Multiple myeloma (MM) is an aggressive cancer that originates from antibody-secreting plasma cells. Although genetically and transcriptionally well characterized, the aberrant gene regulatory networks that underpin this disease remain poorly understood. Here, we mapped regulatory elements, open chromatin, and transcription factor (TF) footprints in primary MM cells. In comparison with normal antibody-secreting cells, MM cells displayed consistent changes in enhancer activity that are connected to superenhancer (SE)-mediated deregulation of TF genes. MM cells also displayed widespread decompaction of heterochromatin that was associated with activation of regulatory elements and in a major subset of patients' deregulation of the cyclic adenosine monophosphate pathway. Finally, building SE-associated TF-based regulatory networks allowed identification of several novel TFs that are central to MM biology. Taken together, these findings significantly add to our understanding of the aberrant gene regulatory network that underpins MM.

Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming TAM populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, FcγRIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed to modify myeloid cells of the TME represent a promising mode of cancer treatment.

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. In this study, RNA sequencing, splice isoform-specific quantitative RT-PCR, lentiviral transduction, and in vivo humanized mouse model studies demonstrated that malignant reprogramming of progenitors into self-renewing blast crisis chronic myeloid leukemia stem cells (BC LSCs) was partially driven by decreased MBNL3. Lentiviral knockdown of MBNL3 resulted in reversion to an embryonic alternative splice isoform program typified by overexpression of CD44 transcript variant 3, containing variant exons 8-10, and BC LSC proliferation. Although isoform-specific lentiviral CD44v3 overexpression enhanced chronic phase chronic myeloid leukemia (CML) progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. Combined treatment with a humanized pan-CD44 monoclonal antibody and a breakpoint cluster region - ABL proto-oncogene 1, nonreceptor tyrosine kinase (BCR-ABL1) antagonist inhibited LSC maintenance in a niche-dependent manner. In summary, MBNL3 down-regulation-related reversion to an embryonic alternative splicing program, typified by CD44v3 overexpression, represents a previously unidentified mechanism governing malignant progenitor reprogramming in malignant microenvironments and provides a pivotal opportunity for selective BC LSC detection and therapeutic elimination.

