1)Treatment of chronically SHIVSF162P3N-infected CyMs with anti-CD4 antibody (CD4R1) results in CD4+T cell depletion in the peripheral blood, BM and LNs
To determine effect of CD4+ T cell depletion on the viral load in chronic SHIV infection, three infected Chinese-origin CyMs were administered (i.v. twice at one-week interval) with the humanized monoclonal antibodies (mAb) CD4R1 (50mg/kg) against CD4 receptor. Three untreated animals were used as the control. All 6 animals had been infected with SHIVSF162P3N for 38 weeks prior to the CD4R1 injection. As shown in Figure 1a, peripheral blood (PB), bone marrow (BM) and inguinal lymph node (Ing LN) specimens were collected at multiple time points throughout the study.
Prior to CD4+ T cell depletion, the baseline % of CD3+CD4+ T cells in the animals of both groups was about 55% ± 5%, and the absolute numbers of CD4+ T cells were 1,500 ± 600 cells/ul blood (mean ± SD). As shown in Figure 1b, c, immediately after the anti-CD4 antibody treatment, the 3 treated animals had a significant decrease (70%–99% relative to baseline) in both percentage and absolute number of circulating CD4+ T cells, which persisted till the end of this study. Among the anti-CD4 antibody-treated animals, animal WCE03 exhibited severer CD4+ T cell depletion (>98%) than other two (WEC02 and WEC05), as it had only a nadir CD4+ T-cell percentage of 1.45% and counts of 5.8 cells/ml (Figure 1b). The same degree of CD4+ T cell depletion was observed in BM, with mean decline of 80% ± 15% (Figure 1d). In the Ing LN, severe depletion (>80%) of CD4+ T cell, however, was not observed till 4th week after the anti-CD4 antibody injection (Figure 1d). In addition, unlike CD4+ T cells in blood and BM, cell numbers in Ing LN at the time of necropsy rebounded to the levels comparable to those at 2nd week after the antibody injection (Figure 1d).
We next examined CD4+ T cell depletion efficiency in the different tissues collected at the necropsy, including peripheral LNs (pLNs), LNs in the gastrointestinal (GI) tract (GI LNs) and spleens. As shown in Figure 1e, CD4+ T cell depletion in PB, BM and spleen (>70%) were severer than that (25-35%) in pLNs and GI LNs. To determine specificity of CD4R1 antibody, we next examined the number of CD8+ T cells in peripheral blood. As shown in Supplemental Figure 1, the number of peripheral blood CD8+ T cells was not affected in the animals treated with CD4R1antibody.
2) CD4+ T cell depletion has little effect on plasma viral load and LN tissues of chronically SHIVSF162P3N-infected CyMs
To determine the impact of CD4+ T cell depletion on the viremia in chronically SHIV-infected CyMs, we examined the kinetics of plasma viral loads of the study animals. As shown in Figure 2, the animal in both groups became infected after SHIV inoculation and had a typical exponential elevation of virus load at acute infection stage. The peak levels of viral load were observed at week 2 post infection (p.i.) or week -36 post depletion (p.d), with the median levels of 7.0×108 (1.1×108~1.8×109) copies/ml and 8.3×108 (1.7×108~1.49×109) copies/ml in control group and CD4+ T cell-depleted group, respectively. Following peak levels of the viremia, the plasma viral loads of SHIV-infected animals gradually decrease and fluctuated between undetectable to 105 copies/ml. At the 38th week p.i. and the 0-week p.d., three animals with chronic SHIV infection and at asymptomatic stage were administered with the anti-CD4 antibody. While all antibody-treated animals showed a significant decrease in both percentage and absolute number of CD4+ T cells, there was little change of plasma viral loads (Figure 2).
