Human peripheral blood mononuclear cells exhibit heterogeneous CD52 expression levels and show differential sensitivity to alemtuzumab mediated cytolysis

Abstract

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.

Figure 1. Phenotypic characterization of lymphoid and myeloid subsets.

Representative polychromatic flow cytometric analysis of lymphoid (A) and myeloid (B) subsets from PBMCs. Mem B (Memory B-cells), CM (Central memory), EM (Effector memory), NK (Natural killer), mDCs (Myeloid Dendritic cells), pDCs (Plasmacytoid dendritic cells).The phenotype of each PBMC subset is detailed in Table 1.

Figure 2. Differential expression of CD52 on human PBMC subsets.

Representative histograms of CD52 expression levels on each of the lymphoid (A) and myeloid (B) cell subsets described in figure 1 were analyzed using Flowjo software v7.2. The histograms and the corresponding median fluorescence intensity value of each of the subsets are shown in the panels. A: Hierarchy of CD52 levels on lymphoid cells. Mem B > CD4−CM > CD4−EM > CD4−Naïve > CD8−Naïve− > Naïve−B > CD8−CM > CD4−Effector > CD16lo Nk > CD16hi NK. B: Hierarchy of CD52 levels on myeloid cells: CD16+ mDC > CD16+ Mono > CD16− Mono > CD16− mDC >pDCs > Basophils. C. Representative plots showing heterogenous expression of CD52 on Naïve B cells and pDCs.

Figure 3. Calibration of Simply Quantum cellular microspheres with alemtuzumab.

Uniformly sized microspheres coated with different numbers (shown above each histogram) of anti-human Fc molecules defined as antibody binding capacity (ABC) are incubated with a saturating concentration of FITC-conjugated alemtuzumab (5 µg/ml). The beads were analyzed by flow cytometry on an LSR-II instrument and the median fluorescence intensity (MFI) values (shown next to each histogram) were plotted against the ABC units (shown on top of each histogram) to generate a standard calibration curve (not shown). The cells were labeled with alemtuzumab-FITC in the same manner as the beads and the MFI of CD52 expression on each cell subset was used to quantify absolute CD52 levels in ABC units or number of CD52 molecules per cell using the calibration curve.

Figure 4. CD52 antigen density on human PBMC subsets.

CD52 expression was determined on subsets of freshly isolated PBMCs from twenty two normal donors using the strategy described in Fig 1. The number of alemtuzumab binding units to CD52 antigen was determined from the calibration curve as described in fig 3. The average CD52 antigen density on lymphoid (A) and myeloid (B) subsets of PBMCs is presented with the error bars representing standard deviation.

Figure 5. Complement-dependent cytolytic effets of alemtuzumab on human PBMCs.

Cells were incubated with 10 µg/ml of alemtuzumab or human IgG1 isotype control in the presence of 10% purified human complement for 3 hrs. Alemtuzumab mediated cytotolysis was assessed by flow cytometry using an LSR-II instrument and a representative dot plot is presented (A). Panel 1 shows the gating strategy. All cells except platelets and counting beads were included into gate H. Panel 2 shows the dot plot analysis of cells from gate H in panel 1. Live cells are negative for Annexin-V and 7AAD. Total lysis was calculated by adding total 7AAD (necrotic) and Annexin V positive (apoptotic) cells. (B). The percentage lysis mediated by alemtuzumab (dark bars) compared to control IgG1(white bars) from each donor is presented. The error bars represent standard deviation (*p<0.05).

Figure 6. PBMC subsets that survived the cytolytic effects of alemtuzumab.

The absolute cell numbers of individual lymphoid (A) and myeloid subsets (B) from PBMCs that survived cytolytic effects (live gate of figure 5) from IgG1 treated (white bars) and alemtuzumab treated (filled bars) of each donor is presented. The error bars represent standard deviation (*p<0.05).

Figure 7. Expression of complement inbitory proteins is higher on myeloid cells than lymphocytes.

Expression levels of CD46, CD55 and CD59 proteins were measured by flow cytometry on individual lymphoid and myeloid cell subsets in PBMCs from donors on whom CDC experiments were conducted. Average median fluorescence intensity values of each CIP along with standard deviations are shown. * Significantly higher levels than the lymphocyte subsets (p<0.02) ^Λ Significantly higher levels than B cells, effector T and NK cells (p≤0.05) # Significantly higher levels than effector T and NK cells (p≤0.02) @ Significantly higher levels than central memory(CM), effector memory(EM), effector T cells and NK cells (p≤0.02) $ Significantly higher levels than CD4-naïve, central memory(CM), effector meory(EM), effector CD4 and CD8 T cells and NK cells (p≤0.01) & Significantly higher levels than CD8 effector T and NK cells (p≤0.02) ∞ Significantly higher levels than CD8 effector memory and CD4 and CD8 effector T cells (p≤0.03) + Significantly higher levels than effector T cells and CD8-naïve T cell subset (p≤0.05).

Figure 8. Alemtuzumab mediates robust cytolysis of purified T cells but not NK cells.

Blocking anti CIP antibodies partially reverse the resistance of monocytes to lysis by alemtuzumab: PBMCs from each of the 4 donors were sorted using a FACS Aria cell sorter into CD3+ T, CD56+ NK and CD14+ CD11c+ monocytes to >95% purity. Each of these purified populations was subjected to a CDC assay in the presence or absence of 15 µg/ml of anti-CD55 and anti-CD59 antibodies. The percent cytolysis was assessed as described in Fig 5.The hatched bars represent lysis with alemtuzumab without the blocking anti-CD55 and anti-CD59 antibodies and the shaded bars in the presence of blocking antibodies. The control IgG1 values were subtracted before plotting the data. (Background IgG1 range for T cells  = 5–10%; NK cells  = 5−15%; and monocytes  = 15−31%). *p≤0.01 (IgG vs Alem alone) **p≤0.001 (Alem alone vs Alem + anti-CIP abs).