Constant Region Primer Research Articles

Abstract 2940: Somatic hypermutation analysis of chronic lymphocytic leukemia research samples by IGH chain next-generation sequencing

Abstract Chronic lymphocytic leukemia (CLL) is a common form of leukemia characterized by clonal expansion of neoplastic B cells and a heterogenous disease course. Somatic hypermutation (SHM) status of the IGHV gene in CLL clinical research samples is important as SHM frequency is an established prognostic biomarker. Conventionally, SHM analysis is performed via Sanger sequencing which is limited by the inability to evaluate more than one rearrangement due to mutations, multiplex constraints, and template input requirements. Ion AmpliSeq™ next generation sequencing (NGS) assays for research in IGH chain SHM are evaluated using multiple cohorts of CLL research samples. These NGS assays employ multiplex primers that target the Leader or FR1 regions of the IGHV gene and the IGHJ gene in either RNA or DNA templates. The robustness of these assays was demonstrated by an evaluation across multiple labs, including comparisons between DNA and RNA input and correlation to orthogonal NGS testing. Ion AmpliSeq™ multiplex primer sets were designed to target the Leader or Framework 1 (FR1) regions of the IGHV gene with joining gene (J) or constant region (C) primers. The Oncomine™ IGHV Leader-J Assay uses Leader primers, while the RNA-input Oncomine™ IGH-LR Assay, and the DNA-input Oncomine™ IGH FR1-J Assay use FR1 variable gene primers in combination with C and J region primers, respectively. Primer panels were designed to amplify all variable gene families, including all individual alleles, listed in the IMGT database. To evaluate accuracy, each assay was used to measure SHM from a panel of gDNA and RNA rearrangements extracted from CLL research samples for clonality testing and SHM analysis. To test the Leader-J and FR1-J DNA input assays, 24 orthogonally characterized CLL samples were evaluated on the Ion Torrent S5 platform. Stereotypy and confirmation of individual software findings assessed by placing FASTA file results into the online ARResT tool. Both RNA and DNA input assays correctly determined the SHM status of CLL rearrangements, with IGHV SHM values concordant between both RNA and DNA approaches. SHM values derived from FR1 variable gene primers delivered concordant results compared to Leader variable gene primers across a range of SHM frequencies, with FR1 primer coverage of the IGHV gene leading to an expected over-estimation of SHM percentage. SHM status was concordant between NGS assays evaluated in over 90% (21/24) of CLL samples tested. Mutational frequency showed excellent concordance, with an R2=0.97, and V-gene usage was 100% concordant. These results support the robustness of long-read NGS assays to quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader-targeting primers using DNA input showing the utility in both priming locations. Orthogonal testing of the Leader-J assay showed excellent concordance for mutation rate, SHM status, and stereotypy. Citation Format: Michelle Toro, Loni Pickle, Shrutii Sarda, Robyn Marshall, Bonnie Pieterse, Tania Venter, Chenchen Yang, Theo Gerdener, Geoffrey Marc Lowman. Somatic hypermutation analysis of chronic lymphocytic leukemia research samples by IGH chain next-generation sequencing [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 2940.

Abstract 511: Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing

Abstract Background Traditional next generation sequencing methods for quantifying somatic hypermutation (SHM) rely on multiplex primers targeting either the framework 1 (FR1) or Leader regions of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA templates. Ion AmpliSeq primer panels for SHM evaluation were compared using both DNA and RNA input. Performance was compared using SHM values obtained from RNA samples amplified using FR1 variable gene primers in combination with constant gene primers to determine each IGH isotype and subtype in a single PCR reaction. Comparison of SHM frequencies measured from matched RNA and DNA samples were used to determine the feasibility for use of RNA in the study of SHM as well as comparison of the performance of Leader and FR1 gene primers in DNA studies. Methods Two multiplex primer panels were developed for DNA templates. These panels target the Leader or FR1 regions of the IGHV gene and the IGHJ gene. RNA samples were surveyed using FR1 and constant region primers. Comparisons of SHM frequency using 200ng of gDNA and 25ng of RNA from B cell lines and clinical research samples. Sequencing was accomplished using the Ion Gene Studio S5, while clonotyping, isotype determination, and somatic hypermutation analysis was performed using Ion Reporter 5.16 analysis software. Results Both RNA and DNA input assay workflows were able to correctly determine the SHM status of all rearrangements tested. IGHV SHM values were highly concordant between both RNA and DNA approaches. Additionally, SHM values derived from FR1 targeting variable gene primers delivered concordant results compared to Leader targeting variable gene primers when using DNA input across a wide range of SHM frequencies tested. Conclusions These results support the ability of highly multiplexed long-read NGS assays to accurately quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader targeting primers using DNA input. RNA based NGS methods benefit from lower sample requirements as well as the addition of isotype (and subtype) identification, opening new research areas for study of the B cell immune repertoire. Citation Format: Michelle Toro, Geoffrey Lowman, Loni Pickle, Stephanie Ostresh, Mark Andersen, Shrutii Sarda, Chenchen Yang. Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing [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 511.

