IgH-V(D)J NGS-MRD measurement pre- and early post-allotransplant defines very low- and very high-risk ALL patients.

Positive detection of minimal residual disease (MRD) by multichannel flow cytometry (MFC) prior to hematopoietic cell transplantation (HCT) of patients with acute lymphoblastic leukemia (ALL) identifies patients at high risk for relapse, but many pre-HCT MFC-MRD negative patients also relapse, and the predictive power MFC-MRD early post-HCT is poor. To test whether the increased sensitivity of next-generation sequencing (NGS)-MRD better identifies pre- and post-HCT relapse risk, we performed immunoglobulin heavy chain (IgH) variable, diversity, and joining (V[D]J) DNA sequences J NGS-MRD on 56 patients with B-cell ALL enrolled in Children's Oncology Group trial ASCT0431. NGS-MRD predicted relapse and survival more accurately than MFC-MRD (P < .0001), especially in the MRD negative cohort (relapse, 0% vs 16%; P = .02; 2-year overall survival, 96% vs 77%; P = .003). Post-HCT NGS-MRD detection was better at predicting relapse than MFC-MRD (P < .0001), especially early after HCT (day 30 MFC-MRD positive relapse rate, 35%; NGS-MRD positive relapse rate, 67%; P = .004). Any post-HCT NGS positivity resulted in an increase in relapse risk by multivariate analysis (hazard ratio, 7.7; P = .05). Absence of detectable IgH-V(D)J NGS-MRD pre-HCT defines good-risk patients potentially eligible for less intense treatment approaches. Post-HCT NGS-MRD is highly predictive of relapse and survival, suggesting a role for this technique in defining patients early who would be eligible for post-HCT interventions. The trial was registered at www.clinicaltrials.gov as #NCT00382109.

CMV reactivation drives posttransplant T-cell reconstitution and results in defects in the underlying TCRß repertoire.

Although cytomegalovirus (CMV) reactivation has long been implicated in posttransplant immune dysfunction, the molecular mechanisms that drive this phenomenon remain undetermined. To address this, we combined multiparameter flow cytometric analysis and T-cell subpopulation sorting with high-throughput sequencing of the T-cell repertoire, to produce a thorough evaluation of the impact of CMV reactivation on T-cell reconstitution after unrelated-donor hematopoietic stem cell transplant. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme B(high)/CD28(low)/CD57(high)/CD8(+) effector memory T cells (Tem) and resulted in a linked contraction of all naive T cells, including CD31(+)/CD4(+) putative thymic emigrants. T-cell receptor ß (TCRß) deep sequencing revealed a striking contraction of CD8(+) Tem diversity due to CMV-specific clonal expansions in reactivating patients. In addition to querying the topography of the expanding CMV-specific T-cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the underlying TCR repertoire. Our results reveal new evidence for significant defects in the underlying CD8 Tem TCR repertoire in patients who reactivate CMV, providing the first molecular evidence that, in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T-cell repertoire. This trial was registered at www.clinicaltrials.gov as #NCT01012492.

Dynamics of the cytotoxic T cell response to a model of acute viral infection.

UNLABELLED: A detailed characterization of the dynamics and breadth of the immune response to an acute viral infection, as well as the determinants of recruitment to immunological memory, can greatly contribute to our basic understanding of the mechanics of the human immune system and can ultimately guide the design of effective vaccines. In addition to neutralizing antibodies, T cells have been shown to be critical for the effective resolution of acute viral infections. We report the first in-depth analysis of the dynamics of the CD8(+) T cell repertoire at the level of individual T cell clonal lineages upon vaccination of human volunteers with a single dose of YF-17D. This live attenuated yellow fever virus vaccine yields sterile, long-term immunity and has been previously used as a model to understand the immune response to a controlled acute viral infection. We identified and enumerated unique CD8(+) T cell clones specifically induced by this vaccine through a combined experimental and statistical approach that included high-throughput sequencing of the CDR3 variable region of the T cell receptor ß-chain and an algorithm that detected significantly expanded T cell clones. This allowed us to establish that (i) on average, ∼ 2,000 CD8(+) T cell clones were induced by YF-17D, (ii) 5 to 6% of the responding clones were recruited to long-term memory 3 months postvaccination, (iii) the most highly expanded effector clones were preferentially recruited to the memory compartment, and (iv) a fraction of the YF-17D-induced clones could be identified from peripheral blood lymphocytes solely by measuring clonal expansion. IMPORTANCE: The exhaustive investigation of pathogen-induced effector T cells is essential to accurately quantify the dynamics of the human immune response. The yellow fever vaccine (YFV) has been broadly used as a model to understand how a controlled, self-resolving acute viral infection induces an effective and long-term protective immune response. Here, we extend this previous work by reporting the identity of activated effector T cell clones that expand in response to the YFV 2 weeks postvaccination (as defined by their unique T cell receptor gene sequence) and by tracking clones that enter the memory compartment 3 months postvaccination. This is the first study to use high-throughput sequencing of immune cells to characterize the breadth of the antiviral effector cell response and to determine the contribution of unique virus-induced clones to the long-lived memory T cell repertoire. Thus, this study establishes a benchmark against which future vaccines can be compared to predict their efficacy.