Introduction Malignant reprogramming, first described in chronic myeloid leukemia (CML), occurs upon activation of the Wnt/b-catenin pathway in granulocyte-macrophage progenitors (GMPs) that gain the capacity to self-renew and contribute to the emergence of BCR-ABL1 tyrosine kinase inhibitor (TKI) resistant blast crisis CML. Deregulation of the Wnt/b-catenin target gene, CD44, plays a vital role in leukemia stem cell (LSC) maintenance in the malignant microenvironment in mouse models of CML. However, extensive alternative mRNA splicing in humans results in expression of multiple CD44 isoforms, some of which have been implicated in cancer invasion and metastasis. In this study we investigated the role of CD44 splice variant expression on human blast crisis LSC maintenance in the malignant niche. Methods and Results CD44 Isoform Expression Analysis To investigate the splice isoform expression pattern of CD44, whole transcriptome RNA sequencing (RNA Seq; Illumina HiSeq 2000) was performed on FACS sorted chronic phase (CP; n=8) and blast crisis (BC; n=8) CML progenitors (CD34 + CD38 + Lin - ) as well as their normal counterparts from cord blood (CB) (n=7) and adult peripheral blood (NPB; n=4). While whole gene expression analysis revealed upregulation of CD44 in blast crisis compared with chronic phase and normal progenitors, a plethora of CD44 transcript variants were also detected including variants 3, 4 (CD44s), 5, 6, 7, 8. Notably, RNA Seq isoform analysis detected a higher expression of CD44 transcript variant 3 in BC compared to CP and CB and NPB. Moreover, CD44 transcript variant 3 gene expression was highly expressed in undifferentied human embryonic stem cells (hESCs) while differentiated hESCs (embryoid bodies) had low expression, suggesting CD44 transcript variant 3 to be important for pluripotency. Lentiviral CD44 Variant 3 Overexpression To directly determine the impact of CD44 variant 3 expression on malignant reprogramming of CP progenitors into self-renewing LSC, we developed a lentiviral human CD44 variant 3 overexpression vector and transduced CP CML progenitors. Transduced CP progenitors harbored increased expression of migration specific markers, such as osteopontin and ICAM1, as well as an upregulation of the pro-survival long isoforms of BCL2 family members BCLX and MCL1, thereby enhancing survival and replating in hematopoietic progenitor assays. Moreover, hESCs transduced with CD44 transcript variant 3 showed upregulation of pro-survival BCL2 isoforms, enhanced proliferation and as well as maintenance of an undifferentiated state, suggesting that CD44 transcript variant 3 promotes pluripotency. Targeted Inhibition of CD44 variant 3 Expressing LSC Humanized RAG2-/-gc-/-mice engrafted with CD34 + BC CML patient samples showed a significant reduction of human progenitor cells post treatment with a clinical grade CD44 mAb, both alone and in combination with Dasatinib in all hematopoietic niches. Bone marrow and spleen samples from primary transplanted mice show a reduced gene expression level of CD44 and CD44 transcript variant 3 upon combination treatment of CD44 and Dasatinib. Most importantly, serial transplantation of progenitors treated with the CD44 mAb as well as in combination with Dasatinib revealed a significant reduction in LSC self-renewal capacity commensurate with a reduction in CD44 variant 3 expression. Conclusions Upregulation of an embryonic splice variant of CD44, variant 3, expands pluripotent stem cell populations and promotes malignant reprogramming of CML progenitors into self-renewing LSC. Treatment with a humanized CD44 specific mAb sensitizes CML LSC residing in malignant niches to Dasatinib. From these results CD44 mAb appears to be an excellent antibody for future combination clinical studies aimed at eradicating therapy resistant blast crisis LSC in CML. In addition, these observations strongly suggest that CD44 transcript variant 3 upregulation serves as a biomarker of progression from CP to BC as well as the generation of TKI resistant LSCs, with the potential of being a more specific target for future combination therapies. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm initiated in hematopoietic stem cells by expression of the BCR-ABL1 fusion oncogene and its protein product, which enhances ABL1 kinase activity. Targeted BCR-ABL tyrosine kinase inhibitors (TKIs), such as Imatinib, and the second generation TKIs like Nilotinib as well as the dual specific SCR and ABL inhibitor Dasatinib have significantly slowed disease progression by eradicating the bulk of BCR-ABL1 expressing cells in the circulation. However, progression to a therapeutically resistant blast crisis (BC) phase is driven by CD34+CD38+Lin- progenitors that co-opt stem cell properties, such as enhanced self-renewal and survival, albeit in a deregulated manner. These BC leukemia stem cells (BC LSC) harbor enhanced BCL2 and beta-catenin expression. Seminal research shows that a key Wnt/beta-catenin target gene, CD44, plays a vital role in cancer stem cell survival and retention in the malignant microenvironment. Because alternative mRNA splicing in humans generates many CD44 isoforms, we investigated CD44 variant expression and retention of human BC LSC in specific hematopoietic niches. Methods and Results We performed whole transcriptome RNA sequencing (RNA Seq) of FACS sorted progenitors (Lin-CD34+CD38+) from chronic phase (CP)(n=8) and blast crisis (BC)(n=8) CML patients as well as from normal cord blood (CB) (n=7) and adult peripheral blood (NPB)(n=4). A number of CD44 transcript variants were detected: 3, 4 (CD44s), 5, 6, 7, 8 plus additional variants. RNA seq analysis uncovered a higher overall CD44 expression in BC compared to CP progenitors. Notably, one isoform of CD44, variant 3, was highly upregulated in BC compared to CP progenitors and minimally expressed by their normal counterparts. Moreover, BC progenitors that engrafted in RAG2-/-γc-/- harbored CD44v3 expression in the bone marrow and the splenic niche and this differential BC LSC isoform expression was reduced after dasatinib treatment. In addition, BC LSC in the spleen showed a reduction of migration specific markers, such as RHAMM, ICAM-1 and Osteopontin, compared with the bone marrow resident BC LSC. Furthermore, a comparative splice isoform specific qRT-PCR analysis of CD44 variants expression in young versus old bone marrow showed no correlation with aging. In fact, human embryonic stem cells harbored variant 3 expression. Conclusions These data suggest that CD44 transcript variant 3 upregulation occurs following malignant reprogramming of hematopoietic progenitors that enables them to adopt features of an embryonic transcriptional program in select microenvironments. Detection of CD44 variant 3 has the potential to more precisely identify patients at risk for relapse and progression, suggest that combined therapeutic strategies involving a BCR-ABL specific TKI and a CD44 monoclonal antibody may decrease relapse and BC transformation rates. Citation Format: Frida L. Holm, Eva Hellqvist, Cayla Mason, Christian Barrett, Kelly A. Frazer, Anil Sadarangani, Catriona HM Jamieson. The niche specific role of CD44 splice isoform expression in blast crisis leukemia stem cell generation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4798. doi:10.1158/1538-7445.AM2014-4798