Defining the Diversity and Specificity of Neoantigen-Specific T Cells in Chronic Lymphocytic Leukemia

Recent studies have highlighted the promise of targeting tumor neoantigens but little is known about the clonal composition of neoantigen-specific T cells. Knowledge of the number and diversity of neoantigen-specific T cell receptors (TCRs), their cognate antigens and how these change over time are informative parameters to consider in the rational design of future immunotherapy. TCR repertoire studies of bulk T cells can provide measures of diversity, but generally mask the antigen-specificity information encoded by TCRαβ pairs, and are insufficient for directly linking particular TCRs to individual antigens.To address these challenges, we developed an approach to discover TCRαβ sequence from single cells, use this information to reconstitute any TCR, and to test the reconstructed TCRs for reactivity against candidate antigens. As a proof-of concept, we sought to identify TCRs against a pool of well-characterized viral peptides (CMV, EBV, Influenza) of defined HLA restriction (called ‘CEF‘). We performed one round of in vitro stimulation of PBMC from 2 healthy volunteers with CEF peptides, and then isolated CEF-specific T cells based on IFN-γ production. These cells underwent single cell targeted sequencing of TCRα/β chains, using 45 Vα and 50 Vβ primers specific for the variable regions and a single constant region primer. Compared to unstimulated PBMCs, the CEF-stimulated PBMCs were clonally expanded. For donors 1 and 2, 57 and 74 total clonotypes were identified, of which 16 and 13 constituted between 1.7-34% and 1.5-18% of total T cells, respectively.To interrogate the antigen-specificity of the identified TCR sequences, we cloned TCRs of interest. As all TCRs share the same modular structure, we designed a plasmid library to include all Vα or β and constant regions. Based on the discovered CDR3-α and -β sequences, oligonucleotides for these regions critical for antigen recognition were custom-synthesized, and cloned by Golden Gate assembly of selected library components. In this fashion, we rapidly cloned the 9 most dominant TCR clones per donor and lentivirally transduced them into a TCR-deficient Jurkat cell line. Upon screening these lines against individual CEF peptides, we confirmed that 4 TCRs per donor had specificities to CEF peptides, previously characterized to stimulate donor-specific reactivity by IFN-γ ELISPOT assay. Screening of the remaining reconstructed TCRs is ongoing.Having demonstrated the ability to link TCRs with cognate antigen, we next applied this approach to study neoantigen-specific T cells from patients with chronic lymphocytic leukemia (CLL). We have previously reported the results of somatic mutation detection from whole-exome sequencing data from 157 CLL patients, from which we predicted 1851 neoantigens with high binding affinity (IC50 < 150 nM). Of these, 326 neoantigens had predicted high binding affinity to HLA-A2. From this collection, we focused on the identification of TCRs specific to neoantigens predicted to be generated by mutation in 2 genes. The first was FNDC3B, a point mutation of which we previously showed to generate a neoepitope that stimulated expansion of CD8+ T cells in a transplanted patient in association with durable remission, and which displayed cytotoxic potential against autologous tumor. The second was MGA, a putative CLL driver, wherein an indel was predicted to generate a novel open reading frame. To identify the number of T cell clones that could be stimulated by these candidate neoantigens, we stimulated HLA-A2+ PBMC with predicted neopeptides arising from these genes, and flow cytometrically isolated peptide-reactive CD8+ T cells for TCR sequencing. 148 of 250 (57%) mut-FNDC3B-specific T cells had the same CDR3 sequences, with 5 dominant clones identified. Three of 5 T cell clones (constituting 57%, 3% and 2% of total T cells) recognized the neopeptide. Similarly, out of 43 MGA-specific CD8+ T cells, 4 dominant clones were identified, 1 of which we confirmed as specific to MGA.Altogether, these studies demonstrate our capability to effectively identify paired TCRαβ sequences, express them on demand and probe antigen specificity. Ongoing studies are focused on further defining the antigen specificity and avidity of engineered T cells, including reactivity against autologous leukemia cells. These studies are expected to provide an assessment of the diversity and function of neoantigen-reactive TCRs. DisclosuresWu:Neon Therapeutics: Consultancy.

