HLA class I, NKG2D, and natural cytotoxicity receptors regulate multiple myeloma cell recognition by natural killer cells by Ennio Carbone, Paola Neri, Maria Mesuraca, Mariateresa T. Fulciniti, Takemi Otsuki, Daniela Pende, Veronika Groh, Thomas Spies, Giuditta Pollio, David Cosman, Lucio Catalano, Pierfrancesco Tassone, Bruno Rotoli, and Salvatore Venuta Blood Volume 105(1):251-258 January 1, 2005 ©2005 by American Society of Hematology

NK recognition of bone marrow, peripheral blood, and pleural effusion-derived myeloma cell line by purified allogeneic NK cells activated with IL-2. NK recognition of bone marrow, peripheral blood, and pleural effusion-derived myeloma cell line by purified allogeneic NK cells activated with IL-2. Close circles represent the percentage of specific lysis of distinct myeloma cell targets: (A and D) KMS12BM, (B) KMS21BM, (C) with cross KMS27PB. Close squares indicate the lysis levels of pleural effusion-derived myeloma target cells: (A) KMS12PE, (B) KMS21PE. In panel C and D with X and open diamond the percentage of specific lysis of KMS11PE and KMS26PE, respectively. The ratios used were 25:1, 12:1, 6:1, 3:1 indicated in abscissa as 1, 2, 3, and 4, respectively. Each panel described 1 representative experiment of at least 3 performed. NK cells preferentially killed BM myeloma lines, while PE-derived cells were less NK susceptible. Ennio Carbone et al. Blood 2005;105:251-258 ©2005 by American Society of Hematology

Reciprocal surface expression of MHC class I and MICA between bone marrow- and pleural effusion-derived myeloma cell lines. Reciprocal surface expression of MHC class I and MICA between bone marrow- and pleural effusion-derived myeloma cell lines. (A) BM, PB, and PE myeloma cell lines were stained with isotypic mAb control (open curves) or with anti-MHC class I antibody (filled area). A clear overexpression of MHC class I molecule is detected in PE cell lines (1 of 5 representative experiments). (B) Staining obtained using BAM195 anti-MICA antibodies: MICA expression is detectable in BM cell lines but not in PE cell lines. Similar results were obtained using the anti-MICA-MICB antibody 6D4. Ennio Carbone et al. Blood 2005;105:251-258 ©2005 by American Society of Hematology

Northern blot and RT-PCR analysis of MHC-I and MICA expression in myeloma cell lines. Northern blot and RT-PCR analysis of MHC-I and MICA expression in myeloma cell lines. (A) Northern blot analysis. Total RNA (10 μg) from T2 (lane 1), KMS21BM (lane 2), KMS12BM (lane 3), KMS12PE (lane 4), KMS26PE (lane 5), KMS11PE (lane 6), and HeLa (lane 7) cell lines was hybridized with MHC-I and MICA cDNA fragments. For the evaluation of gel load and transfer, the same filters were rehybridized with a fragment of GAPDH cDNA. Reciprocal amounts of MHC class I heavy chains and MICA transcripts levels were observed between BM and PE myeloma cell lines. (B) RT-PCR was performed from RNA preparations in duplicate, using specific primers for MHC class I, and MICA (40 cycles). cDNAs were obtained from: KMS12BM (lanes 1-2), KMS12PE (lanes 3-4), KMS21BM (lanes 5-6), and KMS21PE (lanes 7-8). The products were analyzed by agarose gel and were normalized using the levels of GAPDH (20 cycles) as an internal control. The RT-PCR analysis confirmed the inverse correlation between the MHC class I and MICA transcript levels found in BM and PE cell lines. Ennio Carbone et al. Blood 2005;105:251-258 ©2005 by American Society of Hematology

MHC class I inhibits NK cell recognition of pleural effusion-derived myeloma cell lines. MHC class I inhibits NK cell recognition of pleural effusion-derived myeloma cell lines. Mean NK lysis percentage of KMS12BM and of KMS12PE are reported in panel A, while NK lysis of KMS21BM and KMS21PE are reported in panel B. BM and PE myeloma cell lysis are indicated with white and black columns, respectively. Antibody pretreatment of the PE targets are reported on abscisses: 1) complete medium, 2) anti-CD57 (TIB200) isotype control, 3) anti-MHC class I mAb A6 to 136 (IgM). The percentage of lysis reported was calculated with E/T ratio of 12:1. MHC class I masking restored myeloma susceptibility to NK recognition. Ennio Carbone et al. Blood 2005;105:251-258 ©2005 by American Society of Hematology

