Alterations in Peripheral Blood B-cell Populations in SHIV89.6P-infected Macaques (Macacca fascicularis)

Animals.

Adult, Chinese-origin cynomolgus macaques (Macacca fascicularis; weight, 5 to 8 kg) were used in this study. All animals were purchased from National Primate Centers or vendors approved by the Department of Laboratory Animal Research (University of Pittsburgh, PA). Prior to admission to the study, all macaques underwent complete physical examination (pulmonary and cardiac auscultation, thoracic radiographs, computer tomography scanning, tuberculin skin testing, complete blood count, chemistry panel, urinalysis, and flow cytometric analysis of peripheral blood mononuclear and bronchoalveolar lavage cells) and were screened for simian retroviruses (SIV, simian retrovirus, and simian T-lymphocyte associated retrovirus) to verify that they were free of any preexisting disease. Macaques were housed in an AAALAC-accredited, biosafety level 2+ primate facility at the University of Pittsburgh. Animal husbandry and experimental procedures were conducted in accordance with standards set forth by the Guide for the Care and Use of Laboratory Animals¹³ and the provisions of the Animal Welfare Act.² Prior to the initiation of this study, all animal experiments were approved by the IACUC of the University of Pittsburgh.

Study design.

Macaques (n = 29) were intravenously inoculated with 1 × 10^4.9 TCID₅₀ SHIV89.6P (gift of Dr Opendra Narayan, University of Kansas), which induces CD4+ T cell lymphopenia and AIDS-like disease with wasting and opportunistic infections.^32,33 Serial peripheral blood samples were collected for immunoglobulin detection and flow cytometry analysis of T and B lymphocytes¹⁶ at baseline, weekly for the first 8 wk after SHIV infection, and monthly thereafter to 53 wk after infection. Peripheral blood viral levels were determined as previously described.^32,34

Flow cytometric analysis of peripheral blood mononuclear cells (PBMC).

Peripheral blood samples were collected at baseline on all macaques. Serial plasma and PBMC samples from SHIV-infected monkeys were collected weekly for the first 8 wk after SHIV infection and monthly thereafter. Briefly, plasma was isolated from 10 mL EDTA-treated whole blood by centrifugation; PBMC were purified over a Percoll gradient (Amersham Bioscience, Piscataway, NJ) and washed with sterile PBS.³⁴ Plasma aliquots were stored at −80 °C prior to assay. PBMC were counted, stained, and fixed for analysis by flow cytometry, as described.⁶ Antibodies used were: mouse antimonkey CD3–FITC (clone SP34), mouse antimonkey CD4–allophycocyanin (clone L200), mouse antihuman CD21 (clone Bly4)–phycoerythrin, mouse antihuman CD95 (clone DX2)– FITC, and antihuman IgM–FITC [all purchased from BD Pharmingen (San Diego, CA)]; mouse antihuman CD20 (clone 2H7)–Pacific Blue and mouse antihuman CD27 (clone O323)–allophycocyanin [both purchased from eBioscience, San Diego, CA]), and antihuman IgD–biotin (Southern Biotech, Birmingham, AL). A streptavidin–Pacific Orange conjugate (Invitrogen, Carlsbad, CA) was used to detect biotin-conjugated antibodies. Acquisition was performed on BD LSRII flow cytometer (BD Biosciences, San Jose, CA) by using BD FacsDiva software. Forward-/side-scatter dot plots were used to gate the live lymphocyte population. All analyses were performed by using FlowJo flow cytometry analysis software (Tree Star, Ashland, OR). Absolute differential cell counts were determined by Antech Diagnostics (Lake Success, NY), and lymphocyte counts were used to determine absolute numbers of B cells, B cell subsets, and CD4+ T cells.

Determination of plasma SHIV viral load.

Virus loads in plasma and bronchoalveolar lavage fluid supernatant were determined as described elsewhere.³² Briefly, RNA was extracted from plasma and BAL fluid supernatant and was quantified as RNA copies per milliliter by using an adapted protocol for quantitative real-time RT–PCR detecting the SIV gag sequence.

Quantification of total immunoglobulin.