Unbiased RACE-Based Massive Parallel Surveys of Human IgA Antibody Repertoires.

For investigations of human B-cell receptor (BCR) repertoires, we have developed a protocol for large-scale surveys of human antibody heavy chain (VH) rearrangements. Here we study IgA repertoires, as more IgA antibodies are synthesized in the human body on a daily level than all other isotypes combined. In fact, IgA is secreted at all mucosal surfaces, and it is also secreted in the perspiration that coats our cutaneous surfaces. In these studies we can characterize the IgA clonal diversity of B-cell populations obtained from any donor. To recover representative repertoire libraries, we make our libraries from antibody gene transcript templates (i.e., cDNA), as these are closer reflections of the immune repertoire expressed at the antibody protein level. To avoid biases potentially introduced by upstream oligonucleotide primers that hybridize to variable region framework regions, our approach also uses rapid amplification of cDNA ends (RACE) of antibody transcripts. For exploration of human IgA responses, we have designed a duplexing antisense constant region primer that efficiently amplifies, side-by-side, heavy chain transcripts of both the IgA1 and IgA2 subclasses. By these methods we have begun to define the molecular differences in the IgA1 and IgA2 responses occurring simultaneously in different donors. These methods will be used to investigate the effects of microbial virulence factors on host defenses, during autoimmune responses, and in B-cell malignancies.

T Cell Receptor Clonotype Influences Epitope Hierarchy in the CD8+ T Cell Response to Respiratory Syncytial Virus Infection

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.

Immunoglobulin Heavy Chain (IGH) Gene Rearrangement In Waldenström Macroglobulinemia and Other Monoclonal IgM Disorders

