Allelic Inclusion Research Articles

Learning antibody sequence constraints from allelic inclusion.

Antibodies and B-cell receptors (BCRs) are produced by B cells, and are built of a heavy chain and a light chain. Although each B cell could express two different heavy chains and four different light chains, usually only a unique pair of heavy chain and light chain is expressed-a phenomenon known as allelic exclusion . However, a small fraction of naive-B cells violate allelic exclusion by expressing two productive light chains, one of which has impaired function; this has been called allelic inclusion . We demonstrate that these B cells can be used to learn constraints on antibody sequence. Using large-scale single-cell sequencing data from humans, we find examples of light chain allelic inclusion in thousands of naive-B cells, which is an order of magnitude larger than existing datasets. We train machine learning models to identify the abnormal sequences in these cells. The resulting models correlate with antibody properties that they were not trained on, including polyreactivity, surface expression, and mutation usage in affinity maturation. These correlations are larger than what is achieved by existing antibody modeling approaches, indicating that allelic inclusion data contains useful new information. We also investigate the impact of similar selection forces on the heavy chain in mouse, and observe that pairing with the surrogate light chain significantly restricts heavy chain diversity.

ScBCR-seq revealed a special and novel IG H&L V(D)J allelic inclusion rearrangement and the high proportion dual BCR expressing B cells.

Since the initial report of V (D) J "allelic exclusion/inclusion" (allelic exclusion rearrangement or allelic inclusion rearrangement) and the concept of the "dual B cell receptor (BCR)" in 1961, despite ongoing discoveries, the precise proportion and source mechanism of dual BCR under physiological conditions have been puzzling immuologists. This study takes advantage of the single cell B cell receptor sequencing (scBCR-seq) technology, which can perfectly match the heavy and light chains of BCR at the level of a single B cell, and obtain the full length mRNA sequence of the complementary determining region 3 (CDR3). Through analyzing the pairing of functional IGH (immunoglobulin heavy chain) and IGL (immunoglobulin light chain) in single B cell from both human and mouse bone marrow and peripheral blood, it was observed that dual BCR B cells exhibit stable and high levels of expression. Among them, the human bone marrow and peripheral blood contain about 10% dual (or multiple) BCR B cells, while in mouse peripheral blood and bone marrow memory B cells, this proportion reaches around 20%. At the same time, we innovatively found that in each research sample of humans and mice, there are three (or more) functional rearrangements (mRNA level) of a single chain in a single B cell. By analyzing the position, direction and other compositional characteristics of the V(D)J gene family, we found that at least two (or more) of them are derived from over two (or more) specific allelic inclusion rearrangements of a single chromosome (mRNA molecular level evidence), our findings also highlighted the necessity of classified single cell sequencing data based on single, dual (or multiple) and cannotbe assembled into BCR when analyzing the B cell repertoire. The results of this article provides new methods and modeling references for evaluating the proportion and source mechanisms of dual BCR B cells, as well as potential significance of allelic inclusion (exclusion escape) of V(D)J rearrangement.

ScRNA-seq revealed the special TCR β & α V(D)J allelic inclusion rearrangement and the high proportion dual (or more) TCR-expressing cells

Allelic exclusion, one lymphocyte expresses one antigen receptor, is a fundamental mechanism of immunological self-tolerance and highly specific immune responses to pathogens. However, the phenomenon of V(D)J allelic inclusion (incomplete allelic exclusion or allelic escape) rearrangement and dual TCR T cells have been discovered by multiple laboratories. Despite continuous new discoveries, the proportion and underlying mechanism of dual TCR has been puzzling immunologists. In this study, we observed the presence of single T cells expressing multiple TCR chains in all samples, with the proportion of 15%, 10%, and 20% in the human thymus, human peripheral blood, and mouse lymphoid organs, respectively. The proportion of T cells possessing multiple T-cell receptors (TCR) varied significantly in different physiological states and developmental stages. By analyzing RSS category, RSS direction, and V(D)J gene position at TR locus of T cells which contain multiple TCR chains, we creatively found that one of TCR β (or TCR α) should originate from the transcription of V(D)J combination in T-cell receptor excision circle (TREC) formed after the twice successful rearrangement in the same chromosome. Moreover, human V30 (or mouse V31) gene may participate in reverse recombination and transcription to prevent allelic exclusion. In general, high proportion of T cells with multiple TCR at the transcriptome level was first made public, and we proposed a novel mechanism of secondary (or more) TCR rearrangement on a single chromosome. Our findings also indicated that the single-cell sequencing data should be classified according to the single, multiple, and abnormal TCR when analyzing the T-cell repertoire.

