A Bioinformatics Assessment Indicating Better Outcomes With Breast Cancer Resident, Immunoglobulin CDR3-MMP2 Binding.

BACKGROUND/AIM: The recombination of V, D, and J immunoglobulin (IG) gene segments leads to many variations in the amino acids (AAs) encoded at that site, the complementarity determining region-3 (CDR3). Thus, cancer patients may have varying degrees of CDR3 AA binding specificity for cancer proteases, for example, matrix metalloproteinase 2 (MMP2). MMP2 in breast cancer has been found to contribute to metastasis and is used as a marker for tumor staging. Thus, this report evaluated the tumor resident, patient specific IG CDR3 binding affinities to cancer proteases to test the hypothesis that greater binding affinities would be associated with a better outcome. MATERIALS AND METHODS: Using two independent bioinformatics tools, we evaluated the IG CDR3-MMP2 binding affinities throughout the cancer genome atlas breast cancer (TCGA-BRCA) dataset. RESULTS: Results indicated that the better the CDR3-MMP2 binding, the better the survival probability. An analogous evaluation for four other proteases, including calpain-1 and thermolysin, displayed no such associations with survival probabilities. CONCLUSION: This study is consistent with the possibility that patient IG-cancer protease interactions could impact outcomes and raises the question of whether therapeutic antibody targeting of MMP2 would reduce breast cancer mediated tissue destruction and breast cancer mortality rates.

TCR Repertoire as a Novel Indicator for Immune Monitoring and Prognosis Assessment of Patients With Cervical Cancer.

There is increasing evidence that deep sequencing-based T cell repertoire can sever as a biomarker of immune response in cancer patients; however, the characteristics of T cell repertoire including diversity and similarity, as well as its prognostic significance in patients with cervical cancer (CC) remain unknown. In this study, we applied a high throughput T cell receptor (TCR) sequencing method to characterize the T cell repertoires of peripheral blood samples from 25 CC patients, 30 cervical intraepithelial neoplasia (CIN) patients and 20 healthy women for understanding the immune alterations during the cervix carcinogenesis. In addition, we also explored the signatures of TCR repertoires in the cervical tumor tissues and paired sentinel lymph nodes from 16 CC patients and their potential value in predicting the prognosis of patients. Our results revealed that the diversity of circulating TCR repertoire gradually decreased during the cervix carcinogenesis and progression, but the circulating TCR repertoires in CC patients were more similar to CIN patients than healthy women. Interestingly, several clonotypes uniquely detected in CC patients tended to share similar CDR3 motifs, which differed from those observed in CIN patients. In addition, the TCR repertoire diversity in sentinel lymphatic nodes from CC patients was higher than in tumor tissues. More importantly, less clonotypes in TCR repertoire of sentinel lymphatic node was associated with the poor prognosis of the patients. Overall, our findings suggested that TCR repertoire might be a potential indicator of immune monitoring and a biomarker for predicting the prognosis of CC patients. Although functional studies of T cell populations are clearly required, this study have expanded our understanding of T cell immunity during the development of CC and provided an experimental basis for further studies on its pathogenesis and immunotherapy.

T cell receptor assessment in autoimmune disease requires access to the most adjacent immunologically active organ.

Next generation sequencing of T and B cell receptors is emerging as a valuable and effective method to diagnose and monitor hematopoietic malignancies. So far, this approach has not been fully explored in regard to autoimmune diseases. T cells develop in the thymus where they undergo positive and negative selection, and the autoimmune regulator (Aire) is central in the establishment of immunological tolerance. Loss of Aire leads to severe multiorgan autoimmune disease with infiltration of autoreactive T cells in affected organs. Here, we have utilized next generation sequencing technology to investigate the T cell receptor repertoire in autoimmunity induced by immunization of mice with a self-antigen, myeloperoxidase. By investigating the T cell receptor repertoire in peripheral blood, spleen and lumbar lymph nodes from naïve and immunized Aire -/- mice and wild type littermates, changes in the usage of V and J genes were evident. Our results identify TCR clonotypes which could be potential targets for immune therapy. Also, Aire -/- autoimmunity is driven by a variety of autoantigens where the autoimmune response is highly polyclonal, and access to the most adjacent immunologically active tissue is required to identify T cell receptor sequences that are potentially unique to the antigen in Aire-/- immunized mice.

Assessment of B Cell Repertoire in Humans.

The B cell receptor (BCR) repertoire is highly diverse. Repertoire diversity is achieved centrally by somatic recombination of immunoglobulin (Ig) genes and peripherally by somatic hypermutation and Ig heavy chain class-switching. Throughout these processes, there is selection for functional gene rearrangements, selection against gene combinations resulting in self-reactive BCRs, and selection for BCRs with high affinity for exogenous antigens after challenge. Hence, investigation of BCR repertoires from different groups of B cells can provide information on stages of B cell development and shed light on the etiology of B cell pathologies. In most instances, the third complementarity determining region of the Ig heavy chain (CDR-H3) contributes the majority of amino acids to the antibody/antigen binding interface. Although CDR-H3 spectratype analysis provides information on the overall diversity of BCR repertoires, this fairly simple technique analyzes the relative quantities of CDR-H3 regions of each size, within a range of approximately 10-80 bp, without sequence detail and thus is limited in scope. High-throughput sequencing (HTS) techniques on the Roche 454 GS FLX Titanium system, however, can generate a wide coverage of Ig sequences to provide more qualitative data such as V, D, and J usage as well as detailed CDR3 sequence information. Here we present protocols in detail for CDR-H3 spectratype analysis and HTS of human BCR repertoires.

Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice.

BACKGROUND: Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data. RESULTS: We isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors-(i) replicate sequencing and (ii) sequencing depth-are crucial for robust CDR3 and VDJ detection and ranking. CONCLUSIONS: In summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.

An integrated approach to extreme thermostabilization and affinity maturation of an antibody.

Antibodies are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. However, in order to be practically useful, antibodies must be highly stable and bind their target antigens with high affinity. We present a combinatorial approach to generate high-affinity, highly stable antibodies through the design of stable frameworks, specificity grafting and maturation via somatic hypermutation in vitro. By collectively employing these methods, we have engineered a highly stable, high-affinity, full-length antibody with a T(m) over 90°C that retains significant activity after heating to 90°C for 1 h, and has ~95-fold improved antigen-binding affinity. The stabilized IgG framework is compatible with affinity maturation, and should provide a broadly useful scaffold for grafting a variety of complementarity-determining region loops for the development of stable antibodies with desired specificities.

Comprehensive assessment of potential multiple myeloma immunoglobulin heavy chain V-D-J intraclonal variation using massively parallel pyrosequencing.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.

Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures.

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.

A new strategy for the discrimination of mitochondrial DNA haplogroups in Han population.

Mitochondrial DNA (mtDNA) haplogroup discrimination is interesting not only for phylogenetic and clinical but also for forensic studies. We discriminated the mtDNA haplogroups of 570 healthy unrelated Han people from Zhejiang Province, Southeast China, by comprehensive analysis mutations of the hypervariable segments-I sequence and diagnostic polymorphisms in mtDNA coding region using real-time polymerase chain reaction (RT-PCR), which was compared with the widely used PCR and restriction fragment length polymorphism (PCR-RFLP) method. The results showed that in superhaplogroup M, haplogroup D was the most common haplotype within this assay to 24.6%, and in the other superhaplotype N, haplogroup B and F were the most common groups. Samples re-identified by PCR-RFLP showed the consistent results that were got with RT-PCR. In conclusion, the RT-PCR strategy appears to be an accurate, reproducible, and sensitive technique for the discrimination of mtDNA haplogroups, especially for mass screenings quickly and economically.

Comprehensive assessment of the TCRBV repertoire in small T-cell samples by means of an improved and convenient multiplex PCR method.

OBJECTIVE: Overall diversity of the T-cell receptor (TCR) repertoire can be regarded as a recapitulatory signature of a host's immunocompetence status. We aimed to establish a time- and cost-saving multiplex polymerase chain reaction (PCR) method for determining the TCR repertoire of conventional alphabeta T cells in small T-cell samples. MATERIALS AND METHODS: The method estimates the length distribution of the complementarity-determining regions 3 (CDR3) of beta variable (BV) gene segments (TCRBV repertoire) by multiplex PCR, followed by fluorescent run-off reactions to visualize BV-BC and/or BV-BJ rearrangements. Run-off products are separated on a capillary sequencer and subsequently analyzed with GeneScan or Genotyper programs. Detection-limit studies with normal T cells, KMS27 cells, and regulatory T cells were carried out to evaluate sensitivity and reproducibility. RESULTS: Head-to-head comparison of the method with conventional immunoscope assay has shown that it is a time- and cost-saving approach to characterize TCRBV and TCRBJ repertoires, including the presence of oligoclonal T cells in samples containing as few as 1 x 10(5) T cells. CONCLUSION: We have developed a multiplex PCR method that allows comprehensive assessment of the TCRBV repertoire at the BV-BC and BV-BJ levels, and saves a considerable amount of time, reagents, and cell input.

A rapid mtDNA assay of 22 SNPs in one multiplex reaction increases the power of forensic testing in European Caucasians.

We have developed a multiplex mitochondrial (mtDNA) assay of 21 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable region 1 (HVR1) and hypervariable region 2 (HVR2) that can be amplified in a single reverse touchdown polymerase chain reaction. Single base extension using the SNaPshot technique is also carried out as one multiplex. Besides the nine major European haplogroups (i.e. H, I, J, K, T, U, V, W, and X), 16 additional subclades (i.e. N1, X2, X2b, U2'-4/7'-9', J/T, J1, J1c, HV, H1, H1a1, H1c, H3, H4, H6a, H7a H10) can be detected and classified into a phylogenetic mtDNA tree. By analyzing 130 Caucasoid samples from Germany, 36 different haplotypes were found resulting in a power of discrimination of 93.2%. Although 49% of all samples belonged to superhaplogroup H, the most common haplotype, i.e., haplogroup-specific SNPs plus haplogroup unspecific SNPs, had a frequency of only 18%. This assay is applicable for high-throughput mtDNA analysis and forensic mass screening. It will give additional information to the common control region sequencing of HVR1 and HVR2.