AbstractAbstract 4139 Background.The presence of a serum IgM monoclonal component is associated with a spectrum of lymphoid disorders including Waldenström Macroglobulinemia (WM), IgM monoclonal gammopathy of undetermined significance (MGUS) and IgM-related disorders (IgM-RD). Limited information is available on immunoglobulin heavy chain (IGH) gene rearrangement in this setting. Purpose.The aim of this study was to analyze IGHV-D-J rearrangements in a large series of patients (pts) and to compare the gene usage across different monoclonal IgM disorders. Patients and methods.We analyzed 107 pts including 52 WM, 47 IgM MGUS and 8 IgM-RD. Diagnosis was made according to the consensus criteria proposed at the 2nd International Workshop on WM. Mononuclear cells were obtained from bone marrow in all pts. IGHV-D-J rearrangements were amplified and directly sequenced from cDNA using primers specific for each of the leader sequences of HV1-6 subgroups in combination with a joining heavy chain (JH) consensus primer or cμ constant region primer. Sequences were aligned to ImMunoGeneTics sequence directory using the IMGT V-QUEST analysis software. Results.A complete productive monoclonal IGHV-D-J rearrangement was obtained in 84/107 cases (78%). Using a homology cut-off value of 98% to the nearest germline gene, we observed mutated IGHV in 79/84 pts (94%) and unmutated IGHV in 5 (6%). The frequency of IGHV, IGHD and IGHJ gene usage is shown in the table. The most common IGHV subgroup was IGHV3, which was found in 69/84 cases (82%). The individual IGHV genes most frequently used were IGHV3-23 (20/84, 24%) and IGHV3-7 (10/84, 12%). IGHD segments were assignable in 83 out of 84 rearrangements. The most represented IGHD subgroups were IGHD3 (23/83, 28%), IGHD6 (16/83, 19%) and IGHD2 (15/83, 18%). The most common individual IGHD genes were IGHD6-19 (10/83, 12%) and IGHD2-2 (8/83, 10%). The analysis of IGHJ genes showed a preferential usage of IGHJ4 gene (56/84, 67%) and IGHJ4*02 allele (54/84, 64%). The median HCDR3 length was 13 amino acids (range: 5–29). We found an association between IGHV3 and IGHJ4 subgroups (p=0.01), while no association was found between IGHV and IGHD (p=0.8), and between IGHD and IGHJ subgroups (p=0.8).Table 1Distribution of IGVH, IGHD and IGHJ genes in WM, IgM MGUS and IgM-RD.All (n=84)IgM MGUS (n=31)IgM-RD (n=6)WM (n=47)VH (n=84)VH16(7%)303VH21(1%)001VH369(82%)25341VH3-23204214VH3-710505VH3-308206VH3-748404VH3-484112VH3-334400VH46(7%)231VH4-344121VHH51(1%)100VHH61(1%)001DH (n=83)DH110(12%)523DH1-266420DH215(18%)5010DH2-28305DH323(27%)9212DH3-37214DH3-106204DH3-94112DH3-166402DH45(6%)005DH513(15%)625DH5-124112DH5-57313DH616(19%)6010DH6-1910406DH71(1%)001JH (n=84)JH21(1%)001JH39(11%)504JH3*028503JH456(67%)20531JH4*025420529JH511(13%)515JH5*0211515JH67(8%)106JH6*027106We compared IGH rearrangement features in pts with WM, MGUS and IgM-RD. A complete productive monoclonal IGHV-D-J rearrangement was detected in a higher percentage of WM pts (47/52, 90%) as compared to MGUS (31/47, 66%) and IgM-RD pts (6/8, 75%) (p=0.01). The proportion of mutated IGHV cases was similar in the three groups (p=0.6). Regarding specific gene usage, there was a trend toward a higher usage of IGHV3-23 gene in WM (14/47, 30%) as compared to MGUS (4/31, 13%) (p=0.07). On the contrary, there was no difference in distribution of IGHD subgroups (p=0.13) and IGHJ genes (p=0.5). The median HCDR3 length was similar in WM, MGUS and IgM-RD (p=0.6).We also compared IGHV usage with clinical characteristics of pts. Interestingly, we found that autoimmune manifestations were more frequently observed in pts carrying IGHV4-34 gene (3/4, 75%) as compared to pts using alternative genes (8/80 pts, 10%) (p=0.006). Autoimmune phenomena associated with IGHV4-34 gene were represented by cold agglutinin haemolytic anemia in 2 pts with IgM-RD and 1 with WM. Conclusions:the identification of a monoclonal IGHV-D-J rearrangement seems more feasible in WM as compared to MGUS and IgM-RD. In all three groups, the majority of cases are mutated, confirming the derivation of the clone from a post-germinal center cell. This study shows a preferential usage of VH3 subgroup and in particular of VH3-23 gene, with a trend for a higher usage in WM as compared to other IgM disorders. A higher prevalence of autoimmune manifestations was observed in pts carrying VH4-34 gene. Disclosures:No relevant conflicts of interest to declare.

αvβ6 Integrin Regulates Renal Fibrosis and Inflammation in Alport Mouse

The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.

Analysis of Validated Clonotypic Igh VDJ Sequences from a Large Cohort of MM Patients.

