Correspondence 1448 5 Maserati E, Seghezzi L, Pasquali F, Locatelli F. Parental origin of 8 Cross NC, Melo JV, Feng L, Goldman JM. An optimized multi- chromosomes 9 and 22 involved in the Ph chromosome transloca- plex polymerase chain reaction (PCR) for detection of BCR-ABL tion in chronic myelocytic leukemia. Cancer Genet Cytogenet fusion mRNAs in haematological disorders. Leukemia 1994; 8: 1998; 107: 151–152. 186–189. 6 Nakamura H, Itoyama T, Niikawa N, Sadamori N, Tomonaga M. No 9 Jankovic´ GM, Cˇ olovic´ MD, Bogdanovic´ AD, Cˇ olovic´ NR, Jankovic´ parental origin bias for the rearranged chromosomes in myeloid SJ. Parental reciprocation in t(9;22) vs genomic imprinting. leukemias associated with t(9;22), t(8;21) and t(15;17). Leukemia Leukemia 1996; 10: 1402. Res 1998; 22: 793–796. 10 Fioretos T, Heisterkamp N, Groffen J. No evidence for genomic 7 Melo JV, Xiu-Hua Y, Diamond J, Goldman JM. Balanced parental imprinting of the human BCR gene. Blood 1994; 83: 3441–4344. contribution to the ABL component of the BCR-ABL gene in chronic 11 Melo JV, Xiu-Hua Y, Diamond J, Goldman JM. Lack of imprinting myeloid leukemia. Leukemia 1995; 9: 734–739. of the ABL gene. Nat Genet 1994; 8: 318–319. Biochemical pathway of antigen processing by HLA class II molecules in B cell lymphomas Leukemia (2004) 18, 1448–1450. doi:10.1038/sj.leu.2403384 peptide-loaded HLA-DR molecules at the cell surface in various Published online 20 May 2004 types of B cell lymphomas and compared them with normal B lymphocytes from spleen and peripheral blood. Lymph node TO THE EDITOR biopsies were obtained from 16 patients with four diffuse large B cells (DLCL), four mantle cells (MCL), four follicular (FL), and Non-Hodgkin’s B cell lymphomas are characterised by the four lymphocytic (LL) lymphomas. Diagnosis was established expression of both human leukocyte antigen (HLA) class I and according to the WHO classification. B lymphocytes, except for class II molecules. As HLA class II molecules are constitutively peripheral blood (positive purification), were purified by expressed only in professional antigen-presenting cells (APCs), B negative immunomagnetic selection (final population 498% lymphomas are considered as professional APCs. Contrary to the CD19 þ cells). A total of 16 lymphomas expressed HLA-DR majority of tumour cells that usually express only HLA class I molecules at their surface (Table 1). Different populations of molecules, B cell lymphomas can also be considered as an HLA-DR complexes were exposed at the cell surface of B original model for cellular immunotherapy involving the CD4T lymphocytes. These include peptide-loaded HLA-DR complexes lymphocyte component in direct contact with tumour cells. We and HLA-DR/CLIP complexes. Strikingly, four MCLs and three and others previously reported that CD4T lymphocytes may play DLCLs presented no or few HLA-DR/CLIP complexes at their a direct role by cell–cell contact and could not only directly surface when compared to other lymphomas and normal B target tumour cells expressing HLA class II molecules (review by Wang1) but also by differentiation or cell cycle progression. Moreover, modifications of the second messengers in the signalling pathway via class II HLA-DR molecules were Table 1 Purified B lymphocytes were analysed by flow cytometry previously described in B lymphomas.2 This could be of interest in the field of cellular immunotherapy where APCs and Lymphoma DR DR-CLIP Ratio (%) DR-CLIP/DR particularly dendritic cells are used to generate specific antitumour CD4 and CD8T lymphocytes. DLCL 1 750 87 11.6 HLA class II molecules biosynthesis begins in the endoplas- DLCL 2 283 4 1.5 mic reticulum. They are subsequently targeted at compartments DLCL 3 312 13 4.2 intermediately between late endosomes and lysosomes called DLCL 4 1560 10 0.6 MCL 1 2797 12 0.4 MIIC (for MHC class II compartment) where antigen loading can MCL 2 698 0 0 take place, before targeting to the cell surface (review by MCL 3 1067 1 0.1 3 Alfonso and Karlsson ). During this intracellular pathway, the MCL 4 1235 5 0.4 Invariant chain (Ii) blocks the peptide-binding groove of HLA- LL 1 1685 37 2.2 DR molecules and is sequentially degraded until the generation LL 2 1780 66 3.7 of a peptide called CLIP (Class II associated Invariant chain LL 3 488 15 3.1 LL 4 212 10 4.7 peptide). The loading of antigenic peptides instead of CLIP is FL 1 1055 9 0.8 then catalysed by HLA DM in the MIIC, which is in turn FL 2 712 43 6 modulated by HLA DO (selectively expressed in B lymphocytes FL 3 1374 34 2.5 and a specific negative modulator of HLA DM outside the MIIC). FL 4 1086 23 2.1 In this study we explored the key components of the Control 1 899 34 3.8 biochemical pathway leading to the