Statistical analysis of CDR3 length distributions for the assessment of T and B cell repertoire biases.

Complementarity-determining region 3 (CDR3) length distribution analysis explores the diversity of the T cell receptor (TCR) and immunoglobulin (Ig) repertoire at the transcriptome level. Studies of the CDR3, the most hypervariable part of these molecules, have been frequently used to identify recruitment of T and B cell clones involved in immunological responses. CDR3 length distribution analysis gives a clear perception of repertoire variations between individuals and over time. However, the complexity of CDR3 length distribution patterns and the high number of possible repertoire alterations per individual called for the development of robust data analysis methods. The goal of these methods is to identify, quantify and statistically assess differences between repertoires so as to offer a better diagnostic or predictive tool for pathologies involving the immune system. In this review we will explain the benefit of analyzing CDR3 length distribution for the study of immune cell diversity. We will start by describing this technology and its associated data processing, and will subsequently focus on the statistical methods used to compare CDR3 length distribution patterns. Finally, we will address the various methods for assessing CDR3 length distribution gene signatures in pathological states.

An assessment of the hypervariable domains of the 16S rRNA genes for their value in determining microbial community diversity: the paradox of traditional ecological indices.

Amplicon length heterogeneity PCR (LH-PCR) was investigated for its ability to distinguish between microbial community patterns from the same soil type under different land management practices. Natural sagebrush and irrigated mouldboard-ploughed soils from Idaho were queried as to which hypervariable domains, or combinations of 16S rRNA gene domains, were the best molecular markers. Using standard ecological indices to measure richness, diversity and evenness, the combination of three domains, V1, V3 and V1+V2, or the combined V1 and V3 domains were the markers that could best distinguish the undisturbed natural sagebrush communities from the mouldboard-ploughed microbial communities. Bray-Curtis similarity and multidimensional scaling were found to be better metrics to ordinate and cluster the LH-PCR community profiling data. The use/misuse of traditional ecological indices such as diversity and evenness to study microbial community profiles will remain a major point to consider when performing metagenomic studies.

Detection of functional V(H)81X heavy chains in adult mice with an assessment of complementarity-determining region 3 diversity and capacity to form pre-B cell receptor.

Recent reports have indicated that up to 50% of all H chain proteins formed cannot associate with the surrogate L chain complex and therefore fail to form a pre-B cell receptor (pBCR), which is required for allelic exclusion and, in most cases, verifies that the H chain can assemble with the L chain to form an Ab molecule. Certain V(H) genes, such as V(H)81X, appear to be particularly prone to encoding for nonpairing (dysfunctional) H chains. It has been suggested that sequence variability at complementarity-determining region 3, especially when increased by the enzyme TdT, often precludes the ability of V(H)81X-using H chains to form pBCR. To determine whether a motif exists that accounts for the ability of H chains to pair with surrogate L chain complex/L chain, we have bred a mouse line in which H chain recombination can only occur on one allele, allowing us to compile a pool of H chains capable of forming Ab molecules in the absence of dysfunctional H chains. Somewhat unexpectedly, we have found V(H)81X H chains capable of Ab formation and cell surface expression in the presence of TdT. Scrutiny of these H chains has revealed that, although highly prone to encode for dysfunctional H chains, sequence variability is not severely limited among functional V(H)81X H chains. We also demonstrate that surface Ig expression is highly indicative of the capacity of a H chain to form pBCR.

Follicular lymphoma with monocytoid B-cell proliferation: molecular assessment of the clonal relationship between the follicular and monocytoid B-cell components.

Although a number of studies have recognized that follicular lymphomas may be accompanied by a prominent proliferation of monocytoid B-cells, the clonal relationship between these components has not been adequately assessed. Using laser capture microdissection, we isolated the follicular and monocytoid B-cell components from four well-characterized cases of follicular lymphoma with prominent monocytoid B-cells. DNA from each component was analyzed using polymerase chain reaction (PCR)-based methods to assess for clonal rearrangements of the immunoglobulin heavy chain gene (IgH) and for the presence of the bcl-2 gene major breakpoint region/joining region (MBR/JH) DNA fusion products by conventional PCR and fluorescence melting curve analysis. Evidence of clonal identity was established in the follicular and monocytoid B-cell components of three cases by demonstration of IgH gene rearrangements of identical size using IgH PCR, by comparison of complementarity determining region III (CDRIII) DNA sequences, or by detection of bcl-2 MBR/JH fusion products of identical size and/or melting temperature. Molecular analysis of the fourth case revealed a monoclonal and MBR/JH-positive follicular component accompanied by a polyclonal and MBR/JH-negative monocytoid B-cell proliferation. We conclude that the follicular and monocytoid B-cell components of this variant of follicular lymphoma are clonally identical in the majority of cases. However, in a minority of these cases, the monocytoid B-cell component is reactive. Larger studies that assess the prognostic significance of follicular lymphoma with monocytoid B-cells will benefit from molecular studies that assess the clonal relationship of both components.