Light Chain Usage Research Articles

B cell receptor repertoire analysis in primary Sjogren’s syndrome salivary glands identifies repertoire features associated with clinical activity

BackgroundPrimary Sjogren’s syndrome (pSS) is a complex autoimmune disease featuring damage to salivary and lacrimal glands, with the possibility of manifestations across multiple organs. Antibody-producing B cells have long been appreciated to play a significant role in pSS pathogenesis, with a number of autoreactive antibody species having been identified to be elevated in pSS patients. While several studies have attempted to characterize the BCR repertoires of peripheral blood B cells in pSS patients, much remains unknown about the repertoire characteristics of gland-infiltrating B cells.MethodsThrough paired scRNAseq and scBCRseq, we profiled the BCR repertoires of both infiltrating and circulating B cells in a small cohort of patients. We further utilize receptor reconstruction analyses to further investigate repertoire characteristics in a wider cohort of pSS patients previously profiled through RNAseq.ResultsVia integrated BCR and transcriptome analysis of B cell clones, we generate a trajectory progression pattern for infiltrated memory B cells in pSS. We observe significant differences in BCR repertoires between the peripheral blood and labial gland B cells of pSS patients in terms of relative expansion, isotype usage, and BCR clustering. We further observe significant decreases in IgA2 isotype usage among pSS patient labial and parotid gland B cells these analyses relative to controls as well as a positive correlation between kappa/lambda light chain usage and clinical disease activity.ConclusionsThrough BCR repertoire analysis of pSS patient salivary glands, we identify a number of novel repertoire characteristics that may serve as useful indicators of clinical disease and disease activity. By collecting these BCR repertoires into an accessible database, we hope to also enable comparative analysis of patient repertoires in pSS and potentially other autoimmune disorders.

Concurrent Testing of Spatially Separated Tissue Samples and Circulating Tumor DNA Reveals Substantial Intra-Patient Genetic Heterogeneity in Relapsed/Refractory Follicular Lymphoma

Background: Scrutiny of genetic heterogeneity in follicular lymphoma (FL) patients has been mainly limited to the investigation of single-site biopsies in previous studies, while the potential of circulating cell-free DNA (cfDNA) to resolve intra-patient heterogeneity has not been fully exploited in FL. Therefore, we aimed to characterize spatiotemporal heterogeneity in relapsed/refractory (R/R) FL by performing comprehensive genomic profiling of spatially separated tissue samples with matching cfDNA samples collected throughout the disease course. Methods: We performed ultra-deep targeted sequencing of 63 tumor tissue DNA (tisDNA), 28 cfDNA and paired germline DNA samples collected from 22 R/R FL patients. Sequencing libraries were prepared using a SureSelectXT HS (Agilent, USA) panel encompassing 173 genes recurrently mutated in FL, as well as rearrangement regions for immunoglobulin V(D)J repertoire profiling. Sequencing was performed on a NovaSeq6000 or NextSeq2000 (Illumina, USA) platform reaching a median deduplicated depth of 5,419x and 1,695x for cfDNA and tisDNA samples, respectively. We called short somatic variants using GATK Mutect2 (v.4.1.7.0), copy number alterations using PureCN (v.1.20.0) and CNVKit (v.0.9.9), as well as immunoglobulin repertoire using the whole-mark module of ARResT/Interrogate. Results: Median 6 and 8 non-silent variants were detected in cfDNA and tisDNA samples, respectively, affecting KMT2D (52%), CREBBP (45%) and TNFRSF14 (33%) most frequently in both sample types. Non-silent variants were detected in 21 of 24 (87.5%) pretreatment cfDNA samples. Comparing tisDNA samples with matching pretreatment cfDNA samples median 63.6% of variants were shared, yet unique variants were identified in cfDNA in 13/20 sample pairs, involving a median 14.3% of the variants. Studying the phylogenetic evolution of tumor samples pointed to early branching in majority of the patients, with ubiquitously present mutations detected in all samples constituting a median 24.3% of variants, although late branching was also observed in a subset (2/22) of the patients with at least 50% of variants detected in all samples. Most conserved variants were detectable in KMT2D and CREBBP, with notable exceptions of a lower mutation prevalence, but a high conservation rate in ATP6V1B2 and STAT6 genes. Subclonal clustering of variants and hierarchy analysis of samples from patients (n=5) with 2 tisDNA collected at separate lymphoma sites and a matching pretreatment cfDNA indicated that spatially discordant variants formed the same subclone, that could be also recovered in cfDNA in 4/5 patients (80%). An additional patient presented with almost identical tisDNA samples, but further unique mutations in cfDNA (PAT-FL-20)(Figure 1.). Investigating the prognostic significance of ctDNA level in the relapsed setting revealed that higher ctDNA levels were significantly associated with poor response to therapy (p=0.023, Kruskal-Wallis test), yet when we stratified patients by the median pretreatment ctDNA level (1.44x10 5 haploid genome equivalent/mL plasma) progression free survival in the subsequent treatment line was not substantially different between the two arms (p=0.11, log-rank test). Conclusion: Based on our results, R/R FL is characterized by remarkable intra-patient genetic heterogeneity. Although the founder clone harboring pathogenic KMT2D and CREBBP mutations can be identified in majority of the samples, expansion of tumor subclones between lymphoma sites confers divergent evolution, leaving genetic alterations unidentified during single-site based genetic testing. We also provide preliminary evidence, that multitarget high-throughput sequencing of cfDNA can uncover spatially restricted subclones that play a role in disease progression in R/R FL and that pretreatment ctDNA level might have a predictive value in the relapsed setting of FL. Figure 1. Molecular alterations in FL patients with tisDNA samples from two separate lymphoma sites and a matching cfDNA. KMT2D and CREBBP variants show high conservation, yet further variants are often spatially restricted, which can be resolved by cfDNA profiling. Copy number alterations (CNA) show lower concordance, yet recurrent CNA-s can be recovered in a subset of the cases. Identical immunoglobulin heavy and light chain usage can be detected in all samples of 3 patients and in paired tisDNA in 2 patients.

