Kappa Deletion Research Articles

Successful Immune Reconstitution in a Patient with a TYK2 Deficiency after Allogeneic Stem Cell Transplantation from Unrelated Donors.

A boy with primary immunodeficiency, caused by a tyrosine kinase 2 (TYK2) mutation, presented with immune defects and a lifelong history of severe infections. Our aim was to determine whether allogeneic hematopoietic stem cell transplantation (HSCT) could restore the patient's immune defenses and reduce susceptibility to infection. In the absence of a suitable HLA-matched blood relative to act as a donor, the patient received an allogeneic HSCT from unrelated donors. The patient's clinical data were analyzed in the Children's Hospital of Chongqing Medical University (Chongqing, China) before transplantation and during the 4-year follow-up period using a combination of western blotting (e.g., TYK2 and STAT levels), qRT-PCR (e.g., T cell receptor rearrangement excision circles, kappa deletion element recombination circles, and TYK2 transcript levels), and flow cytometry (e.g., lymphocyte subpopulations and CD107α secretion). We found that HSCT significantly reduced the incidence of severe infections, restored normal TKY2 levels, and reversed defects such as impaired JAK/STAT signaling in response to interferon-α or interleukin-10 treatment. Although the patient did not develop acute graft-versus-host disease (GVHD) after transplantation, he did experience chronic GVHD symptoms in a number of organs, which were effectively managed. Our findings suggest that HSCT is a feasible strategy for reconstituting the immune system in TYK2-deficient patients; however, the factors associated with GVHD and autoimmune thyroiditis development in TYK2-deficient patients undergoing HSCT warrant further investigation.

Abstract 2293: Evaluation of multiple myeloma research samples by analysis of B cell heavy and light chain receptors in a single NGS assay

Abstract Introduction: B cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. Methods: A B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. Primers are included to amplify rearrangements involving Kappa deletion element. Reproducibility studies and clonality assessment were conducted using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal via flow cytometry or IHC/ISH in tissue sections. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software. Results: Clonality assessment of MM research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples contained somatic hypermutation with 6 of 45 samples showing mutation rates greater than 10%. Lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Conclusions: These results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify workflow and inclusion of analysis tools such as automated somatic hypermutation rate determination and clonal lineage identification to open new paths for research in B cell disorders. Citation Format: Geoffrey Marc Lowman, Landon Pastushok, Karen Mochoruk, Wayne Hill, Michelle Toro, Loni Pickle, Carolina Gonzalez, Stephanie Ostresh, Shrutii Sarda, Chenchen Yang, Julie Stakiw, Mark Bosch, Hadi Goubran, Ron Geyer, John DeCoteau. Evaluation of multiple myeloma research samples by analysis of B cell heavy and light chain receptors in a single NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2293.

Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

IntroductionB cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction.MethodsThe B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software.ResultsClonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively.ConclusionsThese results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders.For research use only. DisclosuresLowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

Abstract 2779: NGS characterization of multiple immune receptors from a single multiplex PCR reaction

Abstract Background: Immune cell analysis by next-generation sequencing (NGS) has shown utility in the fields of hematology-oncology and immuno-oncology research. In hemato-oncology research, advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, as well as eliminate sample-specific primer design often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR immune cell assays require separate reactions to survey each receptor (TCRB, TCRG, IGH, IGK, IGL,⋯), depleting samples and leading to a longer time-to-answer. Two assays for immune receptor analysis based on Ion AmpliSeq technology have been developed to circumvent these issues, allowing for the effective use of up to thousands of primers in a single reaction. These two highly multiplexed NGS assays provide for efficient single-reaction library preparation for detection of IGH, IGK, and IGL chains and the TCRB and TCRG chains, respectively. Methods: Primer panels target the variable gene and joining gene regions of the individual loci (IGH, IGK, IGL and TCRB/TCRG) for all alleles found within the IMGT database, which allows readout of the complementary-determining region 3 (CDR3) sequence of each receptor. The B cell assay includes primers to amplify IGK loci rearrangements involving the kappa deletion and constant region intronic elements. To evaluate the performance of each assay, clonality detection assessment and limit-of-detection testing used gDNA from a total of 65 research samples representing common B and T cell malignancies. Sequencing was performed using the Ion GeneStudio S5 System and analysis was completed using Ion Reporter 5.16. Results: Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, DLBCL, and T-ALL) show a >90% overall positive detection rate. Measurements of linearity-of-response and limit-of-detection were carried out using T and B cell lines and clinical research samples diluted in PBL gDNA to between 10-2 and 10-6 by mass. Both the TCR and BCR multi-receptor assays perform as expected, with linear response of detection to the sample dilution, including the ability to detect clones of interest at 10-6. Conclusions: These results demonstrate the robustness of newly developed Ion AmpliSeq assays for multi-receptor B and T cell analysis. These assays will cut the time-to-answer for translational research studies as well as simplify clonality assessment and rare clone detection in hematology research. For research use only. Citation Format: Geoffrey Marc Lowman, Michelle Toro, Loni Pickle, Stephanie Ostresh, Shrutii Sarda, Chenchen Yang. NGS characterization of multiple immune receptors from a single multiplex PCR reaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2779.

