Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

<div><p>To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54^high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54^high or B2^high leukemia cells preferentially survived <i>in vivo</i> in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis <i>in vitro</i> and <i>in vivo</i>. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.</p></div

mAbs R54 and B2 both specifically recognize CD43.

<p>(A) FACS plots showing the course of the enrichment of R54-positive cells from YB2/0 cells transduced with a MLL/AF9 leukemia cell–derived cDNA library. (B) FACS analysis of the binding of S11, R54, or B2 mAb to splenocytes from wild-type or CD43-deficient mice.</p

Glycosylation status of CD43 on leukemia cells is associated with sensitivity to CTL-mediated cytolysis.

<p>(A) Gating strategies for FACS-sorting the R54^high and R54^low subpopulations of OVA-expressing MLL/AF9 leukemia cells. (B) FACS analysis of intracellular IFN-γ in OT-1 T cells after co-culture with either R54 ^high or R54^low MLL/AF9 leukemia cells. IFN-γ expression in CD8⁺ T cells is shown. (C) ⁵¹Cr cytotoxicity assay with OT-1 T cells, using either R54 ^high or R54^low leukemia cells as targets. (D) FACS analysis of OVA-IRES-GFP expression levels in MLL/AF9-OVA leukemia clones derived from c-kit⁺ BM cells of the wild type or CD43^-/- mouse (E) ⁵¹Cr cytotoxicity assay with OT-1 T cells, using either the wild type or CD43^-/- leukemia cells as targets (F)⁵¹Cr cytotoxicity assay with OT-1 T cells, using leukemia cells with or without sialidase treatment (E/T ratio = 1).</p

Epitopes for R54 and B2 mAbs, but S11 mAb, are sensitive to O-glycosylation inhibitor or sialidase.

<p>FACS analysis of binding of each CD43-specific mAb to MLL/AF9 leukemia cells or M1 leukemia cells treated with 1 mM benzyl-GalNac for 24 hours (A) or 250 U/ml sialidase for 1 hour (B).</p

The specificities of mAbs R54 and B2 differ from that of the pan-CD43 mAb S11.

<p>(A) FACS analysis for binding of mAb S11, R54, or B2 to B, myeloid, CD4 T, and CD8 T cells in splenocytes from wild-type mice. Gating strategies for each populations are also shown. (B) FACS analysis for binding of mAb S11, R54, or B2 to hematopoietic stem cell (HSC) or myeloid progenitor cell (MP) populations of BM cells from wild-type mice.</p

Glycosylation status of CD43 on leukemia cells is associated with selection of leukemia cells <i>in vivo</i> in the presence of adaptive immune cells.

<p>(A) Scheme showing the experimental design. (B) FACS analysis for binding of mAb S11, R54, or B2 to leukemia cells developed in wild-type or <i>Rag2</i>^-/- recipients (n = 5 for each genotype). Representative FACS plots are shown. (C) Bar graph showing the averages of mean fluorescence intensities (MFI). *:p<0.05, N.S.: not statistically significant.</p

Histopathological changes in lungs of common marmosets inoculated with MERS-CoV.

<p>Common marmosets were euthanized on day 3, 4 or 6 post inoculation and lung tissue was collected and stained with hematoxylin and eosin (H&E; panels <b>A</b>, <b>C</b>, <b>E</b>, <b>G</b>, <b>I</b>) or immunohistochemistry using a polyclonal α-MERS-CoV antibody (IHC; panels <b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>, <b>J</b>). (<b>A</b>) Acute bronchointerstitial pneumonia centered on terminal bronchioles, with influx of inflammatory cells and thickening of alveolar septa in lung tissue collected on 3 dpi. Asterisk indicates essentially normal tissue. (<b>B</b>) IHC staining of sequential section of panel A reveals abundance of MERS-CoV antigen in affected areas. (<b>C</b>) Coalescing bronchointerstitial pneumonia inducing a diffuse lesion on 3 dpi. (<b>D</b>) IHC staining of sequential section of panel C reveals abundance of MERS-CoV antigen in affected areas. (<b>E</b>) Edema, hemorrhage and fibrin (asterisks) fill the alveolar spaces in lung tissue collected on 3 dpi. Arrowhead indicates syncytium. Inset highlights thickened alveolar interstitium with fibrin, edema and inflammatory cells. (<b>F</b>) IHC staining of sequential section of panel <b>E</b>. (<b>G</b>) Type II pneumocyte hyperplasia is visible on 6 dpi, as highlighted further in inset. (<b>H</b>) IHC staining of sequential section of panel <b>G</b> indicates viral antigen has been mostly cleared from remodeling tissue. (<b>I</b>) On 6 dpi, fibrin is consolidating into hyaline membranes (arrows). (<b>J</b>) IHC staining of sequential section of panel <b>I</b>. Magnification: <b>A</b>, <b>B</b>, <b>C</b> and <b>D</b> 4×; <b>E</b>, <b>F</b>, <b>G</b>, <b>H</b>, <b>I</b> and <b>J</b> 40×; inset in panel <b>E</b> and <b>G</b> 100×.</p

Radiographic alterations and lung pathology.

<p>Dorsal-ventral and lateral thoracic x-rays from of common marmosets (CM) imaged prior to MERS-CoV inoculation (day 0) and on days 3 and 4 post-inoculation. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (circle). Positional indicators are included (R – right, L – left). Gross pathology of the lungs from CM5 and CM9 necropsied on 4 dpi are shown below indicating extensive gross lesions. dpi are shown below indicating extensive gross lesions.</p

The cellular tropism of MERS-CoV.

<p>(<b>A</b>) DPP4 is evident on type I pneumocytes (arrow), bronchiolar epithelial cells and smooth muscle cells. (<b>B</b>) <i>In situ</i> hybridization demonstrating the presence of viral RNA in type I pneumocytes (black arrow) and alveolar macrophages (blue arrow). (C) Type I pneumocytes (green) are lost following infection with MERS-CoV (lower right). Viral antigen (red) can be observed in type I pneumocytes (arrow). Magnification: <b>A</b> 500×; <b>B</b> 200×; <b>C</b> 500×.</p

Interaction between the MERS-CoV spike glycoprotein (S1) and its receptor dipeptidyl peptidase 4 (DPP4).

<p>(<b>A</b>) Alignment of the amino acid residues from human, common marmoset and ferret DPP4 that have been identified to interact with the receptor binding domain of the MERS-CoV spike glycoprotein. (<b>B</b>) Interaction model (front, back and side view) of the MERS-CoV S1 and its cognate receptor human DPP4, with amino acid differences in common marmoset DPP4 are highlighted in red.</p