T-cell receptor repertoire variation may be associated with type 2 diabetes mellitus in humans.

BACKGROUND: Recent work in Pima Indians, a population with high rates of obesity and type 2 diabetes mellitus (T2DM), demonstrated that human leukocyte antigen haplotype DRB1*02 carriers have an increased acute insulin response and decreased risk for the development of T2DM, implicating loss of self-tolerance in the pathogenesis of T2DM. Advances in genomic sequencing have made T-cell receptor repertoire analysis a practical mode of investigation. METHODS: High-throughput sequencing of T-cell receptor complementarity-determining region 3 was carried out in male Pima Indians with normal glucose regulation (n = 11; age = 31 ± 8 years; %fat = 30.2 ± 8.7%) and the protective DRB1*02 haplotype versus those with T2DM without DRB1*02 (n = 7; age = 34 ± 8 years; %fat = 31.2 ± 4.7%). Findings were partially replicated in another cohort by assessing the predictive ability of T-cell receptor variation on risk of T2DM in Pima Indian men (n = 27; age = 28.9 ± 7.1 years; %fat = 28.8 ± 7.1%) and women (n = 20; age = 29 ± 7.0 years; %fat = 37.1 ± 6.8%) with baseline normal glucose regulation but without the protective haplotype who were invited to follow-up examinations as frequently as every 2 years where diabetes status was assessed by a 75-g oral glucose tolerance test. Of these subjects, 13 developed diabetes. RESULTS: T-cell receptor complementarity-determining region 3 length was shorter in those with T2DM, and a one-nucleotide decrease in complementarity-determining region 3 length was associated with a nearly threefold increase in risk for future diabetes. The frequency of one variable gene, TRBV7-8, was higher in those with T2DM. A 1% increase in TRBV7-8 frequency was associated with a greater than threefold increase in diabetes risk. CONCLUSIONS: These results indicate that T-cell autoimmunity may be an important component in progression to T2DM in Pima Indians.

Intrathecal BCR transcriptome in multiple sclerosis versus other neuroinflammation: Equally diverse and compartmentalized, but more mutated, biased and overlapping with the proteome.

The mechanisms driving the intrathecal synthesis of IgG in multiple sclerosis (MS) are unknown. We combined high-throughput sequencing of transcribed immunoglobulin heavy-chain variable (IGHV) genes and mass spectrometry to chart the diversity and compartmentalization of IgG-producing B cells in the cerebrospinal fluid (CSF) of MS patients and controls with other neuroinflammatory diseases. In both groups, a few clones dominated the intrathecal IGHV transcriptome. In most MS patients and some controls, dominant transcripts matched the CSF IgG. The IGHV transcripts in CSF of MS patients frequently carried IGHV4 genes and had more replacement mutations compared to controls. In both groups, dominant IGHV transcripts were identified within clusters of clonally related B cells that had identical or related IGHV transcripts in the blood. These findings suggest more pronounced affinity maturation, but an equal degree of diversity and compartmentalization of the intrathecal B-cell response in MS compared to other neuroinflammatory diseases.

The restricted DH gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content.

