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Peripheral and central immune cell reservoirs in tissues from asymptomatic cats chronically infected with feline immunodeficiency virus

View Article: PubMed Central - PubMed

ABSTRACT

Feline immunodeficiency virus (FIV) infection in cats results in life-long viral persistence and progressive immunopathology. We have previously described a cohort of experimentally infected cats demonstrating a progressive decline of peripheral blood CD4+ T-cell over six years in the face of apparent peripheral viral latency. More recently we reported findings from this same cohort that revealed popliteal lymph node tissue as sites for ongoing viral replication suggesting that tissue reservoirs are important in FIV immunopathogenesis during the late asymptomatic phase of infection. Results reported herein characterize important tissue reservoirs of active viral replication during the late asymptomatic phase by examining biopsied specimens of spleen, mesenteric lymph node (MLN), and intestine from FIV-infected and uninfected control cats. Peripheral blood collected coincident with harvest of tissues demonstrated severe CD4+ T-cell depletion, undetectable plasma viral gag RNA and rarely detectable peripheral blood mononuclear cell (PBMC)-associated viral RNA (vRNA) by real-time PCR. However, vRNA was detectable in all three tissue sites from three of four FIV-infected cats despite the absence of detectable vRNA in plasma. A novel in situ hybridization assay identified B cell lymphoid follicular domains as microanatomical foci of ongoing FIV replication. Additionally, we demonstrated that CD4+ leukocyte depletion in tissues, and CD4+ and CD21+ leukocytes as important cellular reservoirs of ongoing replication. These findings revealed that tissue reservoirs support foci of ongoing viral replication, in spite of highly restricted viral replication in blood. Lentiviral eradication strategies will need address tissue viral reservoirs.

No MeSH data available.


Real-time PCR detection and quantitation of vRNA in CD4+, CD8+ and CD21+ leukocyte subsets enriched from blood and tissue compartments.Provirus was detected in all leukocyte subsets from blood, MLN and splenic compartments. Viral RNA was undetectable (represented as ND for not detectable) in blood PBMC-derived CD4+, CD8+ and CD21+ leukocyte subsets harvested from all FIV-infected cats, except from CD4+ cells from cat 165. CD4+ leukocytes derived from MLN and spleen consistently contained detectable vRNA, while it was intermittently detectable in tissue derived CD21+ leukocytes and was rarely detectable in tissue derived CD8+ T cells. Bars represent the median. Student’s t-tests comparing the level of provirus or viral gag RNA between anatomical compartments among leukocytes subsets revealed no statistically significant differences between any tissues. All leukocytes subsets from all compartments in all cats revealed readily detectable GAPDH in both RNA and DNA fractions. Blue = cat 165, black = cat 184, green = cat 186, red = cat 187 (LTNP cat).
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pone.0175327.g003: Real-time PCR detection and quantitation of vRNA in CD4+, CD8+ and CD21+ leukocyte subsets enriched from blood and tissue compartments.Provirus was detected in all leukocyte subsets from blood, MLN and splenic compartments. Viral RNA was undetectable (represented as ND for not detectable) in blood PBMC-derived CD4+, CD8+ and CD21+ leukocyte subsets harvested from all FIV-infected cats, except from CD4+ cells from cat 165. CD4+ leukocytes derived from MLN and spleen consistently contained detectable vRNA, while it was intermittently detectable in tissue derived CD21+ leukocytes and was rarely detectable in tissue derived CD8+ T cells. Bars represent the median. Student’s t-tests comparing the level of provirus or viral gag RNA between anatomical compartments among leukocytes subsets revealed no statistically significant differences between any tissues. All leukocytes subsets from all compartments in all cats revealed readily detectable GAPDH in both RNA and DNA fractions. Blue = cat 165, black = cat 184, green = cat 186, red = cat 187 (LTNP cat).

Mentions: Proviral DNA was detected in enriched CD4+, CD8+, and CD21+ leukocytes from all examined compartments (PBMCs, MLN and spleen, Fig 3). This finding is consistent with previous reports confirming that these leukocyte subsets are susceptible to FIV infection.[11,17,18] Results were variable when blood and tissue-derived enriched leukocyte subsets were interrogated for the presence of vRNA (Fig 3). Viral RNA was detected in CD4+ cells derived from the blood of one animal (cat 165), representing a rarely detectable vRNA blip in the peripheral blood, as previously described.[11] Viral RNA was detected in MLN CD4+ leukocytes from three out of three cats and in splenic CD4+ leukocytes from three out of four FIV-infected cats. Viral RNA was not detected in PBMC-derived CD21+ or CD8+ leukocytes harvested from any of the infected cats, but was detected in MLN and spleen-derived CD21+ leukocytes in 2 out of 3 (cats 184 and 187) and 2 out of 4 cats (cats 165 and 186), respectively. For CD8+ T cells, vRNA was detected in spleen-derived leukocytes from only one infected cat (165). These results support the concept of active viral transcription in central lymphoid tissues, in spite of rare to undetectable viral replication in the peripheral blood. These results also suggest that tissue-associated CD4+ and CD21+ leukocytes support greater active virus replication in the late asymptomatic phase than CD8+ T cells. Viral gag RNA was not detected in any of the enriched leukocytes obtained from the uninfected cats. No statistically significant differences in virus loads were identified between leukocyte subsets or between tissue sites due to the wide variation in viral loads across different cats and different cell subsets.