3) CD4+ T cell depletion results in increased expression of ki67 on CD4+T cells
To determine the impact of CD4+ T cell depletion on the expression of ki67, a T cell proliferation marker correlated with plasma viral load, we measured the number of CD4+ ki67+ T cells in the PB, BM and Ing LNs of the study animals prior to and after anti-CD4 antibody injection. As shown in Figure 3a, there was a significant increase (3.9- to 6.8-fold) in the expression of Ki67 on PB CD4+ T cells in the anti-CD4 antibody-treated animals, which started from the 2nd week and peaked at the 8th week post the antibody injection (Figure 3a). However, the number of CD4+ ki67+ T cells declined to the basal levels at the late stage of the depletion. There was no significant difference in ki67 expression on BM CD4+ T cells between the two groups (Figure 3b). Among three animals with CD4+ T cell depletion, WCE05 had the highest level of ki67 expression in both PB and BM throughout the study. When we examined the correlation between the plasma viral loads and the levels of Ki67 on CD4+ T cells (Figure 3c), although animal WCE05 presented a positive correlation (r=0.7 and P=0.04), no correlation was found in other two animals (WCE02 and WCE03). We also determined the levels of CD4+ Ki67+ T cells in LNs, showing a significant increase of Ki67 expression in inguinal LNs of the depleted animals in the week 2 to week 4 p.d. (Figure 3d). However, there was no difference in all LNs, including inguinal, mesenteric and colon LNs at the time of necropsy (Figure 3e).
4) CD4+T cell depletion has little effect on SHIV specific antibody response.
To determine whether the persistence of low levels of viremia in chronically SHIV infected animals was due to an effective antiviral humoral immune response, we first examined the impact of CD4+ T cell depletion on SHIV-specific antibody response. We found that there was no significant difference in plasma levels of SHIV binding antibody between CD4+ T cell-depleted animals and the those in control group (Table S1). We next measured the in vitro neutralization activity of plasma specimens from two animals (one from depleted group and one from control group). As shown in Figure 4, the plasma neutralization activity to SHIV was absent or very low in both animals.
5) CD4+ T cell depletion results in increased turnover and proliferation (Ki67+) of monocytes
Increased turnover and proliferation of monocytes, particularly the cells with CD14+CD16+ phenotype, predict disease progression to AIDS in SIV-infected Indian RMs [23]. We thus determined the levels of monocyte turnover and Ki67 expression in CD4+ T cell-depleted and control animals. Based on the expression of CD14 and/or CD16, we defined monocytes as either classical (CD14+CD16-), or pro-inflammatory (CD14+CD16+), and or non-classical (CD14- CD16+). As shown in Figure 5, comparing with monocytes from the control animals, the animals with CD4+ T cell depletion had significant higher numbers of pro-inflammatory and non-classical monocytes. There were an average 1.9-fold increase in turnover (1.5~2.2) for pro-inflammatory monocytes and an average 2.1-fold increase (1.7~2.7) for non-classical monocytes relative to baseline levels in the week 2 to week 8 p.d. Of note, pro-inflammatory CD14+CD16+ monocytes exhibited an average 10-fold increase (7.1 to 13.2) of Ki67 expression in the week 2 to week 5 p.d, with a maximum increase of 13-fold at week 4 p.d. However, Ki67 expression on pro-inflammatory monocytes gradually declined to the basal levels at the time of necropsy. No difference was found between the animals in two groups regarding the expression of classical monocytes (CD14+ CD16-).
6) CD4+ T cell depletion has little effect on inflammatory cytokines and CC chemokines
Since the inflammatory cytokines and CC chemokines play an important role in systemic immune activation and HIV infection progression, it is of importance to determine whether these immunologic factors are affected by CD4+ T cell depletion. In examining mRNA expression of the cytokines (IL-1β, IL-10, TNF-a) and chemokines (MCP-1/CCL-2, MIP-1α/CCL3, MIP-1β/CCL4, CXCL-9, CXCL-10) in peripheral blood mononuclear cells (PBMCs), spleen and LNs, we found a significant increase of the levels of IL-10, MCP-1(CCL-2) and MIP-1α (CCL3) in colon LNs of CD4+ T cell depleted animals as compared to those in the control animals (Figure 6). However, no significant differences in the expression of the cytokines and chemokines were observed in PBMC, spleen and ileum LNs of the animals regardless of CD4+ T cell depletion (Figure 6).