Multiple, Independent T Cell Lymphomas Arising in an Experimentally FIV-Infected Cat during the Terminal Stage of Infection

Abstract

Our laboratory has serially reported on the virologic and immunopathologic features of a cohort of experimental feline immunodeficiency virus (FIV)-infected cats for more than eight years. At 8.09 years post infection (PI), one of these animals entered the terminal stage of infection, characterized by undulating hyperthermia, progressive anorexia, weight loss, and pancytopenia; the animal was not responsive to therapeutic interventions, necessitating euthanasia six weeks later (8.20 years PI). Subsequent analyses indicated that neoplastic lymphocytes infiltrated multiple cervical lymph nodes and a band-like region of the mucosal lamina propria within a segment of the intestine. Immunohistochemistry and T cell clonality testing determined that the nodal and intestinal lesions were independently arising from CD3 T cell lymphomas. In-situ RNA hybridization studies indicated that diffuse neoplastic lymphocytes from the cervical lymph node contained abundant viral nucleic acid, while viral nucleic acid was not detectable in lymphocytes from the intestinal lymphoma lesion. The proviral long terminal repeat (LTR) was amplified and sequenced from multiple anatomic sites, and a common clone containing a single nucleotide polymorphism was determined to be defective in response to phorbol myristate acetate (PMA)-mediated promoter activation in a reporter gene assay. This assay revealed a previously unidentified PMA response element within the FIV U3 region 3' to the TATA box. The possible implications of these results on FIV-lymphoma pathogenesis are discussed.

Keywords: FAIDS (feline AIDS); FIV (feline immunodeficiency virus); cat; immunopathology; lymphoma.

Figure 1

Plasma viral copy number, total white blood cells (WBC), and clinical trajectory of cat 165. (a) The plasma viral copy number is plotted as number of copies gag RNA/mL blood vs. weeks post-infection (PI). The limit of detection (LOD) is approximately 800 copies vRNA/mL blood; (b) The total number of WBC (in thousands of leukocytes/microliter blood) is plotted against years PI—8.2 years (red X) is the time of euthanasia PI. The clinical trajectory of FIV for cat 165 is depicted below the x-axis- acute stage (blue), asymptomatic stage (yellow) and terminal stage (red).

Figure 2

The absolute number of peripheral CD4, CD8, and CD21 cells progressively declined over the course of the infection. (a) CD4, (b) CD8, and (c) CD21 cells are graphed in thousands of cells/microliter blood vs. time PI (years). The grey shaded regions represent the mean numbers of leukocytes ± standard deviation for the two uninfected control cats during the eight-year study period. The clinical trajectory of FIV for cat 165 is depicted below the x-axis for Figure 2c, acute stage (blue), asymptomatic stage (yellow) and terminal stage (red).

Figure 3

Clonally distinct, CD3+ T cell lymphomas are present in the cervical lymph nodes and the mucosa of the small intestine (jejunum). (a) The architecture of the cervical lymph node (LN) is effaced by sheets of neoplastic lymphocytes (hematoxylin and eosin stain); (b) The neoplastic lymphocytes within the cervical LN are CD3+ T cells (CD3 IHC); (c) Diffuse neoplastic lymphocytes within the cervical LN contain brown aggregating granules (positive for viral nucleic acid, in-situ hybridization); inset shows high-power magnification of neoplastic lymphocytes demonstrating brown granular material (viral nucleic acid); (d) A monoclonal PCR amplicon derived from the cervical lymphoma lesion is 103 base pairs (bp) long (T cell receptor gamma locus (TRG) clonality); (e) A poorly-delineated band of lymphocytes infiltrates the base of the mucosal villi within a segment of jejunum (hematoxylin and eosin stain); (f) The infiltrating lymphocytes are CD3+ (CD3 IHC); (g) Essentially, no brown granular material is present in these infiltrating lymphocytes (undetectable viral nucleic acid). Inset: high-power magnification; (h) A monoclonal PCR amplicon derived from the intestinal lesion is 87 bp long (TRG clonality).

Figure 4

An atrophied mesenteric lymph node (LN) and bone marrow contain moderate amounts of viral nucleic acid, but are variably positive for CD3+ T cells. (a) The mesenteric LN has a poorly developed cortex lacking follicles (cortical/follicular atrophy, H&E); (b) The remnant cortex and medullary cords have moderate numbers of CD3+ T cells (non-neoplastic lymphocytes, CD3 IHC); (c) Scattered leukocytes in the mesenteric LN contain moderate amounts of brown granular material—inset shows high-power magnification (viral nucleic acid, in-situ hybridization); (d) Bone marrow (BM) is histologically normal (hematoxylin and eosin stain); (e) CD3+ T cells are not detectable in the BM (CD3 IHC); (f) Scattered hematopoietic cells in the BM contain brown granular material (viral nucleic acid, in-situ hybridization).

Figure 5

Proviral DNA (black bars) is detectable by real-time PCR in all of the examined feline tissues while vRNA (cDNA, grey bars) is only detectable in lymphoid tissues (red line).

Figure 6

(a) The FIV promoter (LTR) sequenced from the tissues of cat 165 has multiple single nucleotide point (SNP) mutations. The U3, R, and U5 regions of the inoculating FIV sequence are depicted in black, while the various SNPs are indicated in red; parentheses indicate the number of times the SNP was isolated out of 14 sequenced clones. Known TFB sites are indicated in green, and the putative phorbol myristate acetate (PMA) Response Element (PRE) is denoted by a shaded grey box (nucleotide position −15 to −4, where +1 is the viral transcription start site, R). The _G196_A SNP is emphasized with a red arrow; (b) A β-galactosidase reporter gene assay identifies a novel PMA Response Element in the 3′ aspect of the FIV U3 region. The basal function of the wild type promoter (FIV LTR, green) and FIV promoter with the _G196_A SNP (FIV LTRmut, red) is minimal relative to negative controls (no treatment and pBlue Empty). PMA treatment markedly induces the wild-type FIV promoter expression (FIV LTR + PMA, p < 0.05, green cross-hatch), but has no detectable effect on the FIV promoter with the _G196_A SNP (FIV LTRmut, not significant, red cross-hatch), relative to transfected cells not treated with PMA.