expression of antigenic Control 2 1312 22 1.7 Control 3 1892 40 2.1 Control 4 1190 75 6.3 Correspondence: Dr F Garban, De´partement de cance´rologie et Mean fluorescence intensity for total HLA-DR (mAb L243) and HLA d’he´matologie, CHU de Grenoble, BP 217, 38 043 Grenoble Cedex 9, DR/CLIP (mAb CerCLIP) is presented for each sample and were France; Fax: þ 33 4 76 76 56 61; obtained by subtracting the value of the isotype control antibody to the E-mail: [email protected] sample value. The ratio of HLA-DR/CLIP vs total surface HLA-DR was 3N. Magniez and C. Roucard have equally contributed to this work. calculated. Controls 1 and 2 are from spleen, controls 3 and 4 are from Received 31 July 2003; accepted 12 February 2004; Published online peripheral blood. DLCL, diffuse large cell lymphomas; LL, lymphocytic 20 May 2004 lymphomas; FL, follicular lymphomas; MCL, mantle cell lymphomas. Leukemia Correspondence 1449 Figure 1 Total cell lysates of purified B lymphocytes were analysed by Western blot. Equal amounts of protein were loaded per lane (5 mg for HLA-DR, DM and actin, 15 mg for HLA DO) as demonstrated with actin. SDS stable HLA-DRab complexes (55 kDa) were revealed by incubating the samples for 30 min at room temperature and loading without boiling (NB) and dissociated as a single a chain (34 kDa) after boiling (B). HLA- DRa chain is revealed with DA6-147 mAb (A), HLA-DMa with 5C1 mAb (B), HLA-DOa with polyclonal rabbit serum (B), actin with a specific anti- actin IgM mAb. The corresponding molecular weight of each band is indicated on the right. Control 1 comprises peripheral blood cells and control 2 comprises spleen cells. Figure 2 Purified B lymphocytes adhered onto polyL-lysine-coated slides were fixed by formaldehyde, permeabilised by Triton X-100 and incubated with a polyclonal rabbit serum against HLA-DRab complexes and a mAb against LAMP-1, respectively, revealed with FITC-conjugated swine anti-rabbit antibodies and Cyanin 3 conjugated goat anti-mouse antibodies. Pictures were acquired on an LSM410 Axiovert200 confocal microscope (ZEISS) with LSM410 software from Zeiss. A minimum of 20 pictures were obtained from one sample (80–100 pictures for each lymphoma type). A representative cell for each cell type is shown. lymphocytes (Table 1). As HLA-DR/CLIP complexes are usually Based upon the observation of few or no HLA-DR/CLIP at the found at the cell surface of normal B lymphocytes,4 this raises cell surface in some lymphomas and the fact that CLIP the possibility that differences or abnormalities exist in the dissociates from HLA-DR at acidic pH, we hypothesised that intracellular processing of peptides and loading on HLA-DR HLA-DR molecules could travel via more acidic compartments molecules, leading to complete proteolysis of the invariant on their pathway to the cell surface. Intracellular localisation of chain and complete removal of CLIP. HLA-DR was therefore explored by confocal microscopy and HLA-DR molecules loaded with a stably bound peptide paralleled with a specific marker of lysosomes LAMP-1 remained associated as a 55 kDa ab complex in the presence of (Figure 2). SDS at room temperature, and dissociated into free a and b Two different patterns emerged: one consisting of a group chains after boiling (non-boiled NB and boiled B, Figure 1a). with no colocalisation of HLA-DR and LAMP-1 (FL, LL, DLCL 1, HLA-DR molecules from all lymphomas contained SDS resistant normal B lymphocytes) and a second group (DLCL 2–4 and MCL complexes, demonstrating the presence of peptides-loaded 1–4) where HLA-DR was largely colocalised in acidic compart- complexes (Figure 1a). HLA-DM were also present in all types ments labelled with LAMP-1, confirming our hypothesis. of lymphomas, but were lower expressed in the sample from LL Pictures from nonmalignant B lymphocytes from spleen and (Figure 1b). Remarkably, we observed a constant lower peripheral blood showed a very weak co-localisation (Figure 2). expression or absence of HLA DO in the four MCL compared Unlike classical solid tumours, the constitutive expression of to other samples and normal B lymphocytes (Figure 1b). A HLA class II molecules on their cell surface allow malignant B representative experiment for each type of lymphomas is shown lymphocytes from non-Hodgkin’s B cell lymphomas to interact (Figure 1). with CD4T lymphocytes infiltrating the tumour, and thus Leukemia Correspondence 1450 represent a possible way for the tumour to manipulate the Acknowledgements immune system. To the best of our knowledge, this is the first study of the major components of the biochemical pathway for N Magniez was supported by La Ligue Contre le Cancer Haute- antigen presentation by class II molecules: HLA-DR, DM and Savoie.