Affinity purification of serum-derived anti-IA-2 autoantibodies in type 1 diabetes using a novel MBP-IA-2 fusion protein

Autoantibodies targeting epitopes contained within the intracellular domain (IC) of the protein phosphatase-like islet antigen 2 (IA-2) are a common marker of autoimmune type 1 diabetes (T1D), however the isolation of genuine, serum derived anti-IA-2 autoantibodies has proven challenging due to a lack of suitable bioassays. In the current study, an ELISA format was developed for affinity purification of human anti-IA-2ic autoantibodies utilizing a fusion protein (FP) incorporating maltose binding protein and the full-length IA-2IC domain. Using a T1D patient cohort validated for anti-IA-2ic autoantibodies by commercial ELISA, we demonstrate the MBP-IA-2ic FP ELISA detects serum anti-IA-2IC autoantibodies from 3 of 9 IA-2 positive patients. Further to this, a multi-plate MBP-IA-2ic FP ELISA protocol specifically affinity purifies IgG enriched for anti-IA-2ic autoantibodies. Interestingly, serum derived autoantibodies immobilised on the MBP-IA-2ic FP ELISA demonstrate increased Kappa light chain usage when compared to the respective total IgG derived from donor patients, suggesting a clonally restricted repertoire of anti-IA-2ic autoantigen specific B plasma cells is responsible for autoantibodies detect by the MBP-IA-2ic FP ELISA. This study is the first to demonstrate the generation of specific, genuine human derived anti-IA-2ic autoantibodies, thereby facilitating further investigation into the origin and functional significance of IA-2 autoantibodies in T1D.

Distinct stromal and immune cell interactions shape the pathogenesis of rheumatoid and psoriatic arthritis

ObjectivesImmune and stromal cell communication is central in the pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA), however, the nature of these interactions in the synovial pathology of the...

Structural basis of HLX10 PD-1 receptor recognition, a promising anti-PD-1 antibody clinical candidate for cancer immunotherapy.