Endogenous T Cell Receptor Rearrangement Represses Aggressive Central Nervous System Autoimmunity in a TcR-Transgenic Model on the Non-Obese Diabetic Background.

The T cell response to central nervous system (CNS) antigen in experimental autoimmune encephalomyelitis (EAE) permits one to model the immune aspects of multiple sclerosis. 1C6 transgenic mice on the non-obese diabetic (NOD) background possess a class II-restricted T cell receptor (TcR; Vα5-Vβ7) specific for the encephalitogenic peptide myelin oligodendrocyte glycoprotein (MOG)[35−55]. It remains to be determined what role is played by allelic inclusion in shaping the TcR repertoire of these mice. Here, we show that 1C6 T cells display substantial promiscuity in their expression of non-transgenically derived Vα chains. Further, enforced expression of the transgenic TcR in 1C6 × Rag1−/− mice profoundly disrupted thymic negative selection and led to a sharp decrease in the number of mature peripheral T cells. 1C6 × Rag1−/− mice developed spontaneous EAE at a significant frequency and rapidly developed fatal EAE upon immunization with myelin oligodendrocyte glycoprotein (MOG)[35−55]. Passive transfer of 1C6 × Rag1+/+ CD4+ T cells, but not CD8+ T cells or B cells, partially rescued 1C6 × Rag1−/− mice from severe EAE. FoxP3+ CD4+ Treg cells were present in the CNS of immunized 1C6 mice, as well as immunized 1C6 × Rag1−/− that had been supplemented with 1C6 CD4+ T cells. However, they were not observed in 1C6 × Rag1−/− that did not receive Rag1-sufficient 1C6 CD4+. Further, in vivo blockade of Treg accelerated the onset of symptoms in 1C6 mice immunized with MOG[35−55], indicating the pertinence of Treg-mediated control of autoimmune inflammation in this model. Thus, TcR allelic inclusion is crucial to the generation of FoxP3+ CD4+ T cells necessary for the suppression of severe CNS autoimmunity.

Bayesian Inference of Allelic Inclusion Rates in the Human T Cell Receptor Repertoire.

A small population of αβ Tcells is characterized by the expression of more than one unique Tcell receptor (TCR); this outcome is the result of "allelic inclusion," that is, inclusion of both α- or β-chain alleles during V(D)J recombination. Limitations in single-cell sequencing technology, however, have precluded comprehensive enumeration of these dual receptor Tcells. Here, we develop and experimentally validate a fully Bayesian inference model capable of reliably estimating the true rate of α and β TCR allelic inclusion across two different emulsion-barcoding single-cell sequencing platforms. We provide a database composed of over 51,000 previously unpublished allelic inclusion TCR sequence sets drawn from eight healthy individuals and show that allelic inclusion contributes a distinct and functionally important set of sequences to the human TCR repertoire. This database and a Python implementation of our statistical inference model are freely available at our Github repository (https://github.com/JasonACarter/Allelic_inclusion).

Evidence of Alternative Modes of B Cell Activation Involving Acquired Fab Regions of N-Glycosylation in Antibody-Secreting Cells Infiltrating the Labial Salivary Glands of Patients With Sjögren's Syndrome.

To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Can Concurrent Abnormalities in Free Light Chains and Immunoglobulin Concentrations Identify a Target Population for Immunoglobulin Trials in Sepsis?

Light chains κ and λ are immunoglobulin constituents but also circulate independently in blood as free light chains. We investigated whether a concomitant abnormality in free light chain and immunoglobulin levels could identify a high risk of death sepsis subpopulation to inform future IV immunoglobulin trials. We tested whether light chain allelic inclusion occurs in circulating B cells. Prospective cohort study. Adult general ICUs. Adult sepsis patients without any documented immune comorbidity. None. Serum total free light chain, immunoglobulin G, immunoglobulin A, and immunoglobulin M were measured on ICU days 1, 3, and 7. Population normal ranges defined normal and abnormal categories. Logistic regression models tested any independent relationship between high free light chain, immunoglobulins and hospital mortality. CD19 B-cell subsets expressing cell surface κ and λ were quantified by flow cytometry; their frequencies were compared against healthy subjects and correlation assessed against free light chain concentrations. On ICU day 1, high free light chain λ and high free light chain κ were seen in 46.5% and 75.3% of the study cohort (n = 101). Low immunoglobulin levels were commonplace (45.5%) at ICU admission. ICU admission day free light chain and immunoglobulin concentrations were significantly correlated. Septic patients had significantly more CD19 B cells expressing both κ and λ compared with healthy controls (median [interquartile range] 4.1% [2.4-11.0] vs 1.3% [1.2-2.9], respectively; p = 0.0001); these correlated with free light chain concentrations. To our knowledge, abnormalities and associations of free light chain in critically ill adults with sepsis have not been previously reported. The additional prognostic value of free light chain λ and the significance of allelic inclusion in B cells in sepsis require further investigation.

