In mice with advanced leukemia, CTLs specific for the antigen expressed in leukemia cells were also expanded, but could not suppress disease progression.

<p>(A-C) Analysis of BM and spleen cells from mice with advanced MLL/AF9-OVA leukemia (A) Flow-cytometry analysis of the frequencies of GFP⁺ leukemia cells among the whole BM or spleen cells and the frequencies of H-2K^b/OVA tetramer-positive cells among CD8⁺ T cells. (B) Flow-cytometry analysis of cytokine production by CD8⁺ T cells in BM and spleen with or without SIINFEKL peptide stimulation. (C) Percentages of IFN-γ- and/or TNF-α-producing cells among CD8⁺ BM or spleen T cells, with or without SIINFEKL peptide stimulation (n = 3). *: p < 0.05 (D) Analysis of the expression of T-cell exhaustion–associated markers in H-2K^b/OVA tetramer-positive CD8⁺ T cells. BM cells from non-leukemic mice and mice with advanced leukemia were analyzed (n = 3 for each). Representative flow-cytometry analysis and bar graphs for mean fluorescence intensities (MFI) are shown. Dotted lines represent isotype controls.*: p < 0.05, N.S.: not statistically significant. (E) Analysis of the expression of H-2K^b, GFP, and the presentation of SIINFEKL peptide in leukemia cells that developed in wild-type or <i>Rag2</i>^-/- recipients. Representative flow-cytometry analysis and bar graphs for MFI are shown. Dotted lines represent isotype controls.</p

Spontaneous regression of leukemia was observed in the presence, but not in the absence, of adaptive immunity.

<p>(A) Flow-cytometry analyses of BM cells of non-irradiated wild-type recipients 7 days after transplantation with different numbers (3 × 10³, 3 × 10⁴, or 3 × 10⁵) of MLL/AF9-OVA leukemia cells. (B) FACS analysis of BM from non-irradiated wild-type or <i>Rag2</i>^-/- mice transplanted with 3 × 10⁴ MLL/AF9-OVA leukemia cells. Mice were analyzed 3 weeks after transplant. (C) Kaplan–Meier curves for overall survival of non-irradiated wild-type (n = 7) or Rag2^-/- (n = 3) recipients transplanted with 3 × 10⁴ MLL/AF9-OVA leukemia cells. (D) Percentages of GFP⁺ leukemia cells in BM after transplantation into non-irradiated wild-type mice were examined every week. Each dot and line corresponds to a recipient mouse. The results of four mice in which leukemia spontaneously regressed (Exp. 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144594#pone.0144594.t001" target="_blank">Table 1</a>) are shown. (E) FACS analysis of BM from non-irradiated wild-type or <i>Rag2</i>^<i>-/-</i> mice transplanted with 3 × 10⁴ of MLL/AF9 leukemia cells (OVA-). Mice were analyzed 3 weeks after transplant.</p

Functional CTLs specific for the antigen expressed in leukemia cells were highly expanded in mice that did not develop leukemia.

<p>Analysis of BM and spleen cells from mice 4 weeks (A–C, n = 5) or 6 weeks (D–F, n = 3) after transplant with 3 × 10⁴ MLL/AF9-OVA leukemia cells. (A, D) Flow-cytometry analysis of the frequencies of GFP⁺ leukemia cells in the 7AAD^- whole BM or spleen cells and those of H-2K^b/OVA tetramer-positive cells in CD8⁺ T cells. (B, E) Flow-cytometry analysis of cytokine production by CD8⁺ BM and spleen T cells, with or without SIINFEKL peptide stimulation. (C, F) Percentages of IFN-γ- and/or TNF-α-producing cells in BM or spleen CD8⁺ T cells, with or without SIINFEKL peptide stimulation. *: p < 0.05.</p

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

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

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

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

Apoptotic function of an Ex4a(+)WT1 isoform.

<p>(<b>A</b>) The role of Ex4a(+)WT1 in apoptosis. Ex4a(+) or Mock vector was transfected into WT1-expressing HT-1080 cells. Frequencies (%) of Annexin V-positive apoptotic cells and cells with loss of MMP were determined by flowcytometry after 24 h. Left, Frequencies (%) of Annexin V-positive apoptotic cells are shown. Right, Frequencies (%) of cells with mitochondrial membrane potential (MMP) loss are shown. Results are means and S.D. of three independent experiments. *, p<0.05. (<b>B</b>) Expression of Ex4a(+) and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and analyzed for Annexin-V positive apoptotic cells and expression of Ex4a(+) and major WT1 isoforms by flowcytometry and RT-PCR, respectively. Upper, Frequencies (%) of Annexin V-positive apoptotic cells. Lower, RT-PCR using Ex4-F and Ex6-R primer pair that amplifies both Ex4a(+) and major WT1 isoforms. GAPDH is used as an internal control. Results are representative of three independent experiments. (<b>C</b>) Change of Ex4a(+)WT1 and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and expression of Ex4a(+)WT1 and total WT1 isoforms including both Ex4a(+) and major WT1 isoforms were determined by quantitative real-time RT-PCR using Ex4a-F and Ex6-R primer pair and Ex6-F and Ex7-R primer pair, respectively. Actin is used as an internal control for normalization. Expression levels of Ex4a(+)WT1 and total WT1 in Dox-untreated cells are defined as 1.0. (<b>D</b>) Suppression of Ex4a(+)WT1 inhibits Dox-induced apoptosis. K562 cells were transfected with one μg of either of two WT1 Ex4a-specific siRNAs (si-4a-1 and si-4a-2) or a control siRNA (si-control) together with 2.0 μg of Ex4a(+)WT1 vector or 2.0 μg of empty vector (Mock), cultured for 24 h, treated with 4.0 μM Dox for 12 h, and then analyzed for Annexin-V positive apoptotic cells by flowcytometry. Frequencies (%) of Annexin V-positive apoptotic cells are shown. Results are mean and S.D. of three independent experiments. *, p<0.05.</p