Cancer immunotherapies, such as checkpoint blockade of programmed cell death protein-1 (PD-1), represents a breakthrough in cancer treatment, resulting in unprecedented results in terms of overall and progression-free survival. Discovery and development of novel anti PD-1 inhibitors remains a field of intense investigation, where novel monoclonal antibodies (mAbs) and novel antibody formats (e.g., novel isotype, bispecific mAb and low-molecular-weight compounds) are major source of future therapeutic candidates. HLX10, a fully humanized IgG4 monoclonal antibody against PD-1 receptor, increased functional activities of human T-cells and showed in vitro, and anti-tumor activity in several tumor models. The combined inhibition of PD-1/PDL-1 and angiogenesis pathways using anti-VEGF antibody may enhance a sustained suppression of cancer-related angiogenesis and tumor elimination. To elucidate HLX10's mode of action, we solved the structure of HLX10 in complex with PD-1 receptor. Detailed epitope analysis showed that HLX10 has a unique mode of recognition compared to the clinically approved PD1 antibodies Pembrolizumab and Nivolumab. Notably, HLX10's epitope was closer to Pembrolizumab's epitope than Nivolumab's epitope. However, HLX10 and Pembrolizumab showed an opposite heavy chain (HC) and light chain (LC) usage, which recognizes several overlapping amino acid residues on PD-1. We compared HLX10 to Nivolumab and Pembrolizumab and it showed similar or better bioactivity in vitro and in vivo, providing a rationale for clinical evaluation in cancer immunotherapy.

BRAF Mutations and Inflammatory Gene Expression in Myeloma Cells from Patients with Renal Dysfunction

BRAF Mutations and Inflammatory Gene Expression in Myeloma Cells from Patients with Renal Dysfunction

Clonal IgM-Mediated Disease Cluster in Multiple Family Members

Alterations of IgM is implicated in Chronic Cold Agglutinin Hemolytic Anemia (CAD) and Waldenstrom's Macroglobulinemia (WM). Restricted IgM heavy and light chain usage has been found in both CAD and WM several series of patients but the specific heavy and light gene usage is different in the two disorders. Many patients with CAD develop a lymphoproliferative disorders (LPD), of which some have been diagnosed as WM, but there may be differences in the WM occurring in CAD patients compared to that which occurs sporadically. How the progression from one to another occurs is not known, but the restricted gene expression in WM and CAD offer opportunities to investigate genetic factors which allow or disallow one form of disease or another. However, cases of CAD and WM are usually sporadic. In a single hematology practice, two individuals diagnosed with CAD reported that they were cousins. They later determined that another cousin living in another state may have a similar disorder. Each patient was diagnosed over the age of 50. The third cousin had a small IgM monoclonal protein that rapidly increased and developed into symptomatic WM. The disease responded to cytotoxic chemotherapy. That patient did not have CAD. The IgM clone has reverted to a minimal level and has been stable for several years. The first patient with CAD was controlled by supportive care and relocated to another practice in a nearby city. He developed symptomatic anemia requiring transfusions but then responded to treatment with rituximab. He developed hypo-gammaglobulinemia and requires periodic gamma globulin infusions and is maintained with periodic rituximab infusions. The second patient developed mild hemolytic anemia which require only supportive care, but she has progressive anemia and an increasing IgM monoclonal protein and may soon need treatment with rituximab. Whether the monoclonal IgM is mediating the hemolysis is not yet determined. No other family member has yet to be identified with IgM mediated disease but the deceased father of the second CAD patient was thought to have a low-grade lymphoma. Presuming these patients have an common inherited factor driving the altered expression of IgM, further studies on such an informative family may help to understand these disorders and how each develop and evolve. DisclosuresNo relevant conflicts of interest to declare.

Dysregulation of type I IFN and IL-4R signaling regulates the development of BCR VH and VL repertoire encoding anti-Sm autoantibodies in SLE