Peripheral and central immune cell reservoirs in tissues from asymptomatic cats chronically infected with feline immunodeficiency virus
Real-time PCR detection and quantitation of vRNA in CD4+, CD8+ and CD21+ leukocyte subsets enriched from blood and tissue compartments.Provirus was detected in all leukocyte subsets from blood, MLN and splenic compartments. Viral RNA was undetectable (represented as ND for not detectable) in blood PBMC-derived CD4+, CD8+ and CD21+ leukocyte subsets harvested from all FIV-infected cats, except from CD4+ cells from cat 165. CD4+ leukocytes derived from MLN and spleen consistently contained detectable vRNA, while it was intermittently detectable in tissue derived CD21+ leukocytes and was rarely detectable in tissue derived CD8+ T cells. Bars represent the median. Student’s t-tests comparing the level of provirus or viral gag RNA between anatomical compartments among leukocytes subsets revealed no statistically significant differences between any tissues. All leukocytes subsets from all compartments in all cats revealed readily detectable GAPDH in both RNA and DNA fractions. Blue = cat 165, black = cat 184, green = cat 186, red = cat 187 (LTNP cat).
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Related In: Results  -  Collection

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pone.0175327.g003: Real-time PCR detection and quantitation of vRNA in CD4+, CD8+ and CD21+ leukocyte subsets enriched from blood and tissue compartments.Provirus was detected in all leukocyte subsets from blood, MLN and splenic compartments. Viral RNA was undetectable (represented as ND for not detectable) in blood PBMC-derived CD4+, CD8+ and CD21+ leukocyte subsets harvested from all FIV-infected cats, except from CD4+ cells from cat 165. CD4+ leukocytes derived from MLN and spleen consistently contained detectable vRNA, while it was intermittently detectable in tissue derived CD21+ leukocytes and was rarely detectable in tissue derived CD8+ T cells. Bars represent the median. Student’s t-tests comparing the level of provirus or viral gag RNA between anatomical compartments among leukocytes subsets revealed no statistically significant differences between any tissues. All leukocytes subsets from all compartments in all cats revealed readily detectable GAPDH in both RNA and DNA fractions. Blue = cat 165, black = cat 184, green = cat 186, red = cat 187 (LTNP cat).
Mentions: Proviral DNA was detected in enriched CD4+, CD8+, and CD21+ leukocytes from all examined compartments (PBMCs, MLN and spleen, Fig 3). This finding is consistent with previous reports confirming that these leukocyte subsets are susceptible to FIV infection.[11,17,18] Results were variable when blood and tissue-derived enriched leukocyte subsets were interrogated for the presence of vRNA (Fig 3). Viral RNA was detected in CD4+ cells derived from the blood of one animal (cat 165), representing a rarely detectable vRNA blip in the peripheral blood, as previously described.[11] Viral RNA was detected in MLN CD4+ leukocytes from three out of three cats and in splenic CD4+ leukocytes from three out of four FIV-infected cats. Viral RNA was not detected in PBMC-derived CD21+ or CD8+ leukocytes harvested from any of the infected cats, but was detected in MLN and spleen-derived CD21+ leukocytes in 2 out of 3 (cats 184 and 187) and 2 out of 4 cats (cats 165 and 186), respectively. For CD8+ T cells, vRNA was detected in spleen-derived leukocytes from only one infected cat (165). These results support the concept of active viral transcription in central lymphoid tissues, in spite of rare to undetectable viral replication in the peripheral blood. These results also suggest that tissue-associated CD4+ and CD21+ leukocytes support greater active virus replication in the late asymptomatic phase than CD8+ T cells. Viral gag RNA was not detected in any of the enriched leukocytes obtained from the uninfected cats. No statistically significant differences in virus loads were identified between leukocyte subsets or between tissue sites due to the wide variation in viral loads across different cats and different cell subsets.

View Article: PubMed Central - PubMed

ABSTRACT

Feline immunodeficiency virus (FIV) infection in cats results in life-long viral persistence and progressive immunopathology. We have previously described a cohort of experimentally infected cats demonstrating a progressive decline of peripheral blood CD4+ T-cell over six years in the face of apparent peripheral viral latency. More recently we reported findings from this same cohort that revealed popliteal lymph node tissue as sites for ongoing viral replication suggesting that tissue reservoirs are important in FIV immunopathogenesis during the late asymptomatic phase of infection. Results reported herein characterize important tissue reservoirs of active viral replication during the late asymptomatic phase by examining biopsied specimens of spleen, mesenteric lymph node (MLN), and intestine from FIV-infected and uninfected control cats. Peripheral blood collected coincident with harvest of tissues demonstrated severe CD4+ T-cell depletion, undetectable plasma viral gag RNA and rarely detectable peripheral blood mononuclear cell (PBMC)-associated viral RNA (vRNA) by real-time PCR. However, vRNA was detectable in all three tissue sites from three of four FIV-infected cats despite the absence of detectable vRNA in plasma. A novel in situ hybridization assay identified B cell lymphoid follicular domains as microanatomical foci of ongoing FIV replication. Additionally, we demonstrated that CD4+ leukocyte depletion in tissues, and CD4+ and CD21+ leukocytes as important cellular reservoirs of ongoing replication. These findings revealed that tissue reservoirs support foci of ongoing viral replication, in spite of highly restricted viral replication in blood. Lentiviral eradication strategies will need address tissue viral reservoirs.

No MeSH data available.