in PBMC at this time point.

Figure 8. Significantly different blood cells and cytokines are seen between Puma lentivirus (PLV)‐ infected (PLV) and control cats (SHAM) four weeks after PLV inoculation (at week 0 FIV PI). B220+

CD21+ cells (A) and CD4+MHC II+ cells (B) were decreased in the PLV cats compared to the SHAM Figure 8. +Significantly+ different blood+ cells+ and cytokines are seen between Puma lentivirus (PLV)‐ Figurecats. 8. CD8SignificantlyFAS cells (C different) and CD4 bloodCD134 cellscells (D and) were cytokines increased are in the seen PLV between cats when Puma compared lentivirus to infected (PLV) and control cats (SHAM) four weeks after PLV inoculation (at week 0 FIV PI). B220+ (PLV)-infectedSHAM cats. (PLV) PBMC and IL‐10 control mRNA expression cats (SHAM) (E) was four statistically weeks after increased PLV in inoculation PLV compared (at to week SHAM 0 FIV CD21cats. + cells (A) and CD4+MHC II+ cells (B) were decreased in the PLV cats compared to the SHAM PI). B220+ CD21+ cells (A) and CD4+MHC II+ cells (B) were decreased in the PLV cats compared cats. CD8+FAS+ cells (C) and CD4+CD134+ cells (D) were increased in the PLV cats when compared to + + + + to theSHAM SHAM cats. cats. PBMC CD8 IL‐10FAS mRNAcells expression (C) and (E CD4) wasCD134 statisticallycells increased (D) were in PLV increased compared in to the SHAM PLV cats whencats. compared to SHAM cats. PBMC IL-10 mRNA expression (E) was statistically increased in PLV compared to SHAM cats.

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3.7. Significant Differences Between Co- and FIV-Infected Cats at the Time of FIV Exposure Predict Future OutcomesViruses of 2018 FIV, 10 Infection, x FOR PEER REVIEW 11 of 15

We3.7. performedSignificant Differences linear regression Between Co analysis‐ and FIV‐ toInfected determine Cats at ifthe the Time cell of phenotypesFIV Exposure Predict and cytokines Future that we foundOutcomes to be of differentFIV Infection between the PLV cats (CO) and SHAM cats at the time of FIV inoculation (week zero)We correlated performed with linear the regression immune analysis or viral to determine attributes if of the FIV cell infection. phenotypes Among and cytokines the cells that that we were + + differentfound between to be different FIV and between CO cats the PLV at time cats (CO) zero, and we SHAM found cats that at higher the time CD8 of FIVFAS inoculat(Figureion (week9A) and CD4+zero)CD134 correlated+ (Figure with9B) the cell immune numbers or viral in PLV-infected attributes of FIV cats infection. at time Among 0 could the predict cells that lower were FIV different proviral loadsbetween in blood FIV at oneand weekCO cats FIV at time PI. We zero, also we found found that that higherhigher CD8 B220+FAS+CD21+ (Figure+ (Figure 9A) and9C) CD4 cell+ numbersCD134+ at week(Figure zero could 9B) cell predict numbers lower in PLV FIV‐infected plasma cats viremia at time at0 could one weekpredict FIV lower PI FIV and proviral higher loads IL-10 in expression blood couldat predict one week lower FIV FIVPI. We proviral also found loads that in higher blood B220 two+ weeksCD21+ (Figure FIV PI 9C) (Figure cell numbers9D). Cell at phenotypes week zero could at week 0 werepredict not predictive lower FIV plasma of viral viremia loads, at viremia, one week or FIV circulating PI and higher immunocyte IL‐10 expression populations could predict at four lower weeks FIV proviral loads in blood two weeks FIV PI (Figure 9D). Cell phenotypes at week 0 were not FIV PI. predictive of viral loads, viremia, or circulating immunocyte populations at four weeks FIV PI.