Characterization of the Immunoglobulin Heavy- and Light-Chain Repertoires in a Single Reaction

Background B cell repertoire analysis by next-generation sequencing (NGS) has shown particular utility in the field of hematological oncology research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, and the elimination of specifically designed primers often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR assays require separate PCR reactions to survey each immunoglobulin chain (IGH, IGK, IGL), often leading to a longer time-to-answer for samples in which no marker is initially detected. We have developed an assay for receptor analysis based on Ion AmpliSeq technology to circumvent these issues, allowing the effective use of up to thousands of primers in a single reaction. The highly multiplexed, pan-clonality NGS assay provides for efficient detection of IGH, IGK, and IGL chain rearrangements in a single reaction. Methods We developed a single primer panel targeting the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IGK, IGL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IGK loci rearrangements involving Kappa deletion and C intron elements. To evaluate performance, we conducted clonality assessment and limit-of-detection testing used gDNA from a total of 45 research samples representing common B cell malignancies. We included samples derived from peripheral blood, bone marrow, and FFPE-preserved tissues at input levels ranging from 100ng to 2µg. Finally, we further characterized the samples via a separate AmpliSeq-based multiplex PCR assay targeting rearranged TCRB and TCRG chains. Sequencing and clonality analysis was performed using the Ion GeneStudio S5 System and Ion Reporter 5.16. Results Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, and DLBCL) show a >90% overall positive detection rate. Assessment of linearity-of-response and limit-of-detection was carried out using cell lines diluted in PBL to between 10-3 and 10-6 by mass. The multi-receptor assay performs as expected, with linear response to the cell line frequency across the range tested, including the ability to detect clones of interest at 10-6. Conclusions These results demonstrate the robustness of our newly developed Ion AmpliSeq-assay for B cell receptor heavy and light chains. We expect this assay to simplify the workflow for clonality assessment and rare clone detection in B cell malignancy research. For research use only. Disclosures No relevant conflicts of interest to declare.

Immunoglobulin Light Chain Kappa Deletion Rearrangement as a Marker of Clonality in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) express immunoglobulin light chain h (IgL-h) more frequently than other non-Hodgkin's lymphomas, and IgL-h producing B-cells usually delete one or both alleles of their IgL-K genes. This inactivation is mediated by a rearrangement between the k deletion element (Kde) and the Recombinant Signal Sequence (RSS) in the region between the Joining genes and the Constant region, or the RSS at the 3′-site of a Variable (Vk) segment. This deletion appears as a feasible tool for detecting monoclonality and minimal residual disease by polymerase chain reaction (PCR). Among twelve MCL patients studied, ten presented IgL-K expression, and all but one among these revealed a monoclonal Kde rearrangement by PCR analysis. Six of the nine cases showed a fusion between the Kde and the intron RSS, whilst three with a Vk segment. Since MCL has the worst prognosis of all B-cell lymphomas and high-dose chemotherapy regimens have been proposed, PCR for the Kde rearrangement might be a useful molecular tool to evaluate the ability of the different treatment modalities to eradicate the malignant clones.

Transcriptional activation of the human TNF-alpha promoter by superantigen in human monocytic cells: role of NF-kappa B.

We have studied the transcriptional activation of the human TNF-alpha gene by the superantigen staphylococcal enterotoxin A (SEA) in the human premonocytic cell line THP-1. Nuclear proteins from SEA-stimulated THP-1 cells bound strongly to kappa 3, the most proximal of three putative NF-kappa B binding sites (kappa 1-kappa 3) found in the 5' regulatory region of the TNF-alpha gene, but only weakly to kappa 1, the most distal of the NF-kappa B binding sites, and showed no binding to kappa 2. The mobility of the kappa 3-nucleoprotein complex was identical to that of complexes formed between nuclear proteins and the consensus NF-kappa B seuqence. Moreover, both 5' and 3' mutants of kappa 3 were unable to displace kappa 3 binding, suggesting that the kappa 3 binding complex induced by SEA has the characteristics of NF-kappa B. Studies using Abs directed against the NF-kappa B subunits p50 and p65 suggested that both p50 and p65 bind to the kappa 3 sequence. Reporter gene assays showed that deletion of kappa 3 (-99 to -89 bp) and point mutation of the three 5' guanine bases in the kappa 3 sequence reduced the inducibility of the TNF-alpha promoter by SEA and LPS. These results indicate that superantigen induces NF-kappa B in human monocytic cells and suggest that binding of NF-kappa B to the kappa 3 site of the TNF-alpha promoter plays an important role in the transcriptional activation of the TNF-alpha gene by superantigen.