Abstract Introduction Leukemia stem cells (LSC) in chronic myeloid leukemia (CML) are important in disease progression and relapse. Conventional therapy with tyrosine kinase inhibitors (TKIs), although very effective at reducing bulk CML cells, frequently fail to eliminate LSC residing in the protective bone marrow niche. Thus, there is an unmet need for combination therapy that simultaneously targets bulk disease and eliminates LSC in order to prevent disease progression and relapse. Stem cell niches are often rich in hyaluronic acid. CD44, the main receptor for hyaluronic acid, and some of its splice variants are frequently overexpressed on cancer stem cells, including LSC. In this study we aimed to evaluate the in vivo anti-LSC activity of CD44 monoclonal antibody (mAb) RG7356. Methods and Results For anti-CD44 LSC inhibition studies, immunodeficient RAG2-/-γc-/- neonatal mice were intrahepatically transplanted with human BC LSC from imatinib responsive and resistant patients. Once engraftment was verified, mice were treated with control IgG1 (30mg/kg/week), dasatinib (25mg/kg/day), CD44 mAb (RG7356) (30mg/kg/week) or a combination of CD44 mAb (RG7356) and dasatinib. The mice were sacrificed after two weeks of therapy and bone marrow, spleen, peripheral blood and myeloid sarcomas were analyzed by FACS to assess CML LSC burden. In the imatinib responsive patient, CD44 mAb single agent therapy showed an 85% reduction of LSC in bone marrow, 98% reduction in spleen and 91% reduction in peripheral blood. CD44 mAb therapy thus showed an efficacy equal to that of dasatinib, which showed an 87%, 98% and 98% reduction in bone marrow, spleen and peripheral blood respectively. Combination therapy (CD44 mAb + dasatinib) completely eradicated any trace of CML LSC in all tissues analyzed. In the imatinib resistant patient, dasatinib very effectively reduced the number of myeloid sarcomas and blasts but did not inhibit LSC survival in bone marrow. In contrast, CD44 mAb single agent therapy effectively reduced the number of CML LSC to 51% in bone marrow, 96% in spleen and 93% in peripheral blood. Conclusions This study demonstrates that a human CD44 specific mAb, RG7356, significantly reduces CML LSC survival. Notably, TKI resistance of CML LSC, can be overcome by treatment with a human CD44 specific mAb, RG7356, as it sensitizes CML LSC residing in malignant niches to dasatinib. From these results, RG7356 appears to be an excellent antibody for future combination clinical studies aimed at eradicating CML. Disclosures: Runza: employee of Roche Diagnostics GmbH: Employment. Weigand:Roche Diagnostics GmbH: Employment. Jamieson:J&J: Research Funding; Sanofi: Consultancy; Roche: Research Funding.