Abstract The most specific predictor for systemic lupus erythematosus (SLE) and lupus nephritis (LN) is the presence of anti-Smith (Sm) autoAb. Mechanisms leading to selection bias for anti-Sm development is unclear. We found that anti-Sm levels in SLE correlated with increased endogenous IFN-β and interferon-stimulated genes (ISGs) in combination with low (IL-4R) network genes. Using flow cytometry analysis of 47 SLE patients, we found that anti-Sm+ patients exhibited an increased ratio of IFN-β+ activated naive (aNAV) B cells and a decrease in IL-4R+ resting naive (rNAV) B cells. Using a combined 10X single-cell transcriptomics and BCR usage analysis, anti-Sm+LN+ISGhiIL4Rlopatients (Sm+) were compared with anti-Sm−LN− ISGloIL4Rhi patients (Sm−). BCR heavy chain analysis showed that while B cells from Sm+ patients exhibit greater usage of IGHV1-2, IGHV4-34, and IGHV4-39 genes, Sm− SLE patients exhibit greater usage of IGHV3-30, IGHV3-33, and IGHV4-31 genes. Preferential Ig light chain usage for ISGhi B cells was IGLV1-51, IGLV3-21, IGLV6-57, IGKV1-5, IGKV3-15 and IGKV 6-21. Dominant heavy-light chain pair usages for the Sm+ patients were IGHV4/IGKV1 and IGHV4/IGKV3. IGHV4-34, which is intrinsically autoreactive and the target of the 9G4 anti-idiotype Ab, was found to be expressed by the ISGhiIL4Rlo B cells derived from the same Sm+ patients. The results indicated that increased levels of ISGs and decreased levels of IL-4R pathway genes are the two dominant signatures and the underlying mechanisms leading to selection and activation of anti-Sm+ B cells. Verification that the B-cell IFN-β and IL-4R phenotypes are associated with race (AA vs. EA), repertoire (anti-Sm+) and disease (LN+) would lay the basis for targeted rational therapies.

The Light Chain IgLV3-21 Defines a New Poor Prognostic Subgroup in Chronic Lymphocytic Leukemia: Results of a Multicenter Study

Purpose: Unmutated (UM) immunoglobulin heavy chain variable region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. Interestingly, IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially able to trigger cell-autonomous BCR-mediated signaling. However, this light chain has never been characterized independently of the heavy chain IgHV3-21.Experimental Design: We performed total RNA sequencing in 32 patients and investigated IgLV3-21 prognostic impact in terms of treatment-free survival (TFS) and overall survival (OS) in 3 other independent cohorts for a total of 813 patients. IgLV3-21 presence was tested by real-time PCR and confirmed by Sanger sequencing.Results: Using total RNA sequencing to characterize 32 patients with high-risk CLL, we found a high frequency (28%) of IgLV3-21 rearrangements. Gene set enrichment analysis revealed that these patients express higher levels of genes responsible for ribosome biogenesis and translation initiation (P < 0.0001) as well as MYC target genes (P = 0.0003). Patients with IgLV3-21 rearrangements displayed a significantly shorter TFS and OS (P < 0.05), particularly those with IgHV mutation. In each of the three independent validation cohorts, we showed that IgLV3-21 rearrangements-similar to UM IgHV status-conferred poor prognosis compared with mutated IgHV (P < 0.0001). Importantly, we confirmed by multivariate analysis that this was independent of IgHV mutational status or subset #2 stereotyped receptor (P < 0.0001).Conclusions: We have demonstrated for the first time that a light chain can affect CLL prognosis and that IgLV3-21 light chain usage defines a new subgroup of CLL patients with poor prognosis. Clin Cancer Res; 24(20); 5048-57. ©2018 AACR.

The antibody loci of the domestic goat (Capra hircus)

The domestic goat (Capra hircus) is an important ruminant species both as a source of antibody-based reagents for research and biomedical applications and as an economically important animal for agriculture, particularly for developing nations that maintain most of the global goat population. Characterization of the loci encoding the goat immune repertoire would be highly beneficial for both vaccine and immune reagent development. However, in goat and other species whose reference genomes were generated using short-read sequencing technologies, the immune loci are poorly assembled as a result of their repetitive nature. Our recent construction of a long-read goat genome assembly (ARS1) has facilitated characterization of all three antibody loci with high confidence and comparative analysis to cattle. We observed broad similarity of goat and cattle antibody-encoding loci but with notable differences that likely influence formation of the functional antibody repertoire. The goat heavy-chain locus is restricted to only four functional and nearly identical IGHV genes, in contrast to the ten observed in cattle. Repertoire analysis indicates that light-chain usage is more balanced in goats, with greater representation of kappa light chains (~ 20–30%) compared to that in cattle (~ 5%). The present study represents the first characterization of the goat antibody loci and will help inform future investigations of their antibody responses to disease and vaccination.