Recombination signal sequences shape antigen receptor repertoires and enforce allelic exclusion

Abstract Antigen receptor loci are arrays of variable (V), diversity (D), and joining (J) gene segments that the RAG endonuclease assembles into complete AgR genes. RAG binds to and cleaves at recombination signal sequences (RSSs) that flank gene segments. V(D)J recombination of Tcrb, Igh, and Igk loci is regulated such that functional AgR genes are assembled on and expressed from one allele (allelic exclusion) in most cells. It is assumed that allelic exclusion is achieved by mono-allelic initiation and feedback inhibition of the V-to-(D)J recombination step, and intrinsic features of RSSs have been proposed to mediate mono-allelic V-to-DJ recombination of Tcrb and Igh loci. We established and analyzed mice with the Trbv2 or Trbv31 RSS replaced by an RSS that possesses greater intrinsic recombination activity. Each of these RSS substitutions causes a profound increase in the development of T cells expressing either Trbv2+ or Trbv31+ TCRβ chains, reflecting that the RSSs flanking Trbv segments are major determinants of Trbv recombination frequency. Each RSS replacement also promotes a modest increase in the fraction of cells expressing Trbv2+ or Trbv31+ TCRβ chains along with another type of TCRβ chain (allelic inclusion). Strikingly, mice with Trbv2 and Trbv31 RSS replacements on opposite alleles have a 30-fold higher than normal level of allelic inclusion for Trbv2+ and Trbv31+ TCRβ chains, demonstrating that RSSs flanking Trbv segments enforce TCRβ allelic exclusion. We conclude that the infrequency of Trbv recombination as determined by inherent inefficiencies of Trbv RSSs limits bi-allelic assembly of Tcrb genes within the time window before feedback inhibition permanently cements allelic exclusion.

In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire.

High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 10(6) B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or 'public', VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

L Chain Allelic Inclusion Does Not Increase Autoreactivity in Lupus-Prone New Zealand Black/New Zealand White Mice

L chain allelic inclusion has been proposed as a B cell tolerance mechanism in addition to clonal deletion, clonal anergy, and receptor editing. It is said to rescue autoreactive B cells from elimination by diluting out the self-reactive BCR through the expression of a second innocuous L chain. In autoimmune animals, such as lupus-prone mice, allelically included B cells could be activated and produce pathogenic autoantibodies. We have previously shown that anti-DNA hybridomas from diseased New Zealand Black/New Zealand White F1 mice exhibit nearly perfect allelic exclusion. In the current study, we have analyzed single B cells from these and from nonautoimmune mice. In addition, we have cloned and expressed the Ig variable regions of several L chain-included B cells in cell culture. We find that although the number of L chain-included B cells increases as a result of receptor editing, the majority of such cells do not retain an autoreactive HxL chain combination and, therefore, allelic inclusion in itself does not serve as a B cell tolerance mechanism in these autoimmune mice.

Highly diverse TCRα chain repertoire of pre-immune CD8+T cells reveals new insights in gene recombination

Highly diverse TCRα chain repertoire of pre-immune CD8⁺T cells reveals new insights in gene recombination

Intraclonal Cell Expansion and Selection Driven by B Cell Receptor in Chronic Lymphocytic Leukemia

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.