Studies in B cell malignancies such as chronic lymphocytic leukemia have revealed associations between immunoglobulin V gene characteristics and clinical outcomes. Few studies of this kind have been conducted in multiple myeloma (MM), a B lineage cancer characterized by malignant plasma cells that display somatically hypermutated VDJ, clonal homogeneity, and post switch (IgG or IgA) clinical isotypes. In this study we analyzed the clonotypic VDJ sequences for a large cohort of 115 MM patients to define associations with clinical parameters such as staging at diagnosis (e.g. clinical isotype, percent PC, M protein, beta-2-microglobulin) and overall survival. A key feature of this analysis is the rigorous validation of the experimentally derived clonotypic VDJ sequence by confirming its presence in the majority of single MM PC, to exclude any VDJ sequences arising from expanded but unrelated clones of normal PC that are also present in MM bone marrow (BM) samples. This process involves the amplification of patient specific clonotypic VDJ using sets of VH family and constant region primers, direct sequencing of VDJ fragments, and CDR2 / CDR3 patient specific primer design. The proposed VDJ is sequence is confirmed by one of several single cell assays, preferably RT-PCR on CD138+ sorted PC, but also including in-situ RT-PCR and limiting dilution analysis. The set of confirmed clonotypic VDJ sequences were analyzed for several parameters including: VH, DH, and JH gene usage, VH and CDR3 length, percent mutation in the CDR1-CDR3 region, and CDR3 amino acid sequence. We found that V gene usage amongst MM patients was not significantly different from expected values based on the frequency of functional germline alleles: VH3 (52.2%), VH4 (19.1%), VH1 (17.4%), VH2 (7.8%), VH5(3.5%). Frequently used VH alleles included VH3-30 (10.4%), VH1-69 (9.6%), VH3-23 (7.8%). Amongst VH5 alleles, VH5-51 was observed exclusively. VH4-34, which has been associated with autoimmune diseases and is not present in normal plasma cells was observed in a single case. DH and JH usage from most to least frequent was as follows: JH4, JH6, JH3, JH5, JH1, and JH2; DH3, DH2, DH1, DH6, DH5, and DH4. Sequence analysis of the CDR1-CDR3 region revealed 1.8–18.8% mutation with a mean of 9.4%. Using mutation frequency as a guideline, we identified three groups, high (12–18.8%), medium (6–11.9%) and low (1.8–5.9%) frequencies of mutation, and compared their clinical characteristics. In a similar fashion, CDR3 length, selected VH, DH, and JH alleles were also evaluated in the context of clinical parameters. In a preliminary analysis, there were no detectable correlations between any of these V gene characteristics and overall survival. Future studies with this database will include CDR3 sequence analysis to determine if subsets of recurrent amino acid patterns, suggestive of specific antigen involvement are detectable. Unlike other B lineage malignancies, such as B-CLL or Waldenstrom's macroglobulinemia, the V gene usage of MM appears to be a largely random representation of the normal VH gene repertoire, with no selection for particular VH gene families or individual VH genes, and a broad range of mutation frequencies. This type of analysis has meaning only if the IgH VDJ sequences analyzed are derived from the MM clone itself, making validation of experimentally derived clonotypic sequences a critical priority. Overall, this is the largest analysis to date of rigorously validated IgH VDJ gene sequences for MM.

Aptamers and SELEX: the technology

DNA and RNA microarrays have become an important analytical technique in the understanding and characterisation of genomes and transcriptomes. A recent development in this field of combinatorial chemistry has concentrated on using artificial DNA/RNA sequences––aptamers––as the screening ligand. In reviewing this technology, the article covers the topic, following a general introduction, under the headings: generating an aptamer library, aptamer library complexity, aspects of nucleotide chemistry, constant region primer design, and the SELEX protocol for exposing an aptamer library to the desired target.

T Cell Receptor β Chain Gene Usage in Endemic Pemphigus Foliaceus (Fogo Selvagem)

The trimolecular complex comprised of the major histocompatibility complex, peptide antigen, and the T cell receptor is a requisite for T cell activation in normal and autoimmune responses. T cell receptor analysis is critical to further our understanding regarding mechanisms of T cell epitope selection and autoimmune initiation and progression and may help to identify targets for immunotherapy. Pemphigus foliaceus is an autoimmune blistering skin disease characterized by intraepidermal blisters and circulating autoantibodies directed against desmoglein 1, a 160 kDa transmembrane desmosomal molecule expressed in keratinocytes. As tissue damage is mediated by anti-desmoglein 1 antibodies, an initial T cell response is a likely requirement for autoantibody generation in this disease. To elucidate the role of pathogenic T cells in autoimmunity further, we have directly characterized the T cell receptor of T cells derived from pemphigus foliaceus patients. Complementary DNA was isolated from 17 desmoglein 1 specific T cell clones generated from pemphigus foliaceus patients by clonal expansion in vitro. To analyze the T cell repertoire, a panel of primers, collectively specific for the known human T cell receptor beta variable region (TCRBV) families were paired with a constant region primer to polymerase chain reaction to amplify one distinct T cell receptor beta variable region allele for each T cell clone studied. Polymerase chain reaction products were sequenced to determine exact beta chain gene usage. In the 17 clones tested, 10 distinct T cell receptor beta variable region usages and nine T cell receptor beta joining gene segment usages were identified. Furthermore, T cell receptor beta variable region and beta joining usage did not appear to be random, but oligoclonal in nature, with some preference shown for T cell receptor beta variable region 5S1 and T cell receptor BJ2S5.