Propagation of Waldenström’s Macroglobulinemia Cells In Vitro and in Severe Combined Immune Deficient Mice: Utility as a Preclinical Drug Screening Model

Abstract Waldenstrom's macroglobulinemia (WM) represents an indolent incurable human B-cell tumor. We have successfully established a permanent cell line, WSU-WM, without growth factors or viral transformation, from the pleural effusion of a 60-year-old man with IgM kappa WM. Phenotypic characterization of WSU-WM shows IgM lambda and expression of other B- cell markers. Karyotypic analysis shows a male chromosome complement with several clonal aberrations, including t(8;14)(q24;q32). Molecular characterization shows deletion of kappa and rearrangement of lambda light chain genes indicating a class switching. Both the secretory (s mu) and membrane (m mu) components of IgM are expressed. In addition, the breakpoint on 8q24 is downstream of exon 3 of the c-myc oncogene. WSU-WM grows in liquid culture and soft agar. When cells were injected subcutaneously in immune deficient mice, six of seven SCID mice developed subcutaneous tumors as opposed to three of seven in the athymic nude mice. When a WSU-WM SCID tumor was passaged in vivo in the SCID mice, the take rate was 100%. This xenograft model and a soft agar disk-diffusion assay were used to test the efficacy of standard chemotherapy agents against this tumor in vivo and in vitro, respectively. The cell line and the assays described herein can be used as a model to facilitate the discovery of new therapeutic agents or modalities for this disease.

Propagation of Waldenstrom's macroglobulinemia cells in vitro and in severe combined immune deficient mice: utility as a preclinical drug screening model

Waldenström's macroglobulinemia (WM) represents an indolent incurable human B-cell tumor. We have successfully established a permanent cell line, WSU-WM, without growth factors or viral transformation, from the pleural effusion of a 60-year-old man with IgM kappa WM. Phenotypic characterization of WSU-WM shows IgM lambda and expression of other B-cell markers. Karyotypic analysis shows a male chromosome complement with several clonal aberrations, including t(8;14)(q24;q32). Molecular characterization shows deletion of kappa and rearrangement of lambda light chain genes indicating a class switching. Both the secretory (s mu) and membrane (m mu) components of IgM are expressed. In addition, the breakpoint on 8q24 is downstream of exon 3 of the c-myc oncogene. WSU-WM grows in liquid culture and soft agar. When cells were injected subcutaneously in immune deficient mice, six of seven SCID mice developed subcutaneous tumors as opposed to three of seven in the athymic nude mice. When a WSU-WM SCID tumor was passaged in vivo in the SCID mice, the take rate was 100%. This xenograft model and a soft agar disk-diffusion assay were used to test the efficacy of standard chemotherapy agents against this tumor in vivo and in vitro, respectively. The cell line and the assays described herein can be used as a model to facilitate the discovery of new therapeutic agents or modalities for this disease.

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.

Deletions of immunoglobulin C kappa region characterized by the circular excision products in mouse splenocytes.

We have identified circular DNAs containing the kappa light chain constant region (C kappa), as well as the excision products of V kappa-J kappa and V lambda-J lambda joining in adult mouse splenocytes. Analysis of C kappa-positive circular DNA clones revealed two recombination sites (intron recombining sequence [IRS]1 and -2) within the germline J kappa-C kappa intron region and the recombining sequence (RS) located downstream of the C kappa exon. While RS contains a conserved heptamer and nonamer separated by a 23-bp spacer on the 5' side, IRS1 sequence is an isolated heptamer without an obvious nonamer, and IRS2 contains a variant heptamer, CACAAAA. Since IRS1 and IRS2 recombined with both RS (23-bp spacer signal) and V kappa (12-bp spacer signal) with significant frequency, intron recombination sites seem to have dual recombination signals. These findings provide direct evidence that C kappa deletion preceding lambda gene rearrangement can occur by looping out and excision. Increased accessibility of inefficient recombinational loci within the intron may enable recombinase to accept wide signal sequence variation.

Deletions of kappa chain constant region genes in mouse lambda chain-producing B cells involve intrachromosomal DNA recombinations similar to V-J joining.

We isolated and characterized the germ-line counterpart of a DNA segment designated RS (for recombining sequence), that is frequently recombined in mouse lambda light chain-producing B lymphocytes. Using Southern blot analyses of myelomas and mouse-Chinese hamster fusion cell lines, we found that RS DNA sequences are located on mouse chromosome 6, evidently more than 15 kilobases downstream of the kappa light-chain locus. We find that a typical recognition site for Ig gene recombination is situated within germ-line RS sequences near the recombination points observed in at least two lambda chain-producing cell lines. This represents a complete and functional Ig recognition site that is not directly associated with Ig genes. We also characterized a recombined RS segment isolated from the cell line BM18-4.13.9. This recombined segment has a variable region kappa light chain gene (V kappa) joined directly to RS sequences. Our results suggest that the deletion of the kappa light chain constant region (C kappa) exon in many lambda chain-producing B cells is the result of RS recombination and that C kappa deletion may be mediated by the same processes as antibody gene V-J joining (J = joining segment gene). We discuss the potential biological significance of RS DNA recombination in B-cell maturation.