The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection. To study how the Ab repertoire is selected, we analyzed one part of diversity generation that cannot be driven by the rearrangement mechanism: the reading frame usage of DH genes. We have used two high-throughput sequencing methodologies, multiple subjects and advanced algorithms to measure the DH reading frame usage in the human Ab repertoire. In most DH genes, a single reading frame is used predominantly, and inverted reading frames are practically never observed. The choice of a single DH reading frame is not limited to a single position of the DH gene. Rather, each DH gene participates in rearrangements of differing CDR3 lengths, restricted to multiples of three. In nonproductive rearrangements, there is practically no reading frame bias, but there is still a striking absence of inversions. Biases in DH reading frame usage are more pronounced, but also exhibit greater interindividual variation, in IgG(+) and IgA(+) than in IgM(+) B cells. These results suggest that there are two developmental checkpoints of DH reading frame selection. The first occurs during VDJ recombination, when inverted DH genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is expressed. The second checkpoint implies that DH reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host-specific DH reading frame usage bias.

Fractal organization of the human T cell repertoire in health and after stem cell transplantation.

T cell repertoire diversity is generated in part by recombination of variable (V), diversity (D), and joining (J) segments in the T cell receptor ß (TCR) locus. T cell clonal frequency distribution determined by high-throughput sequencing of TCR ß in 10 stem cell transplantation (SCT) donors revealed a fractal, self-similar frequency distribution of unique TCR bearing clones with respect to V, D, and J segment usage in the T cell repertoire of these individuals. Further, ranking of T cell clones by frequency of gene segment usage in the observed sequences revealed an ordered distribution of dominant clones conforming to a power law, with a fractal dimension of 1.6 and 1.8 in TCR ß DJ and VDJ containing clones in healthy stem cell donors. This self-similar distribution was perturbed in the recipients after SCT, with patients demonstrating a lower level of complexity in their TCR repertoire at day 100 followed by a modest improvement by 1 year post-SCT. A large shift was observed in the frequency distribution of the dominant T cell clones compared to the donor, with fewer than one third of the VDJ-containing clones shared in the top 4 ranks. In conclusion, the normal T cell repertoire is highly ordered with a TCR gene segment usage that results in a fractal self-similar motif of pattern repetition across levels of organization. Fractal analysis of high-throughput TCR ß sequencing data provides a comprehensive measure of immune reconstitution after SCT.

Tumor-infiltrating lymphocytes in colorectal tumors display a diversity of T cell receptor sequences that differ from the T cells in adjacent mucosal tissue.

Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor-infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison with the TCRs from adjacent healthy mucosal tissue. In parallel, we captured TIL counts using standard immunohistochemistry. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent mucosal tissue, and the number of shared clones is not dependent on distance between the samples. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.

Polymorphisms of the IL1-receptor antagonist gene (IL1RN) are associated with multiple markers of systemic inflammation.

BACKGROUND: Circulating levels of acute phase reactant proteins such as plasma C-reactive protein (CRP) are likely influenced by multiple genes regulating the innate immune response. METHODS AND RESULTS: We screened a set of 16 inflammation-related genes for association with CRP in a large population-based study of healthy young adults (n=1627). Results were validated in 2 independent studies (n=1208 and n=4310), including a pooled analysis of all 3 studies. In the pooled analysis, the minor allele of IL1RN 1018 (rs4251961) within the gene encoding interleukin (IL)-1 receptor antagonist (IL-1RA) was significantly associated with higher mean plasma log(CRP) level (P<1 x 10(-4)). The same IL1RN 1018 allele was associated with higher mean plasma log(IL-6) levels (P=0.004). In the pooled analysis, the minor allele of IL1RN 13888 (rs2232354) was associated with higher fibrinogen, (P=0.001). The IL1RN 1018 and 13888 variant alleles tag a clade of IL1RN haplotypes linked to allele 1 of an 86-bp VNTR polymorphism. We confirmed that the IL1RN 1018 variant (rs4251961) was associated with decreased cellular IL-1RA production ex vivo. CONCLUSIONS: Common functional polymorphisms of the IL1RN gene are associated with several markers of systemic inflammation.

Identification of patient-specific and tumor-shared T cell receptor sequences in renal cell carcinoma patients.