FigureFigure 9. Linear 9. Linear regression regression analysis analysis to to determine determine cellcell phenotypes and and cytokine cytokine expression expression that thatwas was different between the PLV (CO) and SHAM cats at week zero or time of FIV inoculation could predict different between the PLV (CO) and SHAM cats at week zero or time of FIV inoculation could predict future outcomes of FIV infection in CO cats. Higher CD8+FAS+ (A) and CD4+CD134+ (B) cell numbers future outcomes of FIV infection in CO cats. Higher CD8+FAS+ (A) and CD4+CD134+ (B) cell numbers in the blood of CO cats (those previously infected with PLV) at week 0 could predict lower FIV in theproviral blood of loads CO catsin blood (those at one previously week FIV infected‐PI, and withthat higher PLV) atB220 week+CD21 0 could+ (C) cell predict numbers lower in blood FIV proviral at + + loadsweek in blood zero atcould one weekpredict FIV-PI, lower andFIV plasma that higher viremia B220 at CD21one week(C )FIV cell PI. numbers Higher inIL‐ blood10 mRNA at week zero couldexpression predict in PLV lower‐cats FIV at week plasma 0 predicted viremia lower at one FIV week proviral FIV loads PI. Higher at two weeks IL-10 mRNAFIV PI (D expression). in PLV-cats at week 0 predicted lower FIV proviral loads at two weeks FIV PI (D).

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4. Discussion In previous studies, we documented that prior exposure to apathogenic PLV infection resulted in the preservation of CD4+ T cells and increased PBMC IFN-γ mRNA expression when compared with cats that were infected solely with FIV [7]. Increased pro-inflammatory cells and mediators [10], and attenuated proviral loads in the CO vs. FIV cats [7] were seen three weeks FIV PI. However, adaptive immune responses raised against PLV were not implicated in these differences, and G to A substitutions, due to cytidine deaminase activity, were also not found to be responsible for the bottleneck of FIV infection three weeks post-PLV infection [11,12]. The current study was designed to identify immunologic phenotype variations between CO and FIV cats during acute viral replication and to gain a better understanding of the potential mechanisms underlying the differences between these two scenarios. We again noted a blunting of FIV viral loads in blood and peripheral tissues and a dampening of CD4 depletion when PLV infection preceded FIV challenge. We hypothesized this could be due to changes in the susceptible target cell population at the time of FIV challenge in PLV versus naïve cats, and/or this impact could be due to the immunological state induced by PLV infection. While we found evidence of peripheral cell subset changes on the day of challenge, the preponderance of evidence suggests that the innate immune landscape at the time of challenge contributed to different infection outcomes. Circulating CD4+CD134+ cells (activated CD4 T lymphocytes) and B cells varied between PLV and SHAM cats at the time of FIV challenge. CD4+CD134+ cells were higher in PLV versus SHAM cats at the time of challenge (Figure8), a finding that correlated with lower FIV viral loads one week FIV PI (Figure9). While CD4 +CD134+ cells represent the primary cell type infected during acute FIV infection [7,16,17], the expansion of this population during PLV infection did not result in higher viral loads following FIV exposure. It is possible that this subset of activated CD4+ lymphocytes possessed anti-viral activity that contributed to a lower viral setpoint [18]. PLV-infected cats had statistically lower numbers of B cells in circulation on the day of FIV challenge than the SHAM cats. It is possible PLV triggered B cell apoptosis or B cell shifting from peripheral to central sites. FIV has been shown to infect CD21+ B cells, so lower circulating B cells in PLV could result in the decreased susceptibility to FIV challenge, though B cell tropism is typically considered to occur late in FIV infection [19]. CD4+CD45RA+ cells rapidly decreased in the FIV cats by two weeks FIV PI, but transient preservation was seen in CO cats. These findings might be explained by the fact that CD4+CD45RA+ cells are preferentially infected during acute FIV infection that is similar to acute syncytium-inducing variants of HIV infection [20,21]. None of the CD4+ cell subsets that were examined in this study (CD4+CD45RA+, CD4+CD134+, and CD4+ MHCII) could be attributed to global CD4+ maintenance in CO cats. Thymic CD4+CD45RA+ cells trended lower in the CO versus FIV cats. Peripheral naïve T cells were shown to be replenished by naïve thymic T cells in mice [22], and similar mechanisms may underlie our observations in this system. The lower thymic double positive cell percentages in the CO versus FIV cats might indicate a greater infection in the thymus of FIV compared with CO cats, but this did not correlate with our findings of no difference in proviral loads between the two groups. By 26 days post simian/human immunodeficiency virus, infection in rhesus macaques marked decreases in double positive cells were seen in the thymus [23]. However, differences could be due to differential changes in other thymic cell types. CD8+FAS+ T cells were increased at the time of FIV inoculation in PLV when compared to SHAM cats, and this increase is inversely correlated with viral loads at one week PI (Figure9A) suggesting an acute impact of these cells on the establishment of viral infection. We previously determined that CD8+FAS+ cells were analogous to LGLs in FIV and that these cells correlated with dampening of FIV viremia [24,25]. We believe that these cells are analogous to anti-viral CD8+ T cells in HIV-infected individuals [26] and CD8+ T cells in SIV-infected long-term non-progressing (LTNP) rhesus macaques [27]. Our findings bolster the conclusion that CD8+ T cells represent an innate immune cell type [27]. Viruses 2018, 10, 210 13 of 15