Alternative mechanisms of receptor editing in autoreactive B cells

Pathogenic anti-DNA antibodies expressed in systemic lupus erythematosis bind DNA mainly through electrostatic interactions between the positively charged Arg residues of the antibody complementarity determining region (CDR) and the negatively charged phosphate groups of DNA. The importance of Arg in CDR3 for DNA binding has been shown in mice with transgenes coding for anti-DNA V(H) regions; there is also a close correlation between arginines in CDR3 of antibodies and DNA binding. Codons for Arg can readily be formed by V(D)J rearrangement; thereby, antibodies that bind DNA are part of the preimmune repertoire. Anti-DNAs in healthy mice are regulated by receptor editing, a mechanism that replaces κ light (L) chains compatible with DNA binding with κ L chains that harbor aspartic residues. This negatively charged amino acid is thought to neutralize Arg sites in the V(H). Editing by replacement is allowed at the κ locus, because the rearranged VJ is nested between unrearranged Vs and Js. However, neither λ nor heavy (H) chain loci are organized so as to allow such second rearrangements. In this study, we analyze regulation of anti-DNA H chains in mice that lack the κ locus, κ-/κ- mice. These mice show that the endogenous preimmune repertoire does indeed include a high frequency of antibodies with Arg in their CDR3s (putative anti-DNAs) and they are associated mainly with the editor L chain λx. The editing mechanisms in the case of λ-expressing B cells include L chain allelic inclusion and V(H) replacement.

Autoreactive Anti-DNA Transgenic B Cells in Lupus-Prone New Zealand Black/New Zealand White Mice Show Near Perfect L Chain Allelic Exclusion

Recent work on B cell tolerance and autoimmunity has suggested the L chain allelic inclusion is a property of autoreactive B cells and is closely linked to receptor editing. Allelic inclusion could rescue autoreactive B cells from clonal deletion by reducing their effective BCR surface density. We have investigated this phenomenon in anti-DNA producing hybridomas, derived from different strains of Ig gene-targeted, lupus-prone NZB/NZW mice. Our results indicate that isotype and allelic exclusion was strictly maintained in most high- and low-affinity, edited and nonedited, anti-DNA transgenic B cells. However, a substantial fraction of the anti-DNA hybridomas expressed a very restricted set of nonproductively rearranged L chain mRNA, in addition to the productive anti-DNA L chain. The aberrant L chains could have a role in the selection and survival of autoreactive B cells in these autoimmune mice.

Allelic and Isotypic Light Chain Inclusion in Peripheral B Cells from Anti-DNA Antibody Transgenic C57BL/6 and BALB/c Mice

Most mature B lymphocytes express one BCR L chain, kappa or lambda, but recent work has shown that there are exceptions in that some B lymphocytes express both kappa and lambda and some even bear two different kappa L chains. Using the anti-DNA H chain-transgenic mouse, 56R, we find that B cells with pre-existing autoreactivity are especially subject to L chain inclusion. Specifically, we show that isotypic and allelic inclusion enables autoreactive B cells to bypass central tolerance giving rise to B cells that retain dangerous features. One receptor in dual receptor B cells is an editor L chain, i.e., neutralizes or alters self-reactivity of the 56R H chain transgene. We compare the 56R mouse when on the C57/BL/6 background, a strain prone to autoimmunity, with that of 56R when on the BALB/c background, a strain that resists autoimmunity. In the B6.56R mouse, polyreactive B cells with dual L chain move to the follicular B cell compartment. Their localization in the follicular compartment may explain the ease with which B cells in the B6.56R differentiate into autoantibody-producing plasma cells. Likewise, in the BALB/c.56R mouse, dual L chain B cells are found in the follicular B cell compartment. Yet, the lack of autoantibody-producing plasma cells in the BALB/c.56R suggests that postfollicular tolerance checkpoints are intact. The Jkappa usage in dual kappa L chain B cells suggests increased receptor editing activity and is consistent with the expected distribution of Jkappa genes in our computational model for random selection of Jkappa.

Receptor editing and receptor revision in rheumatic autoimmune diseases

Receptor editing is a key mechanism of B cell tolerance that modifies the B cell receptor (BcR) specificity of self-reactive lymphocytes. It acts through initiation of secondary immunoglobulin rearrangements, through generation of newly rearranged endogenous lambda chains that displace kappa chains, or through isotypic and allelic inclusion of dual BcRs (kappa(+)/lambda(+) or kappa(+)/kappa(+) B cells). Mounting evidence indicates that receptor editing is either impaired or accelerated in patients suffering from rheumatic autoimmune diseases. Remarkably, both alterations can promote the pathogenesis of autoimmune disorders by favoring the uncontrolled emergence and/or persistence of autoreactivity. Whereas impaired secondary rearrangements might result in ineffective silencing of B cells, exacerbation of receptor editing can give rise to autoreactive receptors from clones that were initially devoid of autoreactivity.