Rearranged T lymphocyte antigen receptor genes as markers of malignant T cells

We have recently cloned a number of canine T cell receptor (TCR) Vβ genes using degenerate oligonucleotides. From the DNA sequences of the resulting clones and the canine Vβ gene sequences in the literature, seven distinct canine TCR Vβ genes were identified. Vβ specific PCR primers were designed for each of the seven TCR Vβ genes such that under defined conditions, each primer could only amplify a specific TCR Vβ gene in conjunction with the same 3′ constant region (Cβ) primer. By performing RT-PCR on RNA derived from a source containing T lymphocytes, the presence and expansion of T cells expressing a particular Vβ gene could be detected. Moreover, the clonality or diversity of a T cell population under analysis could be easily determined by the VDJ junctional sequence of the amplified Vβ PCR product, in the form of a “DNA fingerprint”. These findings have been used to detect canine T cell lymphoma, and could potentially be used to monitor the remission of T cell malignancies in response to treatment.

Rapid screening of T-cell receptor (TCR) variable gene usage by multiplex PCR: application for assessment of clonal composition.

The selection of various T-cell receptor (TCR) gene families and complex rearrangements during intra-thymic differentiation provide the basis for the expression of antigen specificity by mature T cells. TCR beta variable (TCRBV) transcripts can be identified by RT-PCR, but multiple reactions are required to detect all genes of the TCRBV subfamilies. We describe here a multiplex PCR method that amplifies 46 functional genes comparing 23 TCRBV families in 5 reactions where each reaction contains 4 to 7 specific primers together with a single fluorescence-tagged TCR beta constant region primer. Between 8 and 10 distinct subtypes within each of the 23 TCRBV families can be identified by analysis of the CDR3 length. Multiplex PCR products isolated from agarose gels can be subjected to direct sequencing for confirmation and definitive clonotyping if necessary. The data illustrated here show that the multiplex PCR technique is useful for screening TCRBV usage and can be easily adapted for analysis of clonal composition in T-cell populations.

Caprine T-cell receptor variable beta-chain (TCRV beta) repertoire analysis and potential applications in cowdriosis immune response studies.

Anchor polymerase chain reaction has been applied to the study of caprine TCR V beta-chain repertoire at the mRNA level in peripheral blood of a Saanen goat. Single stranded, g-tailed cDNA synthesized from total RNA was PCR-amplified using a sheep V beta constant region primer (3') and a poly(dC) anchor primer (5') at the variable region end of the TCR V beta-chain. The obtained amplicon was subsequently cloned into Bluescript plasmid vector. A total of 72 recombinant clones whereof 61 contained an insert of appropriate size were harvested. Up to now, the full length sequences of a total of 55 clones have been obtained. Nine clones were rearranged but not functional due to stop codons. Forty-five sequences were functionally rearranged and further analyzed. They were classified into 15 different V beta families on the basis of V-region sequence homology with their human counterpart. V beta families corresponding to the 9 published bovine families were represented in our library. This complexity enables us to develop V beta family-specific primers in order to study the TCR V beta repertoire of other goat breeds of interest, i.e., the Creole goat. There the TCR V beta repertoire analysis and kinetic study of the cowdriosis model will provide insight into the type of the immune response and the status of protection during the immunization process and challenge.

Cloning and expression of the human tumor-specific antibody GM4.