Plasma samples were heat-inactivated at 56 °C for 30 min and then diluted in blocking buffer (5% nonfat milk in PBS) prior to assay. Microtiter plates (Immunolon 4HBX, Thermo Fisher Scientific, Waltham, MA) were coated with affinity-purified antimonkey IgG (1 μg/mL; Rockland Immunochemicals, Gilbertsville, PA) at 4 °C overnight. The next day, 100 µL plasma was plated in triplicate into antimonkey IgG-coated wells. Goat antimonkey immunoglobulin-conjugated horseradish peroxidase (1:10,000 for IgG, 1:2000 for IgM; Nordic Immunology, Tilburg, Netherlands) was used for detection, and plates were developed by standard methods. Normal (uninfected) macaque plasma was used as a plate control. Serial dilutions of whole-molecule monkey IgG (Rockland Immunochemicals) were used to generate a standard curve.

Statistical analyses.

Statistical analyses were performed by using Prism software (GraphPad, La Jolla, CA). Comparisons among multiple time points were made by using repeated-measures ANOVA with Bonferroni post tests. In addition, a paired 2-tailed Student t test was used to compare data from time points after SHIV infection with baseline values. A P value of less than 0.05 was considered significant.

Total B cell populations and percentages of CD95+ B cells in peripheral blood after SHIV infection.

Changes in B cell levels induced by SHIV_89.6P infection of cynomolgus macaques were analyzed serially by flow cytometry. Significant changes in numbers (repeated-measures ANOVA, P < 0.0001; Figure 1 B) and proportions (repeated-measures ANOVA, P = 0.0004; Figure 1 C) of total B (CD20+) of lymphocytes, compared with baseline values, were present after SHIV infection. Comparison of individual time points revealed significant declines in numbers (week 2, P = 0.0008; Figure 1 B) and percentages (week 2, P = 0.026; Figure 1 C) of CD20+ cells early after SHIV infection. Absolute numbers of CD20+ cells remained depressed throughout the experiment (repeated-measures ANOVA, P < 0.0001; Figure 1 B) and at week 53 were significantly (P = 0.0019, Figure 1 B) decreased from baseline. Percentage of CD20+ cells returned to near baseline levels by approximately 1 y after infection (week 53; paired Student t test, P = 0.23), which result is likely a reflection of the decline in CD4+ T cells (Figure 1 D). Peak viral loads in SHIV-infected monkeys were detected between 1 to 2 wk after SHIV infection, with a mean of 1.86 × 10⁷ ± 8 × 10⁶ viral RNA copies per milliliter plasma (Figure 1 E). The early decline in CD20+ lymphocytes coincided with the peak plasma viral load (Figure 1 E). Although mean viral loads declined below 10⁴ RNA copies per milliliter of plasma by 6 wk after SHIV infection and remained at this level for the duration of the experiment, CD20+ cells numbers did not return to baseline levels (Figure 1 E).

B cells from HIV-infected humans exhibit increased expression of CD95 (Fas),³⁷ an activation marker, and greater susceptibility to CD95-mediated apoptosis. We investigated whether peripheral blood CD95+ B cells (CD95+ CD20+) similarly were increased in a subset of SHIV-infected macaques (n = 17). Absolute numbers of CD95+ B cells declined with total B cell numbers after SHIV infection (Figure 2 A) but by approximately 7 mo after SHIV infection had returned to baseline levels (week 28; paired Student t test, P = 0.96) and remained near baseline levels at approximately 1 y after infection (week 58; paired Student t test, P = 0.38), despite the fact that total B cell numbers remained decreased at this point (Figure 1). The percentage of CD95+ B cells was increased early after SHIV infection (week 2; paired Student t test, P < 0.0001; Figure 2 B), increasing from 12.7% ± 8.1% at baseline to 41.8% ± 13.7% at week 2 after infection; percentages of CD95+ B cells remained significantly (P < 0.0001) increased at 51.1% ± 10.0% of total B cells by 58 wk after SHIV infection (Figure 2 B).

Surface expression of CD21 on peripheral blood B cells in SHIV-infected macaques.

