Lymphoid organs as a major reservoir for human T-cell leukemia virus type 1 in experimentally infected squirrel monkeys (Saimiri sciureus): provirus expression, persistence, and humoral and cellular immune responses

Abstract

The aim of this study was to investigate the distribution of human T-cell leukemia virus type 1 (HTLV-1) in various organs of serially sacrificed squirrel monkeys (Saimiri sciureus) in order to localize the reservoir of the virus and to evaluate the relationship between viral expression and the humoral or cellular immune response during infection. Six squirrel monkeys infected with HTLV-1 were sacrificed 6, 12, and 35 days and 3, 6, and 26 months after inoculation, and 20 organs and tissues were collected from each animal. PCR and reverse transcription-PCR (RT-PCR) were performed with gag and tax primers. Proviral DNA was detected by PCR in peripheral blood mononuclear cells (PBMCs) of monkeys sacrificed 6 days after inoculation and in PBMCs, spleens, and lymph nodes of monkeys sacrificed 12 and 35 days and 3, 6, and 26 months after inoculation. Furthermore, tax/rex mRNA was detected by RT-PCR in the PBMCs of two monkeys 8 to 12 days after inoculation and in the spleens and lymph nodes of the monkey sacrificed on day 12. In this animal, scattered HTLV-1 tax/rex mRNA-positive lymphocytes were detected by in situ hybridization in frozen sections of the spleen, around the germinal centers and close to the arterial capillaries. Anti-HTLV-1 cell-mediated immunity was evaluated at various times after inoculation. Anti-p40(Tax) and anti-Env cytolytic T-cell responses were detected 2 months after infection and remained detectable thereafter. When Tax peptides were used, this response appeared to be directed against various Tax epitopes. Our results indicate that squirrel monkeys represent a promising animal model for studying the early events of HTLV-1 infection and for evaluating candidate vaccines against HTLV-1.

FIG. 1

Detection of HTLV-1 tax sequence (202 bp) (first line) and SRY sequence of Y chromosome (second line) by PCR in PBMCs of monkeys 90047 (female), 92038 (male), and 90083 (female) sacrificed 6, 12, and 35 days after inoculation. C− and C+ represent negative and positive controls, respectively.

FIG. 2

Detection of HTLV-1 provirus expression (tax/rex mRNA) by RT-PCR. (A) Detection of tax/rex mRNA (first line) and GAPDH sequence (second line) in PBMCs of monkeys 90047, 92038, and 90083 sacrificed 6, 12, and 35 days after inoculation. (B) Detection of tax/rex mRNA (first line) and GAPDH sequence (second line) in spleens of different monkeys sacrificed at various times after inoculation. C− and C+ represent negative and positive controls; respectively.

FIG. 3

In situ hybridization with HTLV-1 tax riboprobe on frozen spleen sections from an infected squirrel monkey sacrificed 12 days after inoculation. (A and B) Positive signals in the spleen observed with antisense ³³P-labeled riboprobe; arrows indicate positive spleen cells after 10 days of in situ hybridization. (C) Negative control (PBMCs from squirrel monkey not infected with HTLV-1). (D) Positive control (EVO/798 HTLV-1 monkey-transformed cell line).

FIG. 4

Antibody responses of monkeys against HTLV-1, tested by ELISA after inoculation with homologous HTLV-1 monkey-transformed cell lines. Monkey 90083 was sacrificed at 35 days, monkey 92106 was sacrificed at 3 months, monkey 92039 was sacrificed at 6 months, and monkey 1657 was sacrificed at 26 months. O.D., optical density.

FIG. 5

Cultured CD4⁺-depleted CD8⁺-enriched cells obtained from PBMCs or splenocytes, used as effectors in cytolytic chromium release assays. Target cells were autologous B-lymphoblastoid cell lines either uninfected (n.i.), vaccinia virus infected, or Tax peptide loaded. In some cases, a 25- or 50-fold excess of cold autologous vaccinia virus-infected B-lymphoblastoid target cells was added to reduce background. (A) Monkey 90047 sacrificed day 6 after inoculation; CD4-depleted splenocytes by day 12 of culture. (B) Monkey 92038 sacrificed day 12 after inoculation; CD4-depleted splenocytes by day 18 of culture. (C) Blood from monkey 92039 sampled at 2 months; CD4-depleted PBMCs by day 18 of culture. (D) Blood from monkey 92039 sampled at 2 months; CD4-depleted PBMCs by day 16 of culture. (E) Monkey 92039 sacrificed at 6 months; CD4-depleted splenocytes by day 24 of culture. (F) Blood from monkey 1491 sampled 20 and 30 months after inoculation; CD4-depleted PBMCs by days 23 and 15 of culture, respectively. (G) Blood from monkey 1491 sampled 20 and 30 months after inoculation; CD4-depleted PBMCs obtained from blood sample at 30 months after inoculation, by day 19 of culture. (H) Blood from monkey 1657 sampled 20 months after inoculation; CD4-depleted PBMCs by day 26 of culture. (I) Monkey 1657 sacrificed 26 months after inoculation; CD4-depleted splenocytes by day 21 of culture.

FIG. 5

Cultured CD4⁺-depleted CD8⁺-enriched cells obtained from PBMCs or splenocytes, used as effectors in cytolytic chromium release assays. Target cells were autologous B-lymphoblastoid cell lines either uninfected (n.i.), vaccinia virus infected, or Tax peptide loaded. In some cases, a 25- or 50-fold excess of cold autologous vaccinia virus-infected B-lymphoblastoid target cells was added to reduce background. (A) Monkey 90047 sacrificed day 6 after inoculation; CD4-depleted splenocytes by day 12 of culture. (B) Monkey 92038 sacrificed day 12 after inoculation; CD4-depleted splenocytes by day 18 of culture. (C) Blood from monkey 92039 sampled at 2 months; CD4-depleted PBMCs by day 18 of culture. (D) Blood from monkey 92039 sampled at 2 months; CD4-depleted PBMCs by day 16 of culture. (E) Monkey 92039 sacrificed at 6 months; CD4-depleted splenocytes by day 24 of culture. (F) Blood from monkey 1491 sampled 20 and 30 months after inoculation; CD4-depleted PBMCs by days 23 and 15 of culture, respectively. (G) Blood from monkey 1491 sampled 20 and 30 months after inoculation; CD4-depleted PBMCs obtained from blood sample at 30 months after inoculation, by day 19 of culture. (H) Blood from monkey 1657 sampled 20 months after inoculation; CD4-depleted PBMCs by day 26 of culture. (I) Monkey 1657 sacrificed 26 months after inoculation; CD4-depleted splenocytes by day 21 of culture.