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface membrane receptors determines whether the cells will be proliferating, anergic or apoptotic. To better understand the role of antigen in leukemogenesis, CLL cell lines producing monoclonal antibodies (mAbs) will facilitate structural analysis of antigens and supply DNA for genetic studies. We present here a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA/short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA.

Abstract Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the BCR-ABL fusion oncogene whose protein product has greatly increased ABL1 kinase activity. Although specific BCR-ABL tyrosine kinase inhibitors (TKIs) and the second generation TKIs like Nilotinib and the dual specific SCR and ABL inhibitor Dasatinib/Sprycel have dramatically improved CML therapy and significantly slowed disease progression by eradicating the bulk of CML cells in the circulation, they frequently fail to eliminate quiescent leukemic stem cells residing in the protective bone marrow niche. Leukemia stem cells (LSC) are able to drive disease relapse and may eventually contribute to the emergence of TKI resistant blast crisis (BC) CML, which is the final phase in the evolution of CML with rapid progression and short survival. CD44 is an adhesion molecule that promotes retention in the niche through adhesion to extracellular matrix components, such as hyaluronic acid and osteopontin. It plays an important role in wound healing and cell migration as well as in tumor invasion and metastasis. Through alternative mRNA splicing several CD44 isoforms exist, some of which are frequently overexpressed by cancer stem cells, including LSCs. The CD44 variant expression pattern on human blast crisis CML LSC, however, had not been elucidated. In this study we aimed to investigate the CD44 transcript variant expression of human blast crisis CML LSC. Methods and Results We performed whole transcriptome RNA sequencing of FACS sorted CML LSCs (Lin-CD34+CD38+) from chronic phase (CP)(n=8) and blast crisis (BC)(n=8), CML patients as well as the normal counterpart from cord blood (CB) (n=3) and adult peripheral blood (NPB)(n=3). A number of CD44 transcript variants were detected: v3, v4 (CD44s), v5, v6, v7, v8 plus additional variants. Earlier data suggest a total of 16 different isoforms of CD44. We found a higher overall variant gene expression of CD44 in BC compared to CP. A higher expression of CD44 transcript variant 3 and CD44 transcript variant 5 was detected in both CP and BC compared to CB and NPB. Specific CD44 transcript variant expression patterns distinguished BC progenitors from CP samples. Using splice isoform specific PCR, we were able to confirm isoform variants that were upregulated in the BC CML samples. We also compared the expression of the CD44 transcript variants in young versus old bone marrow in order to exclude that LSC-specific isoform variant expression was expressed in aged patients. We could not detect such a correlation. Conclusions These observations suggest that unique CD44 isoform expression patterns predict progression from CP to BC as well as the generation of TKI resistant LSCs and may be used as biomarkers of response to LSC targeted therapy. Disclosures: Jamieson: J&J, Roche: Research Funding; Sanofi: Membership on an entity’s Board of Directors or advisory committees.