RNA-Seq De Novo Assembly of Clonal Immunoglobulin Rearrangements Identifies Interesting Biology and Uncovers Prognostic Features in Multiple Myeloma

IntroductionAlthough monoclonal immunoglobulin (Ig) production by myeloma cells is one of the central features of the disease, genotypic identification of the clonal Ig sequence remains understudied in multiple myeloma (MM). Here, using extensive RNA-seq data, we study molecular features of clonal Ig rearrangements, as well as their association with other MM markers and patient outcome.MethodsWe performed deep RNA-seq on purified CD138+ MM cells from 429 newly-diagnosed uniformly-treated patients with long clinical follow-up. For each sample, we performed de novo assembly using sequences that appeared in the library with a frequency of at least one in a million. Germline V and J genes were then BLASTed against the assembled contigs to determine the clonal germline genes and pinpoint mutations. Using the sequences reconstructed from the Ig contigs and the BLAST output, we ran IgBLAST to fully characterize the predominant Ig V(D)J sequence.ResultsWe tested the accuracy of our approach by looking at 24 technical duplicates and one triplicate. In all cases, the predicted gene and gene allele were consistent across replicates. Next, we evaluated our large patient cohort, identifying IGHV3 as the most common clonal VH gene subgroup (53.3%), followed by IGHV4 (17.8%) and IGHV1 (15.6%). Importantly, we observed a significant association between poorer prognosis and IGHV3, both for progression-free survival (PFS) (p=0.0019) and overall survival (OS) (p=0.012). IGHV3-30 (11%, the most commonly rearranged VH gene) and IGHV3-9 (4.8%) were the drivers behind this poor prognosis (IGHV3-30: PFS p=0.021; OS p=0.013) (IGHV3-9: PFS p=0.002). IGHV3-30 was even more preferentially rearranged than in normal B-cell VH repertoires from previous studies (8.5%, 6.3%) and ours (2%). Remarkably, these results sharply contrast with what has been observed in CLL. In this malignancy, IGHV3-30 use has been seen to be underrepresented and usually characterizes an indolent clinical course, while IGHV3-21 and possibly IGHV3-23 carry poor prognosis.We predicted light chain usage through the presence of clonal VL sequences. The most frequent VL genes were from the κ locus (69.4% total): IGKV1-33 (12.4%), IGKV1-5 (11.3%), IGKV3-20 (9.9%) and IGKV1-39 (8.0%). Del(22q) was observed more frequently in patients with IGλ (OR=10.0, p=6e-15) and, within this group, del(22q) was more frequent if Vλ belonged to the more centromeric V-clusters C or B, in contrast to cluster A (OR=8.4, p=5e-4). Remarkably, patients with Vλ gene from cluster A presented worse OS (vs. Vk: p=0.0079; vs. Vλ B,C: p=0.067).The proportion of mutated bases was higher in the heavy chain than in the light chain (mean 7.0% vs. 4.8%, max 14.6% vs. 14.3%), and it was associated with OS (heavy p=0.0020, light p=0.036, both=0.0056), but not PFS. Interestingly, mutated Ig in CLL results in a more benign clinical course. We further found that 24.9% and 22.7% of the mutations lay within WRCY or RGYW AID motifs in the light and heavy chains respectively (enrichment p<1e-16), while AID mutations in a TW or WA context accounted for 22.9% and 25.7% (p=0.14, p=0.64). Higher ratios of mutations in WRCY vs. RGYW motifs within the light chain were highly predictive of poor prognosis (PFS p=0.0019, OS p=6.3e-4). Strikingly, IGλ usage was linked to higher ratios (p=3e-6), an association not explained by germline sequence variability (p=0.24).The usage of IGHV3 genes and the AID WRCY/RGYW motif ratio were independent markers of each other (p=1) and of other markers of poor prognosis in MM, such as presence of either t(4;14) or del(17p) (IGHV3 p=0.10; motif ratio p=0.49). In conclusion, de novo Ig heavy and light chain assembly using RNA-seq identifies interesting biology, may provide MM markers and highlights a novel application of high-throughput genomics. DisclosuresAnderson:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Avet-Loiseau:sanofi: Consultancy; celgene: Consultancy; amgen: Consultancy; janssen: Consultancy.