The human monoclonal antibody GM4 was generated by fusing pooled lymphocytes from cancer patients with the lymphoblastoid cell line SHFP-1. Immunohistochemical staining of tumor and normal tissue indicated that this human IgG4 antibody preferentially reacted with melanomas and neuroblastomas. In this study, we demonstrate that GM4 recognizes a "vimentin-like" peptide sequence that we have termed AgGM4. To generate a recombinant derivative of this human antibody, we isolated and expressed the complete heavy and light chain genes. The entire coding sequence for both the heavy and light chains was isolated by RT-PCR using a set of degenerate 5' signal sequence specific primers and a 3' constant region primer. High level antibody synthesis and secretion was achieved in Chinese hamster ovary (CHO) cells using a vector designed to maximize expression. Western blot and FACS analysis indicated recombinant GM4 reacted with human tumor cell lines and AgGM4 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cloning.

TCR Vβ Usage of TSH Receptor-specific CD4+T Cells in Graves' Disease Patients and Healthy Humans

Healthy humans have CD4+T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+cells present in healthy persons. We studied CD4+cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves’ disease (GD) patients.We used short-term CD4+cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject.We determined their TCR Vβ usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vβ region family, in conjunction with a constant region primer. Six lines preferentially used one Vβ family (42–94%), different for each line. In all lines, three or less Vβ families accounted for approximately 60% or more of the Vβ usage. Different Vβ regions were used by each subject. There was no obvious difference between the Vβ usage of the lines from GD patients and healthy controls.These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4+cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4+cells specific for the same autoantigen in healthy subjects.

Construction, expression, and activities of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation.

The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.

Fetal development of T cell repertoire diversity and clonal expansion. • 1296

Introduction. An effective cellular immune response involves the host's ability to recognize and respond to a variety of antigens. The third complementary region (CDR3) of the T-cell receptor (TCR) is the site of antigen recognition. Recombination events within the genetic elements coding for CDR3 lead to the generation of diversity for specific antigen recognition. These recombinations result in different sizes of the mRNA/cDNA encoding CDR3. The preterm infant is unusually susceptible to certain T-cell mediated infections. The ability of the preterm infant's TCR repertoire to diversify has not been previously characterized. Objective. To describe the developmental changes of T-cell receptor diversity generation during gestation. Design. Reverse transcriptase/polymerase chain reaction was performed on umbilical cord or peripheral blood lymphocytes using a constant region primer and 26 Vβ family specific primers. The final PCR products were analyzed on a 5% acryalmide gel. Each V-specific amplification results in a number of bands representing rearrangements of the TCR Vβ chain gene segments. Analysis of the absolute CDR3 size was accomplished with an oligonucleotide primer directed against a conserved region of the 26 Vβ families and a constant region primer by PCR amplification. Results. The CDR3 length from 25 week gestation neonates was six base pairs (two amino acids) shorter than corresponding CDR3 length of adults. Term gestation neonates had CDR3 lengths which were three base pairs (one amino acid) shorter than those of adults. Samples from 24-28 week gestation had no limitation of Vβ family expression. Oligloclonal expansion was more common in infants as compared to adults (p=0.03). Beyond 29 weeks gestation most infants had more than one oligoclonal expansion with concomitant restricted CDR3 expression within that Vβ family. Conclusions. T-cell receptors undergo maturation during gestation and postnatally which is reflected by a lengthening of the antigen binding region of the TCR. The naive repertoire found in most infants 24-28 weeks gestation appears diverse; however, in some infants oligoclonal expansions and marked restrictions of other CDR3s within the same Vβ family may be due to in utero antigenic challenge. Limitation of T-cell repertoire in response to antigenic challenge differs from that of the adult. We speculate that the shorter CDR3 region and unusual response to antigen may contribute to the neonate's predisposition to T-cell mediated infection.