A major requirement for cancer immunotherapy is the development of biomarkers for prognosis and for monitoring therapy response. In an attempt to evaluate the immune response of renal cell carcinoma (RCC) patients, tumor lesions and / or blood samples from 12 RCC patients underwent deep T cell receptor (TCR) sequencing. Despite the low number of samples, different TCR distribution patterns could be detected. Most of the RCC patients presented "patient-specific" TCR sequences, and those clonotypes were present at higher frequency in tumor lesions suggesting a specific extravasation from the blood. Comparison among the tumor samples revealed also "patient-shared" TCR patterns. Indeed, a central core of 16 different TCRs were shared by 3 patients, whereas other 6 patients shared between 4 and 6 TCR sequences, with two sub-groups sharing 12 to 17 different clonotypes. The relative frequencies of shared clonotypes were very different varying from < 1% to a maximum of 37% of the total TCR repertoire. These data confirm the presence of tumor-specific TCR within the cancer tissue and suggest the existence of shared epitopes among different patients that might be used as targets for tumor immunotherapy.

Tracking the Fate and Origin of Clinically Relevant Adoptively Transferred CD8+ T Cells In Vivo.

Adoptively transferred tumor-specific cells can mediate tumor regression in cancers refractory to conventional therapy. Autologous polyclonal tumor-specific cytotoxic T cells (CTL) generated from peripheral blood and infused into patients with metastatic melanoma show enhanced persistence, compared to equivalent numbers of more extensively expanded monoclonal CTL, and are associated with complete remissions (CR) in select patients. We applied high-throughput T cell receptor Vß sequencing (HTTCS) to identify individual clonotypes within CTL products, track them in vivo post-infusion and then deduce the pre-adoptive transfer (endogenous) frequencies of cells ultimately responsible for tumor regression. The summed in vivo post-transfer frequencies of the top 25 HTTCS-defined clonotypes originally detected in the infused CTL population were comparable to enumeration by binding of antigen peptide-HLA multimers, revealing quantitative HTTCS is a reliable, multimer-independent alternative. Surprisingly, the polyclonal CTL products were composed predominantly of clonotypes that were of very low frequency (VLF) in the endogenous samples, often below the limit of HTTCS detection (0.001%). In patients who achieved durable CRs, the composition of transferred CTLs was dominated (57-90%) by cells derived from a single VLF clonotype. Thus, HTTCS now reveals that tumor-specific CTL enabling long-term tumor control originate from endogenous VLF populations that exhibit proliferative/survival advantages. Along with results indicating that naïve cell populations are most likely to contain cells that exist at VLF within the repertoire, our results provide a strong rationale for favoring T cells arising from VLF populations and with early-differentiation phenotypes when selecting subset populations for adoptive transfer.

Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire.

An individual's T cell repertoire dynamically encodes their pathogen exposure history. To determine whether pathogen exposure signatures can be identified by documenting public T cell receptors (TCRs), we profiled the T cell repertoire of 666 subjects with known cytomegalovirus (CMV) serostatus by immunosequencing. We developed a statistical classification framework that could diagnose CMV status from the resulting catalog of TCRß sequences with high specificity and sensitivity in both the original cohort and a validation cohort of 120 different subjects. We also confirmed that three of the identified CMV-associated TCRß molecules bind CMV in vitro, and, moreover, we used this approach to accurately predict the HLA-A and HLA-B alleles of most subjects in the first cohort. As all memory T cell responses are encoded in the common format of somatic TCR recombination, our approach could potentially be generalized to a wide variety of disease states, as well as other immunological phenotypes, as a highly parallelizable diagnostic strategy.

Common clonal origin of central and resident memory T cells following skin immunization.

Central memory T (TCM) cells in lymph nodes (LNs) and resident memory T (TRM) cells in peripheral tissues have distinct roles in protective immunity. Both are generated after primary infections, but their clonal origins have been unclear. To address this question, we immunized mice through the skin with a protein antigen, a chemical hapten, or a non-replicating poxvirus. We then analyzed antigen-activated T cells from different tissues using high-throughput sequencing (HTS) of the gene encoding the T cell receptor (TCR) ß-chain (Trb, also known as Tcrb) using CDR3 sequences to simultaneously track thousands of unique T cells. For every abundant TRM cell clone generated in the skin, an abundant TCM cell clone bearing the identical TCR was present in the LNs. Thus, antigen-reactive skin TRM and LN TCM cell clones were derived from a common naive T cell precursor after skin immunization, generating overlapping TCR repertoires. Although they bore the same TCR, TRM cells mediated rapid contact hypersensitivity responses, whereas TCM cells mediated delayed and attenuated responses. Studies in human subjects confirmed the generation of skin TRM cells in allergic contact dermatitis. Thus, immunization through skin simultaneously generates skin TRM and LN TCM cells in similar numbers from the same naive T cells.