Bone marrow basophils enhance immature B cell survival by recruitment and contact dependent signals

Abstract B cell development in the bone marrow relies upon an organized tissue structure. Maturing progenitors migrate between several distinct niches and towards the sinusoidal vessels before entering circulation. Microenvironments specific for early developmental stages are provided by stroma expressing CXCL12 (pre-pro-B), IL-7 (pro-B), or Galactin-1 (pre-B); however, the identity of a defined niche for immature B cells remains unclear. A DX5+Thy1lo cell has been shown to provide IgM+ progenitors survival signals through direct cell-cell contact; but the cell type responsible, factors provided, and the influence this help may have on B cell function remains unknown. Here, utilizing in vitro B cell selection assays and in vivo depletion experiments, we identify the DX5+Thy1lo cells as basophils. We demonstrate that this population produces chemotactic factors, which act to recruit immature B cells and therefore enable provision of pro-survival signals directly via cell-cell contact. Furthermore, the bone marrow basophil population expanded during the first 24 hours following induction of inflammation by injection of Freund’s incomplete adjuvant. This response correlated with an increase in the number of immature B cells within the marrow, as well as, a reduction of lambda light chain usage in the immature but not mature B cell population. These results reveal that bone marrow basophils actively provide a supportive microenvironment for immature B cells and that the sensitivity of basophils to inflammatory stimuli may modulate BCR repertoire development.

Antigen nature and complexity influence human antibody light chain usage and specificity

Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies.

Systemic adjuvant inflammation rapidly reorganizes bone marrow B cell populations, and their niche, to shape B cell function and repertoire development (HEM1P.224)

Abstract Maturing B cells migrate through the organized bone marrow parenchyma toward sinusoid vessels before entering circulation. Niche components rigorously control this process. Infecting mice with pathogens, or injecting inflammatory agents, is known to produce perturbations in B lymphopoiesis, however; it is poorly understood how the B cell niche responds to these challenges. Moreover, the impact on subsequent B cell function remains unknown. Here, we report that injection of Freund’s incomplete adjuvant in mice induced a large and rapid (within hours) mobilization of B cells to the bone marrow, mediated by increased sensitivity to the chemokine CXCL12. Notably, localization within the marrow is altered with all B cell subsets showing a reduced affinity for the sinusoids. Reorganization of B cell positioning occurred together with relevant modification of their supportive niche including increased expression of BAFF and MIF mRNA, expansion of DX5+Thy1loAPRIL+ basophils, and elevation of several important cytokines. This altered marrow milieu may have a role in shaping B cell fate, as we observed a decrease in lambda light chain usage in immature cells. Further, disruption of homing dynamics with a CXCR4 inhibitor attenuated the formation of IgM secreting cells. Together these results demonstrate an exquisite sensitivity of the marrow B cell compartment to peripheral inflammation and suggest that initial inflammatory signaling regulates subsequent B cell effector responses.

Antibody Light-Chain-Restricted Recognition of the Site of Immune Pressure in the RV144 HIV-1 Vaccine Trial Is Phylogenetically Conserved

In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognition prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. These data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.

Prevalence, specificity and functionality of anti-ganglioside antibodies in neuropathy associated with IgM monoclonal gammopathy

IgM antibodies against gangliosides and their complexes were studied in sera from 54 patients with polyneuropathy and IgM monoclonal gammopathy (IgM-PNP) without anti-MAG antibodies. Anti-ganglioside antibodies were found in 19 (35%) patients. Five (9%) patients had antibodies against ganglioside complexes. IgM antibodies against gangliosides activated complement in vitro. Light chain usage was restricted to kappa or lambda in most, but not all patients.In conclusion, anti-ganglioside antibodies in IgM-PNP are common, display pathogenic properties and do not always arise from a monoclonal B cell proliferation.

SLAP deficiency decreases dsDNA autoantibody production

Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP(-/-) mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP(-/-) 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection.