VH gene usage is multiple myeloma: complete absence of the VH4.21 (VH4- 34) gene

The immunoglobin heavy chain variable region (VH) gene usage in multiple myeloma (MM) has not been reported, although a few studies have incidentally identified the VH gene rearranged in small cohorts of MM patients. We used a reverse transcriptase-polymerase chain reaction based technique to analyze the VH gene usage in MM. The VH sequences were obtained after amplification of bone marrow cDNA using the seven VH family-specific and constant region primers. The VH sequences of 72 patients were successfully identified. The frequency of VH family usage in decreasing order was VH3>VH4>VH1>VH5>VH2>VH6>VH7 and corresponded to the functional germline complexity of the VH families. Individual VH genes (VH1-69, VH3-9, VH3-23, and VH3-30) were overrepresented in our cohort of MM patients; some VH genes [VH3-49, VH3-53, and VH4.21 (VH4-34)], which are rearranged with increased frequency in normal circulating B cells, autoimmune diseases, and other B-cell malignancies, were not detected in any MM patient. Compared with germline sequences, an average of 8.8% (range, 2.7% to 16.5%) of the nucleotides had evidence of mutation within each VH sequence. Based on these results, we conclude that (1) the VH gene usage in MM is unique compared with other malignant and nonmalignant B-cell populations, (2) the physiologic process of clonal deletion functions to remove clones that have rearranged VH genes (VH4.21) capable of expressing antibodies, which recognize self-antigens, and (3) the complete lack of VH4.21 gene rearrangement may help to partially explain the paucity of autoimmune phenomena in MM.

Profile of immunoglobulin heavy chain variable gene repertoires and highly selective detection of malignant clonotypes in acute lymphoblastic leukemia

The predominant B cell immunoglobulin heavy chain variable gene (IgH-V) usage and the uniquely rearranged, clonotype-specific variable-diversity-joining region gene (VDJ) sequences were identified in patients with B cell acute lymphoblastic leukemia (B-ALL) using a novel DNA-based gene amplification strategy. The approach allows a thorough and sensitive determination of the number of clonal leukemic IgH rearrangements and their precise V gene usage. This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease-free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones. An initial primary polymerase chain reaction (PCR), directed by an IgH-J generic primer and a complement of family-specific IgH-V primers, defined the major B cell IgH-V gene usage. Use of an IgH-J generic primer supplanted the use of a constant region primer anchor and thus eliminated the need to target mRNA by the traditional RNA reverse transcription-PCR amplification method. Monoclonality of rearranged VDJ bands was further substantiated by high-resolution denaturant gel electrophoretic analysis. The predominant amplified bands were subcloned and sequenced. By sequencing through VDJ juxtaposed regions, that is, the third complementarity-determining region, clonotype-specific primers were developed and used in a secondary clonotype primer-directed PCR (CPD-PCR) to detect, with extreme sensitivity and specificity, a unique B cell clone. Analysis of the products of the CPD-PCR permitted the detection of a single malignant cell among 1 million polyclonal cells and superseded the constraints of prior studies that have provided a limited evaluation of family variable gene repertoire usage. Leukemic clonal rearrangements were detected in 100% of the eight cases of pediatric and two cases of adult B-ALL studied. Two or more clonal IgH-VDJ amplified sequences were observed in 50% of the B-ALL bone marrows analyzed. In two cases, clonotype-specific oligodeoxynucleotide primers, derived from B-ALL VDJ sequences, directed the secondary CPD-PCR, and disease activity was monitored after chemotherapy and allogeneic bone marrow transplantation.

A rapid and reliable PCR method for detecting clonal T cell populations

Aims-To establish a reverse transcription polymerase chain reaction (RT-PCR) for the detection of clonal T cell populations, and to evaluate the sensitivity and reliability of the technique.Methods-After reverse transcription of the target RNA with a consensus T cell receptor (TCR) beta constant (C) region primer, consensus C, variable (V), diversity (D) and joining (J) region primers were used to amplify across various portions of the TCRbeta V-D-J-C junction.Results-In normal T cells the polyclonal rearrangements produce a ladder of PCR bands representing the different sized junction fragments. The presence of a T cell clone leads to over-representation of one junction fragment, hence a disproportionately brighter band in the PCR ladder. In a series of 16 patients the RT-PCR detected nine of nine shown to have a clonal TCRbeta rearrangement by Southern blotting and for six of seven patients, it confirmed the presence of a clone indicated by histology or immunophenotyping with FACS analysis, but which was undetectable (five patients) or not investigated (two patients) by Southern blotting. Investigations mixing RNA from normal lymphocytes and the Jurkat TCR-Vbeta8 T cell line suggested that the method was more sensitive than Southern blotting.Conclusions-All PCR methods are faster and easier than Southern blotting, but RT-PCR also improves detection of clonal T cell populations, is reliable and produces results that are easy to interpret.