Virus Protein-Specific Immune Responses in Selective Depletion of Lymphocyte Populations Using Monoclonal Antibodies in Bolivian Squirrel Monkeys (Saimiri boliviensis boliviensisv)

Abstract

The increasing use of immune suppressive monoclonal antibodies in the treatment of organ transplant recipients and patients with oncologic, neurological, and autoimmune diseases can lead to serious morbidity and mortality from the reactivation of viral agents that persist in humans. The squirrel monkey polyomaviruses are naturally found in Bolivian squirrel monkeys (SQM) and may be a useful model for the study of polyomavirus-associated pathogenesis and experimental treatment and prevention strategies. Two diverse groups of squirrel monkeys were given, a single dose of an anti-B cell antibody (rituximab) resulting in complete depletion of B cells (CD20+), while an anti-CD8 monoclonal antibody (7 pt-3F9) resulted in a transient depletion of CD8+ lymphocytes compared with control animals (group with no infusion with either of the monoclonal antibodies). The animals remained clinically healthy, with no pathological symptoms suggesting that the intensity and/or duration of immune suppression were inadequate to trigger pathogenic reactivation of the latent polyoma and herpes viruses. We observed a transient reduction in circulating plasma cytokines, IL-2, IFN-γ, and IL-12 reduced JC and BK viral protein-specific proliferative responses in both the CD8 and CD20 depletion groups. This study clearly elucidates the consequences of the use of depletion monoclonal antibodies in immune suppression modalities in the treatment of human malignancies and during transplantation, and SQM acts as a good model in the selection of dosage at which activation of latent viruses is at a minimum, with no pathological consequences.

Keywords: 7Pt-3F9; CD20; CD8; immune suppression; rituximab; squirrel monkey.

FIG. 1.

Reactivity of CD8 (clone 7Pt-3F9) and CD20 (rituxan) antibodies used for depletion assessed under in vitro conditions using SQM whole blood. Whole blood (100 uL) is incubated with 1 ug antibody for 30 min, excess antibody removed by a single wash, and incubated with a conjugated secondary antibody for another 30 min. Red blood cells (RBCs) were lysed using BD lysis buffer (1×) washed one time with FACS buffer and the pellet of cells fixed in 300 uL 1× paraformaldehyde, to acquire cells using FACS Celesta. Histogram plots (C and D) show cells incubated with a secondary antibody alone (light shaded) and testing antibodies followed by a secondary antibody (dark shaded). The dot plots (A and B) show lymphocyte gating and CD8+ and CD20+ lymphocytes.

FIG. 2.

Flow cytometric analysis of whole blood of 52 squirrel monkeys. Absolute numbers of lymphocyte subsets CD3+, CD4+, CD8+, CD20+, and CD16+ are shown.

FIG. 4.

Expression of NK cells in control saline-treated group (A), in monoclonal mouse anti-CD8 antibody (7 pt-3F9) group (B), and in the monoclonal mouse/human chimeric anti-human CD20 antibody (rituximab) group (C).

FIG. 5.

A multiplex nonhuman primate cytokine Plex panel was used to measure analytes in plasma of CD8- and CD20-depleted SQM according to the procedure described in the kit. Acquisition and data were analyzed by the Bio-Plex manager 5.0 (Bio-Rad, Hercules, CA).

FIG. 6.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood from controls, CD8 antibody-, and CD20 antibody-treated monkeys, and were in vitro stimulated with Con A (A), LPS (B), and PWM (C), JC virus protein (D) and BK virus protein (E) (each at 1/mL/mL) in triplicate wells of 96-well plates for 6 days. On day 5 of incubation 1uci per well of 3Htritum was added in each well and harvested on a semiautomatic cell harvester (Skatron) and the filter was counted on b-counter. Cell proliferation is expressed as CPM (counts per minute) above medium as background.

FIG. 7.

PBMCs (10⁵/well) were incubated with 1 ug/mL of protein of BK and JC virus and seeded in triplicate wells of 96-well plates (polyvinylidene difluoride-backed plates, MAIP S 45, Millipore Bedford, MA), precoated with the primary IFN-γ antibody. After incubation for 36 h at 37°C, the cells were removed, and the wells were thoroughly washed with PBS and developed and counted using the KS-ELISpot automatic system (Carl Zeiss, Inc., Thornwood, NY) as per protocol provided by the manufacturer. Responses were considered positive when the numbers of spot-forming cells (SFCs) with the test antigen were at least five above the background control values from cells cultured in the medium alone or with the negative control peptide.

FIG. 3.

Intravenous infusion of the monoclonal mouse anti-CD8 antibody (7 pt-3F9) or monoclonal mouse/human chimeric anti-human CD20 antibody (rituximab) alone or in saline in the controls in squirrel monkeys. In peripheral blood from squirrel monkeys, expression of absolute numbers of CD3 (A), CD4 (B), CD8 (C), and CD20 (D) in control group (upper panel). Absolute numbers of CD3 (E), CD4 (F), CD8 (G), and CD20 (H) in CD8 depletion group (middle panel). Absolute numbers of CD3 (I), CD4 (J), CD8 (K), and CD20 (L) in CD20 depletion group (lower panel).