Immune activation and a 9-year ongoing complete remission following CD40 antibody therapy and metastasectomy in a patient with metastatic melanoma.

Direct immune activation via agonistic mAbs is a potentially complementary approach to therapeutic blockade of inhibitory immune receptors in cancer. Here, we provide genetic analysis of the immunologic consequences associated with the use of an agonistic CD40 mAb in a patient with metastatic melanoma who responded, underwent a single metastasectomy, and then achieved a complete remission ongoing for more than 9 years after starting therapy. Tumor microenvironment after immunotherapy was associated with proinflammatory modulations and emergence of a de novo T-cell repertoire as detected by next-generation sequencing of T-cell receptors (TCR) in the tumor and blood. The de novo T-cell repertoire identified in the posttreatment metastasectomy sample was also present-and in some cases expanded-in the circulation years after completion of therapy. Comprehensive study of this "exceptional responder" highlights the emerging potential of direct immune agonists in the next wave of cancer immunotherapies and a potential role for TCR deep sequencing in cancer immune assessment.

T cell repertoire following autologous stem cell transplantation for multiple sclerosis.

Autologous hematopoietic stem cell transplantation (HSCT) is commonly employed for hematologic and non-hematologic malignancies. In clinical trials, HSCT has been evaluated for severe autoimmunity as a method to "reset" the immune system and produce a new, non-autoimmune repertoire. While the feasibility of eliminating the vast majority of mature T cells is well established, accurate and quantitative determination of the relationship of regenerated T cells to the baseline repertoire has been difficult to assess. Here, in a phase II study of HSCT for poor-prognosis multiple sclerosis, we used high-throughput deep TCRß chain sequencing to assess millions of individual TCRs per patient sample. We found that HSCT has distinctive effects on CD4+ and CD8+ T cell repertoires. In CD4+ T cells, dominant TCR clones present before treatment were undetectable following reconstitution, and patients largely developed a new repertoire. In contrast, dominant CD8+ clones were not effectively removed, and the reconstituted CD8+ T cell repertoire was created by clonal expansion of cells present before treatment. Importantly, patients who failed to respond to treatment had less diversity in their T cell repertoire early during the reconstitution process. These results demonstrate that TCR characterization during immunomodulatory treatment is both feasible and informative, and may enable monitoring of pathogenic or protective T cell clones following HSCT and cellular therapies.

Intramuscular therapeutic vaccination targeting HPV16 induces T cell responses that localize in mucosal lesions.

About 25% of high-grade cervical intraepithelial neoplasias (CIN2/3) caused by human papillomavirus serotype 16 (HPV16) undergo complete spontaneous regression. However, to date, therapeutic vaccination strategies for HPV disease have yielded limited success when measured by their ability to induce robust peripheral blood T cell responses to vaccine antigen. We report marked immunologic changes in the target lesion microenvironment after intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens, in subjects with CIN2/3 who had modest detectable responses in circulating T lymphocytes. Histologic and molecular changes, including markedly (average threefold) increased intensity of CD8(+) T cell infiltrates in both the stromal and epithelial compartments, suggest an effector response to vaccination. Postvaccination cervical tissue immune infiltrates included organized tertiary lymphoid-like structures in the stroma subjacent to residual intraepithelial lesions and, unlike infiltrates in unvaccinated lesions, showed evidence of proliferation induced by recognition of cognate antigen. At a molecular level, these histologic changes in the stroma were characterized by increased expression of genes associated with immune activation (CXCR3) and effector function (Tbet and IFNß), and were also associated with an immunologic signature in the overlying dysplastic epithelium. High-throughput T cell receptor sequencing of unmanipulated specimens identified clonal expansions in the tissue that were not readily detectable in peripheral blood. Together, these findings indicate that peripheral therapeutic vaccination to HPV antigens can induce a robust tissue-localized effector immune response, and that analyses of immune responses at sites of antigen are likely to be much more informative than analyses of cells that remain in the circulation.

Regression of metastatic Merkel cell carcinoma following transfer of polyomavirus-specific T cells and therapies capable of re-inducing HLA class-I.