NCR and NKG2D are the main activating receptors involved in NK recognition of bone marrow-derived myeloma cells. NCR and NKG2D are the main activating receptors involved in NK recognition of bone marrow-derived myeloma cells. KMS12BM (A) and KMS21BM (B) killing by resting purified NK cells. Target cells were treated with control antibody (○), anti-NCR (▪), anti-NKG2D (▴), and anti-NCR + NKG2D (•). One representative experiment of 4 performed is reported. E/T ratio indicated on abscissa are 12:1 (1) and 6:1 (2). (C-D) Average NK lysis and SEM are calculated from 4 experiments with KMS12BM and KMS21BM, respectively. The target treatment with different antibodies is reported on the abscissa: untreated target cell percentage of killing is indicated as cnt. Separately, anti-NCR and anti-NKG2D treatment of NK cells inhibited BM myeloma cells recognition; when used together, a clear synergistic inhibitory effect emerged. Ennio Carbone et al. Blood 2005;105:251-258 ©2005 by American Society of Hematology

Heterogeneous MHC class I molecule expression on freshly isolated CD38+ and CD138+/CD38+ bone marrow myeloma cells. Heterogeneous MHC class I molecule expression on freshly isolated CD38+ and CD138+/CD38+ bone marrow myeloma cells. Staining profile of MHC class I expression on patients 2, 3, 4, 5, 6, and 10 derived CD38+ cells are reported in panels i, ii, iii, iv, v, and vi, respectively (A). It should be noted that in patients 2 and 3 the myeloma cells were stained equally well by either anti-CD38 or anti-CD138 mAbs; for p10 MHC class I expression was tested on immunomagnetic-purified CD38+/CD138+ myeloma cells, and for patients 4, 5, and 6 CD38bright cells were gated. At least 10 000 events were analyzed within the CD38+bright or the CD38+/CD138+ gated cells. Open curves are the isotypic control; close curves are W6-32 staining. The correlation between myeloma clinical stages and MHC class I expression was analyzed (B). Stage IIA (n.4), stage IIB (n.2), stage IIIA (n.5), and stage IIIB (n.3). The differences between stage IIB and IIIB reached statistical significance with P of 05 or less. The analysis was performed via gating on living MHC class I-positive myeloma cells. Ennio Carbone et al. Blood 2005;105:251-258 ©2005 by American Society of Hematology

Autologous NK cells recognize freshly explanted bone marrow myeloma cells. Autologous NK cells recognize freshly explanted bone marrow myeloma cells. (A) Freshly explanted autologous NK and myeloma cells were used in cytotoxicity assays. The results obtained in patients 1, 7, 8, 9, 10, and 11 are shown. ♦ indicates recognition by autologous fresh NK cells; •, percentage of lysis obtained using as effectors IL-2 activated autologous NK cells. The E/T ratios used were 25:1 (1), 12:1 (2), 6:1 (3), and 3:1 (4). Myeloma cells from patient 10 were obtained by immunomagnetic selection using BB-4 (anti-CD138) mAb and kept without exogenous IL-6 treatment. Five of 6 patients showed a very high autologous NK recognition of BM-derived myeloma cells, while patient 1 had less efficient autologous myeloma elimination. In patient 1, 10, and 11, IL-2 activation of NK cells had a beneficial effect on autologous myeloma recognition. (B) CD38+ cells from bone marrow aspirates were double stained with anti-MHC class I, W6-32 (i-iii) or anti-MICA, BAM195 (iv-vi) antibodies. Open areas are isotype controls; filled areas are specific antibody staining. Patient 1 had higher MHC class I expression than patient 7 and patient 8, while MICA was not detectable. Patient 7- and patient 8-derived myeloma cells were readily stained with anti-MICA antibody. After subtracting the background value the MHC class I surface expression median value for the patients 1, 7, and 8 were, respectively, 30, 8, and 10, while the MICA median values were 3, 10, and 8. For patients 9, 10, and 11 it was not possible to evaluate the complete phenotype because of the limited number of cells obtained. Ennio Carbone et al. Blood 2005;105:251-258 ©2005 by American Society of Hematology