Relative Stability Of Plasma Cells Immunophenotype In Multiple Myeloma Over Time

Emerging evidence indicates that multiple myeloma (MM) can follow a number of evolutionary pathways over a patient's disease course. Recently, genomic data have shown the coexistence in many patients of several tumour types, which could be either genetically stable, evolving linearly or be constituted of heterogeneous clonal mixtures with predominant clones shifting over time (Keats, Blood 2012;120:1067-1076). Little is known however of immunophenotypic evolution of the myeloma cells (MM cells) during MM progression.In a retrospective single-centre analysis of tumoral immunophenotypes, we examined sequential bone marrow or peripheral blood samples from 51 MM patients. Multiparameter flow cytometry was performed to assess MM phenotype using CD38 and CD138 expression as well as intracytoplasmic light chain usage restriction in most cases. The presence or absence of CD19, CD20, CD27, CD28, CD56 or CD117 on the MM cells'surface was investigated additionally. A median of 4 of the latter antigens was examined per sample. Most MM cells were homogeneous in the expression or absence of expression of the differentiation antigens investigated. Immunophenotype changes were defined as the loss or gain of at least 1 antigen. Whenever partial expression of one or several antigens was observed on MM cells, indicating MM subsets, the presence of subclones was suspected.Samples were obtained at diagnosis and relapse for 33 MM patients, and during consecutive relapses for 18. The total amount of analysed samples was 109, with a median of 2 samples per patient (range: 2-3). A modification of immunophenotype occurred in 22 patients (43%), with one antigen change (68%), two antigens changes (18%) or three antigens changes (14%). Immunophenotypic changes are summarized in table 1. For 20% of the cases or more, this involved CD28 (n=11, balanced gain or loss) or CD27 (n=7, mostly loss). CD56 expression, scarcely negative on the first assessment, was gained at relapse in three cases. Other antigens were expressed in a stable fashion over time. Eighteen (35%) of the patients had at least a subclone on the first immunophenotype, which was lost and/or modified in 14 / 18 patients at time of subsequent relapse.Table 1Evolution of MM immunophenotype at relapses.Positive at first ImmunohenotypeNb of patients tested at follow-upStableGainLossChange (%)CD193/51510000CD208/423934145 (13)CD2716/362518167 (20)CD2825/4847366511 (23)CD5643/515148303 (6)CD1178/362119022 (10)CD335/282120011 (5)There was no significant impact of immunophenotypic modifications on overall survival rates Similarly, the presence of a subclone at first examination, or the immunophenotypic change of this subclone over time was not associated with differences in overall survival rates.In conclusion, this retrospective single centre study demonstrates that the immunophenotype of MM cells is mostly stable during the course of the disease. When changes occur, they do not seem to affect the clinical course of the patients. These preliminary results need confirmation on a larger and prospective trial. Disclosures:No relevant conflicts of interest to declare.

Immunoglobulin Light Chain Use and Characteristics in the Chlamydophila Psittaci negative MALT Lymphomas of the Ocular Adnexa