The surface marker CD21 is part of the B cell coreceptor complex, which serves to strengthen the signal resulting from antigen recognition.³⁶ Signaling pathways activated by CD21 amplify antibody responses and induce costimulatory molecules on the B cell, thereby increasing effectiveness at eliciting T-cell help.³⁶ B cells from HIV-infected patients demonstrate diminished CD21 expression,^14,28 which may result in reduced B cell responsiveness to antigen and subsequent proliferation. To examine this phenotype in SHIV-infected monkeys, we evaluated expression of the CD21 surface marker on peripheral blood B cells (CD20+CD21+) of macaques (n = 29). The absolute number of CD21+ B cells in the peripheral blood was significantly (repeated-measures ANOVA, P < 0.0001) decreased after SHIV infection (Figure 3 A, B). Acute significant declines in the absolute number (week 2; paired Student t test, P = 0.0007; Figure 3 A) and percentage (week 2; P = 0.0012, paired Student t test; Figure 3 B) of CD20+CD21+ cells occurred after SHIV infection. Both number (paired Student t test; P = 0.0003) and percentage (paired Student t test; P < 0.0001) of CD21+ B cells remained significantly depressed by approximately 1 y after SHIV infection.

Peripheral blood memory and naïve B cells in SHIV-infected macaques.

Memory B cells were assessed by evaluating CD27 surface marker expression.⁴⁰ Flow cytometry of PBMC revealed significant (repeated-measures ANOVA, P < 0.0001) changes in the number of CD27+ B cells (Figure 4 A). Numbers of CD27+ B cells declined significantly during acute SHIV infection (week 2; P = 0.037; Figure 4 A) but rebounded thereafter and, at 1 y after SHIV infection, remained near baseline levels (P = 0.18, week 0 compared with week 53; Figure 4 A).

Subsets of memory B cells may be differentially affected during HIV infection, especially with regard to class-switched (CD27+IgM–IgD– B cells) compared with IgM+ memory (CD27+IgM+) B cells, with IgM+ memory cells reportedly significantly reduced in HIV+ patients.¹² Because we noted an early loss of total memory (CD27+) B cells in our macaques, we further investigated whether SHIV infection was associated with declines in various subsets of memory B cells. After SHIV infection, numbers of class-switched (IgM–IgD–) memory (CD27+) B cells were significantly (repeated-measures ANOVA, P = 0.0003) reduced (Figure 4 C). There were also significant (repeated-measures ANOVA, P < 0.0001) declines after SHIV infection in the percentages of CD27+ B cells that were class-switched (Figure 4 B). However, by 41 wk after SHIV infection, numbers (P = 0.16) and percentages (P = 0.2) of class-switched CD27+ B cells returned to baseline levels (Figure 4 B, C). In addition, numbers (repeated-measures ANOVA, P < 0.0001; Figure 4 D) and percentages (repeated-measures ANOVA, P < 0.0001; Figure 4 E) and of IgM memory B cells (IgM+CD27+) were decreased after SHIV infection. Numbers and percentages of IgM+ CD27+ B cells significantly declined by 16 wk after SHIV infection, and, in contrast to the rebound observed for class-switched memory B cells by 45 wk after SHIV infection, remained decreased for the duration of the experimental infection (week 0 versus 53; numbers, P = 0.007; percentages, P = 0.014; Figure 4 D, E).

We also examined the naïve B cell (CD20+CD27–) population in PBMC of SHIV-infected macaques (n = 29). In the current examination of SHIV infection of cynomolgus macaques, we noted significant declines in numbers (repeated-measures ANOVA, P < 0.0001; Figure 5 A) and percentages (repeated-measures ANOVA, P < 0.0001; Figure 5 B) of CD27– B cells. Numbers of CD27– B cells (Figure 5 B) declined significantly early after SHIV infection (week 2, P = 0.0006) and did not return to baseline levels for the duration of the experiment (week 53, P = 0.0011). Percentages of CD27– B cells (Figure 5 A) declined from baseline by week 12 (P = 0.0038).

Evidence of hypergammaglobulinemia in the plasma of SHIV-infected macaques.

Chronic immune activation and hypergammaglobulinemia are well-documented in HIV infection.^19,31 The polyclonal response is composed of both virus-specific and nonspecific antibodies. Several isotypes are increased, but IgG is affected predominantly, and serum IgG levels may represent a prognostic marker of disease progression.^8,9 To further assess whether SHIV infection of macaques induces a similar response, we examined macaques (n = 12) for evidence of plasma hypergammaglobulinemia at baseline and at serial time points after SHIV infection. Plasma IgG levels varied significantly (repeated-measures ANOVA, P = 0.0041) from baseline over the course of SHIV infection (Figure 6). Therefore, SHIV infection of cynomolgus macaques was associated with hypergammaglobulinemia.