IL-10 expression at the time of FIV infection was greater in the PLV when compared to SHAM cats (Figure9E), and the predicted suppression of proviral loads two weeks FIV PI in the CO cats. IL-10 is a complex cytokine that is induced during acute HIV infection and has potent anti-inflammatory properties in chronic HIV [28]. Higher IFN-γ expression was identified in CO when compared to FIV cats at several points post-FIV infection. Activated CD8+ T cells, CD4+ T cells, NK cells, and CD8+FAS+ cells primarily produce IFN-γ during HIV-1 and FIV infection [29–33]. This type II interferon has been associated with variable expression during acute and chronic HIV infection, and has not been correlated with a significant control of disease [34]. In SIV-infected sooty mangabey monkeys (SMM), IFN-γ elevation was more transient, and IL-10 was more prominent during acute control of SIV infection compared SIV infected macaques that did not control infection [35]. Although we were unable to detect differences between CD4+CD25+ between FIV and CO cats in this study, in comparison to SMM infection indicates that IL-10 expression during acute infection may be more relevant to subsequent viral control than IFN-γ. Similarly, regulatory T cell-induced IL-10 expression was shown to be responsible for decreased IFN-γ expression following the stimulation of CD4+ and CD8+ HIV-1-specific T cell immune responses of HIV-1-exposed-uninfected infants [36]. Overall, these findings show a complex interplay of cytokines that vary in intensity and at the stage of lentivirus infection, providing evidence that innate immune processes are associated with the early suppression of viral replication during co-infection. This study reinforced previous observations that prior exposure to an apathogenic lentivirus infection can diminish the effects of acute infection with a second, more virulent, viral exposure. In addition, it investigated immunocyte phenotypes and cytokines that could distinguish the characteristics between PLV/FIV and FIV during the acute phases of the infection. Overall, circulating blood cell types at the time of infection are less relevant to FIV susceptibility than the inflammatory immune environment that is induced by prior lentiviral exposure. Additionally, as virus attenuation in HIV and SIV long-term non-progression is at least partly due to innate viral immunity [27,37], we have also identified non-virus-specific entities at play at the time of FIV inoculation in PLV-infected cats. These innate immune parameters (i.e., cytokines, LGLs/CD8+FAS+ cells) that are present during the first week of infection are key to defining differences in infection outcomes and could be used in therapeutic interventions to reduce disease severity in susceptible populations. Follow-up studies will evaluate non-infectious means to produce these biologically relevant and resistance phenotypes.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1999-4915/10/4/ 210/s1. Acknowledgments: We would like to thank the CSU Retroviral Research Laboratory Animal Care Staff for excellent animal care and support. This study was funded by a grant from the National Institute of Health (NIH HL092791). Author Contributions: Wendy S. Sprague and Susan VandeWoude conceived and designed the experiments; Wendy S. Sprague performed the multicolor flow cytometry and analysis, performed differential counts on the blood smears, did final analysis on all the data, including t-tests, and wrote the manuscript; Xin Zheng ran the cytokine and viral proviral and viral load experiments; Britta A. Wood and Martha Macmillian helped with bleeding the animals and performing necropsies and taking samples; Martha Macmillian performed the PCVs, red blood and white blood cell counts; Ryan M. Troyer helped with the flow cytometry and with interpreting the data; Scott Carver performed the detailed repeated measures ANOVA and other statistical analyses. Conflicts of Interest: The authors declare no conflict of interest.

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