Abstract Abstract 3886 ROR1 is a receptor-tyrosine kinase like protein expressed on the surface of chronic lymphocytic leukemia (CLL) B cells, but not on normal mature B cells, suggesting that it may be a promising therapeutic target. We have generated a chimeric monoclonal antibody (mAb), UC99961, which binds to an intradomain epitope of human ROR1 (hROR1). UC99961 binds the same epitope as the murine anti-hROR1 mAb, UC D10–001, which has direct cytotoxic effects on hROR1 positive CLL cells. In this study we investigated the in-vivo anti-leukemic activity and tolerability of UC99961 on ROR1+ primary patient CLL cells and human cord-blood-derived B cells and T cells, respectively. For these studies, immunodeficient RAG2−/−γc−/− neonatal mice were reconstituted with a human immune system by intrahepatic xenotransplantation of 1×105 CD34+ human cord blood progenitor cells. Eight to ten weeks post transplantation, cord blood engraftment was verified by peripheral blood screening, at which point the mice received an intraperitoneal transplantation of 2×107 primary patient ROR1+ CLL cells. Twenty-four hours after CLL transplantation, five animals per group were each treated with a single intraperitoneal injection (10mg/kg) of UC99961, UC D10–001, or control IgG. Seven days following mAb treatment, the animals were sacrificed and marrow, spleen, thymus, and peritoneal lavage samples were collected and analyzed by flow cytometry for CLL cells, as well as normal cord-blood-derived B cells and T cells. To confirm mAb administration according to the study design, serial residual ROR1 plasma antibody levels were determined by ELISA. Results from three consecutive experiments using leukemia cells from two different patients showed that the vast majority of CLL B cells remained in the peritoneal cavity of the animals and did not migrate to other hematopoietic organs. Both anti-hROR1 mAbs UC99961 and UC D10–001 significantly reduced the average number of harvested CLL cells in the peritoneal lavage compared to control IgG (99% and 71% reduction respectively), while cord-blood-derived T cells (CD45+3+) in thymus remained unaffected by the mAb treatment. For the majority of cord-blood-derived B cells in marrow and spleen, no significant reduction could be observed after UC99961 or UC D10–001 mAb treatment. A small CD19+ROR1+CD34− cord-blood-derived B cell population was identified in marrow and spleen that was reduced after UC99961 and UC D10–001 mAb treatment. This study demonstrates that the anti-human ROR1 specific mAbs have in vivo anti-leukemic activity with minimal impact on human cord-blood-derived B cells and T cells. From these results, UC99961 appears to be an excellent candidate antibody for future clinical studies for patients with CLL. Disclosures: No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) cells express the receptor for Epstein-Barr virus (EBV) and can be infected in vitro. Infected cells do not express the growth-promoting set of EBV-encoded genes and therefore they do not yield LCLs, in most experiments. With exceptional clones, lines were obtained however. We describe a new line, HG3, established by in vitro EBV-infection from an IGHV1-2 unmutated CLL patient clone. All cells expressed EBNA-2 and LMP-1, the EBV-encoded genes pivotal for transformation. The karyotype, FISH cytogenetics and SNP-array profile of the line and the patient's ex vivo clone showed biallelic 13q14 deletions with genomic loss of DLEU7, miR15a/miR16-1, the two micro-RNAs that are deleted in 50% of CLL cases. Further features of CLL cells were: expression of CD5/CD20/CD27/CD43 and release of IgM natural antibodies reacting with oxLDL-like epitopes on apoptotic cells (cf. stereotyped subset-1). Comparison with two LCLs established from normal B cells showed 32 genes expressed at higher levels (> 2-fold). Among these were LHX2 and LILRA. These genes may play a role in the development of the disease. LHX2 expression was shown in self-renewing multipotent hematopoietic stem cells, and LILRA4 codes for a receptor for bone marrow stromal cell antigen-2 that contributes to B cell development. Twenty-four genes were expressed at lower levels, among these PARD3 that is essential for asymmetric cell division. These genes may contribute to establish precursors of CLL clones by regulation of cellular phenotype in the hematopoietic compartment. Expression of CD5/CD20/CD27/CD43 and spontaneous production of natural antibodies may identify the CLL cell as a self-renewing B1 lymphocyte.

The aim of the work presented in this thesis was to elucidate B cell interaction with antigen in the two B cell proliferative disorders chronic lymphocytic leukemia (CLL) and monoclonal gammopathy ...