AbstractAbstract 1597Ocular adnexal mucosa associated lymphoid tissue lymphomas (OAMALTL) are the most common lymphomas of the eye. The potential roles for specific antigens in these lymphomas are still controversial. Previously we examined the usage and mutations of the IGVH in Chlamydophila (C) psittaci-negative OAMALTL, demonstrating biased use of the IGHV4 family and IGHV4–34 gene and evidence for antigen selection (PLoS One. 2011;6(12):e29114). However, there have been no previous reports characterizing the Ig light chain (IGKV/IGLV) usage and properties in OAMALTL. Herein, we examined IGKV/IGLV gene usage and mutations in 34 C. psittaci-negative OAMALTL originating from the orbit (15), conjunctivae (14) and lacrimal gland (5).Clonal potentially functional IGKV/IGLV gene sequences were identified in 30 tumors (18 kappa and 12 lambda). Among the 7 kappa light chain families, IGKV1 (n=5, 28%), IGKV2 (n=1, 6%), IGKV3 (n=8, 44%) and IGKV4 (n=4, 22%) were observed. Among the 8 lambda chain families, IGLV2 (n=5, 42%), IGLV3 (n=1, 8%), IGLV6 (n=1, 8%), IGLV7 (n=3, 25%) and IGLV8 (n=2, 17%) were detected. In comparison with the IGKV and IGLV repertoire employed in normal peripheral blood B-lymphocytes, an overrepresentation of the IGKV4 family (P<0.01) was observed in the analyzed cohort of tumors. No statistically significant difference in the use of other kappa and lambda light chain families was observed. In the most commonly used IGKV3 family, only 2 (IGKV3–20*01 (n=6) and IGKV3–15*01 (n=2)) of the 7 family members were used by the OAMALTL. The IGKV3–20*01 allele was used at a greater frequency than in normal peripheral blood B-lymphocytes (p=0.02). The observed frequency of the allele in OAMALTL (20%) is higher than the previously reported frequency in other non-MALT lymphoma types. Of the six cases which utilized the IGKV3–20*01 allele, corresponding heavy chains were available for five cases. Three of the IGKV3–20*01 alleles paired with the IGHV4–34 allele, one paired with the IGHV3–23 allele and one paired with the IGHV1–69 allele. Interestingly, each of these three alleles has been previously implicated in autoreactivity. The IGKV4-1*01 allele was observed to pair with IGHV3–74 (n=2), IGHV1–24 (n=1) and IGHV2–5 (n=1), respectively. Of the 30 clonal tumor sequences, 27 displayed mutations from germline. The average percent homology to germline was 96.35%. Most mutated cases (14 out of 27, 52%) exhibited less than a 2% difference from their original germline sequence. Four of the six tumors utilizing the IGKV3–20*01 allele exhibited more than 2% mutation away from germline, with the remaining two sequences unmutated. Three of the four tumors employing IGKV4-1*01 exhibited greater than 2% mutation away from germline. No acquired N-glycosylation sites were observed in the OAMALTL. To analyze for potential selective pressure exerted by antigens, we utilized the BASELINe algorithm, which assesses for both presence and strength of antigen selection. 23 sequences exhibited evidence of selection in the CDR regions, while 26 sequences exhibited evidence of selection in the FR regions. Of the 26 sequences for which paired light and heavy chains were available, 9 heavy chains exhibited strong evidence of selection (8 in FR, 1 in CDR) and 5 light chain exhibited strong evidence of selection (all in FR). Two patients’ tumors exhibited strong evidence of selection in both heavy and light chains. A recurrent mutation was observed in the FR2 region of sequences derived from the IGKV3–20*01, with three of six cases showing an acquisition of histidine in place of glutamine at position 39. An analysis of the tumor derived CDR3 regions did not reveal stereotyped sequence determinants and showed no homology to other known bacterial binding antibodies. Our findings demonstrate that C. psittaci negative OAMALTL exhibit biased usage of IGKV families and genes with evidence of antigen selection. Combined with our previous findings, we demonstrate the frequent use and common pairing of IGVH and IGKV genes, which are usually used by autoantibodies, suggesting that the C. psittaci negative OAMALTL encoded immunoglobulins may be directed to autoantigens. Indeed, generated recombinant immunoglobulins derived from multiple OAMALTLs react to self and not to bacterial antigens. These findings and the identity of the autoantigens will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

Organization, complexity and allelic diversity of the porcine (Sus scrofa domestica) immunoglobulin lambda locus

We have characterized the organization, complexity, and expression of the porcine (Sus scrofa domestica) immunoglobulin lambda (IGL) light chain locus, which accounts for about half of antibody light chain usage in swine, yet is nearly totally unknown. Twenty-two IGL variable (IGLV) genes were identified that belong to seven subgroups. Nine genes appear to be functional. Eight possess stop codons, frameshifts, or both, and one is missing the V-EXON. Two additional genes are missing an essential cysteine residue and are classified as ORF (open reading frame). The IGLV genes are organized in two distinct clusters, a constant (C)-proximal cluster dominated by genes similar to the human IGLV3 subgroup, and a C-distal cluster dominated by genes most similar to the human IGLV8 and IGLV5 subgroups. Phylogenetic analysis reveals that the porcine IGLV8 subgroup genes have recently expanded, suggesting a particularly effective role in immunity to porcine-specific pathogens. Moreover, expression of IGLV genes is nearly exclusively restricted to the IGLV3 and IGLV8 genes. The constant locus comprises three tandem cassettes comprised of a joining (IGLJ) gene and a constant (IGLC) gene, whereas a fourth downstream IGLJ gene has no corresponding associated IGLC gene. Comparison of individual BACs generated from the same individual revealed polymorphisms in IGLC2 and several IGLV genes, indicating that allelic variation in IGLV further expands the porcine antibody light chain repertoire.