Igkappa allelic inclusion is a consequence of receptor editing.

The discovery of lymphocytes bearing two light chains in mice carrying self-reactive antibody transgenes has challenged the “one lymphocyte–one antibody” rule. However, the extent and nature of allelically included cells in normal mice is unknown. We show that 10% of mature B cells coexpress both Igκ alleles. These cells are not the result of failure in allelic exclusion per se, but arise through receptor editing. We find that under physiological conditions, editing occurs both by deletion and by inclusion with equal probability. In addition, we demonstrate that B lymphocytes carrying two B-cell receptors are recruited to germinal center reactions, and thus fully participate in humoral immune responses. Our data measure the scope of allelic inclusion and provide a mechanism whereby autoreactive B cells might “escape” central tolerance.

Quantitation of Minimal Residual Disease in Patients with Chronic Lymphocytic Leukemia Using LNA-Modified Fluorescently Labeled Probes and Real-Time PCR Technology.

Background: Patients with chronic lymphocytic leukemia (CLL) relapse even after aggressive therapies and stem cell transplantation. As the therapeutical goal today is to clear off the tumor cell burden as much as possible, highly sensitive assays for minimal residual disease (MRD) evaluation and monitoring are needed. At present, many patients with not only germline IgVH sequences, but also with hypermutated IgVH genes are being treated, with the need for a sensitive and specific MRD monitoring. The original notion of MRD follow-up in CLL was based on the usage of JH-gene specific TaqMan hybridization probes. At present, due to the vast diversity of B-clonal rearrangements to be detected, the original idea has been challenged and the methodology should be modified.Aims: Since the hypermutation process does not restrict itself to the VH segments only and might afflict the JH segment as well, the molecular tools for the monitoring of CLL clonal rearrangements must be versatile enough to allow for the detection and quantitation of virtually any sequence possible. Moreover, the technique must meet the criteria for high sensitivity and specificity. We present here a novel methodology for MRD monitoring in CLL, based on LNA technology (Locked Nucleic Acids) and quantitative Real-Time PCR.Methods: 59 patients with the diagnosis of CLL were enrolled into our MRD study (22 females, 37 males, median age 59.1 yrs). 33 out of 59 individuals had unmutated IgVH genes (4 females, 29 males), 26 out of 59 patients had mutated IgVH genes (15 females, 11 males). For each patient, clone-specific primers were designed and their clonal VH sequences were molecularly cloned to construct the quantitation standards. In one patient, allelic inclusion has been identified (VH1–8 and VH3–30, both mutated), and for this individual, clone-specific primers and standards have been constructed for both rearrangements. To quantify the individual clonal VH transcripts, LNA-modified fluorescently labeled probes targeted against individual gene segments were employed. For any of 6 (7) IgVH families with unmutated VH genes, family-specific consensus LNA-modified probes were used. For those CLL cases with heavily hypermutated genes, ProbeLibrary™ was employed. For quantitation experiments, ABL was used as the control gene.Results: The LNA-modified probes are distinguished by a very high specificity and sensitivity (reaching to 10−8, in contrast to flow cytometry with its detection limit being 10−4). The LNA-based assays allow for precise monitoring of the residual tumor cell burden in CLL patients, especially during those periods of time, when other, less sensitive techniques fail to trace the malignant clone.Conclusions: LNA-modified probes and Real-Time PCR technology represent a highly versatile, specific and extremely sensitive methodology for the monitoring of MRD in chronic lymphocytic leukemia. We strongly advocate their usage in the molecular follow-up of MRD in the setting of CLL (and possibly other B-cell malignancies with hypermutated VH gene sequences as well).

A Model for TCR Gene Segment Use

The TCR alpha-chain is assembled by somatic recombination of variable (V) and joining (J) gene segments at the CD4+ CD8+ stage of development. In this study, we present the first analytical model for deletional rearrangement and show that it is consistent with almost all available data on Valpha Jalpha use in mice and humans. A key feature of the model is that both "local" and "express service" models of rearrangement can be obtained by varying a single parameter that describes the number of gene segments accessible at a time. We find that the window is much larger for Valpha segments than Jalpha segments, which reconciles seemingly conflicting data for the former. Implications for the properties of the repertoire as a whole and experiments that seek to probe them are discussed. Special considerations for allelic inclusion are treated in the Appendices.