BACKGROUND:Monoclonal gammopathy of undetermined significance of immunoglobulin M isotype is a condition with clonally expanded B cells, recently suggested to have an infectious origin. This monoclonal gammopathy is frequently associated with polyneuropathy and antibodies against myelin protein zero, whereas the role of the T cells remains largely unknown. We analyzed protein zero-specific B cells, as antigen-presenting cells, and their capacity to activate T helper cells. DESIGN AND METHODS:We used a well-characterized monoclonal gammopathy of undetermined significance-derived B-cell line, TJ2, expressing anti-protein zero immunoglobulin M. The ability of TJ2 cells to bind, endocytose, process, and present protein zero was investigated by receptor-clustering and immunofluorescence. The activation of protein zero-specific autologous T cells was studied by measuring interleukin-2 and interferon-gamma with flow cytometry, immunobeads, and enzyme-linked immunospot assays. RESULTS:Surface-receptor clustering and endocytosis of receptor-ligand (immunoglobulin M/protein zero) complexes were pronounced after exposure to protein zero. Naturally processed or synthetic protein zero peptide (194-208)-pulsed TJ2 cells significantly induced interleukin-2 secretion from autologous T cells compared to control antigen-pulsed cells (P<0.001). The numbers of interferon-gamma-producing T helper cells, including CD4(+)/CD8(+) cells, were also significantly increased (P=0.0152). Affinity-isolated naturally processed myelin peptides were potent interferon-gamma stimulators for autologous peripheral blood mononuclear cells, but not for control peripheral blood mononuclear cells. CONCLUSIONS:We show for the first time that myelin protein zero is naturally processed in B cells from monoclonal gammopathy of undetermined significance of immunoglobulin M isotype, acting as aberrant antigen-presenting cells in activation of a patient's T helper cells. Our findings cast new light on the important role of autoreactive protein zero-specific B cells in the induction of the pathogenic T-cell responses found in nerve lesions of patients with monoclonal gammopathy of undetermined significance with peripheral neuropathy.

There is a demand for more efficient and tissue-specific MRI contrast agents and recent developments involve the design of substances useful as molecular markers and magnetic tracers. In this study, nanoparticles of gadolinium oxide (Gd2O3) have been investigated for cell labeling and capacity to generate a positive contrast. THP-1, a monocytic cell line that is phagocytic, was used and results were compared with relaxivity of particles in cell culture medium (RPMI 1640). The results showed that Gd2O3-labeled cells have shorter T-1 and T-2 relaxation times compared with untreated cells. A prominent difference in signal intensity was observed, indicating that Gd2O3 nanoparticles can be used as a positive contrast agent for cell labeling. The r(1) for cell samples was 4.1 and 3.6s(-1) mm(-1) for cell culture medium. The r(2) was 17.4 and 12.9s(-1) mm(-1), respectively. For r(1), there was no significant difference in relaxivity between particles in cells compared to particles in cell culture medium, (p(r1) = 0.36), but r(2) was significantly different for the two different series (p(r2) = 0.02). Viability results indicate that THP-1 cells endure treatment with Gd2O3 nanoparticles for an extended period of time and it is therefore concluded that results in this study are based on viable cells. Copyright (c) 2008 John Wiley & Sons, Ltd.

The restricted immunoglobulin (Ig) repertoire found in B-cell chronic lymphocytic leukemia (CLL) implies a role for antigen(s) in the leukemogenesis. The nature of the antigens has, however, not been characterized, although examples of autoantigens have been demonstrated. We have analyzed a panel of 28 CLL cell lines and primary cultures, producing monoclonal Ig with different Ig heavy-chain variable region gene usage and mutational status, including several complementarity determining region 3 homology subset members. Using mass-spectrometry, immunoassays, or protein macroarrays, we have discovered novel antigens binding to CLL Igs. These antigens included cytoskeletal proteins vimentin, filamin B, and cofilin-1, but also phosphorylcholine-containing antigens (eg, Streptococcus pneumoniae polysaccharides and oxidized low-density lipoprotein [oxLDL]). Additional new antigens identified were cardiolipin and proline-rich acidic protein-1. Remarkably, these antigens represent molecular motifs exposed on apoptotic cells/blebs and bacteria, and several CLL Igs bound to apoptotic Jurkat cells. In conclusion, these intriguing data, showing a limited target structure recognition, indicate that CD5+ CLL B cells are derived from a cell compartment that produces "natural antibodies," which may be instrumental in elimination and scavenging of apoptotic cells and pathogenic bacteria.