Merkel cell carcinoma (MCC) is an aggressive skin cancer that typically requires the persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins that can serve as ideal immunotherapeutic targets. Several immune evasion mechanisms are active in MCC including down-regulation of HLA class-I expression on tumor cells and dysfunctional endogenous MCPyV-specific CD8 T cell responses. To overcome these obstacles, we combined local and systemic immune therapies in a 67-year-old man, who developed metastatic MCPyV-expressing MCC. Intralesional IFNß-1b or targeted single-dose radiation was administered as a pre-conditioning strategy to reverse the down-regulation of HLA-I expression noted in his tumors and to facilitate the subsequent recognition of tumor cells by T cells. This was followed by the adoptive transfer of ex vivo expanded polyclonal, polyomavirus-specific T cells as a source of reactive antitumor immunity. The combined regimen was well-tolerated and led to persistent up-regulation of HLA-I expression in the tumor and a durable complete response in two of three metastatic lesions. Relative to historical controls, the patient experienced a prolonged period without development of additional distant metastases (535 days compared to historic median of 200 days, 95% confidence interval = 154-260 days). The transferred CD8(+) T cells preferentially accumulated in the tumor tissue, remained detectable and functional for >200 days, persisted with an effector phenotype, and exhibited evidence of recent in vivo activation and proliferation. The combination of local and systemic immune stimulatory therapies was well-tolerated and may be a promising approach to overcome immune evasion in virus-driven cancers.

Estimating the ratio of CD4+ to CD8+ T cells using high-throughput sequence data.

Mature T cells express either CD8 or CD4, defining two physiologically distinct populations of T cells. CD8+ T cells, or killer T-cells, and CD4+ T cells, or helper T cells, effect different aspects of T cell mediated adaptive immunity. Currently, determining the ratio of CD4+ to CD8+ T cells requires flow cytometry or immunohistochemistry. The genomic T cell receptor locus is rearranged during T cell maturation, generating a highly variable T cell receptor locus in each mature T cell. As part of thymic maturation, T cells that will become CD4+ versus CD8+ are subjected to different selective pressures. In this study, we apply high-throughput next-generation sequencing to T cells from both a healthy cohort and a cohort with an autoimmune disease (multiple sclerosis) to identify sequence features in the variable CDR3 region of the rearranged T cell receptor gene that distinguish CD4+ from CD8+ T cells. We identify sequence features that differ between CD4+ and CD8+ T cells, including Variable gene usage and CDR3 region length. We implement a likelihood model to estimate relative proportions of CD4+ and CD8+ T cells using these features. Our model accurately estimates the proportion of CD4+ and CD8+ T cell sequences in samples from healthy and diseased immune systems, and simulations indicate that it can be applied to as few as 1000 T cell receptor sequences; we validate this model using in vitro mixtures of T cell sequences, and by comparing the results of our method to flow cytometry using peripheral blood samples. We believe our computational method for determining the CD4:CD8 ratio in T cell samples from sequence data will provide additional useful information for any samples on which high-throughput TCR sequencing is performed, potentially including some solid tumors.

Minimal residual disease monitoring with high-throughput sequencing of T cell receptors in cutaneous T cell lymphoma.

Mycosis fungoides (MF) and the leukemic presentation Sézary syndrome (SS) are clonal T cell lymphomas arising from the skin and are considered noncurable with standard therapies. To develop a specific and sensitive monitoring tool, we tested the ability of high-throughput sequencing (HTS) of T cell receptors (TCRB) to monitor minimal residual disease (MRD) after allogeneic hematopoietic cell transplantation. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) or skin samples. The rearranged TCRß loci were amplified using Vß- and Jß-specific primers, followed by HTS, to generate up to 1,000,000 reads spanning the CDR3 region of individual cells. Malignant clones were identified in diagnostic samples in all cases by a dominant CDR3 sequence. Before transplant, four patients had circulating Sézary cells by the routine flow cytometry, which was confirmed by TCRB HTS. Although the flow cytometry found no detectable Sézary cells, malignant clones were detected by TCRB HTS in all other six cases. Five patients achieved "molecular remission" in blood between +30 and +540 days after transplant. Four of these patients also achieved molecular clearance in skin after transplant. Experiments using blood samples spiked with purified Sézary cells demonstrated that TCRB HTS can detect Sézary cells at the level of 1 in 50,000 PBMCs, which is more sensitive than standard diagnostics. We have thus demonstrated the utility of TCRB HTS to assess MRD with increased sensitivity and specificity compared to other current methodologies, and to monitor response to therapy in this MF/SS patient population.

Using synthetic templates to design an unbiased multiplex PCR assay.

T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.