Abstracts submitted by Adaptive Biotechnologies

Carlson, C., et al. 2012. Immune Profiling Suggests an IGH Signaling-Dependent Subtype of Aggressive B-ALL. Blood. 120:1428 (Abstr).

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The majority of B-cell acute lymphocytic leukemias (ALL) in childhood are derived from precursor B cells. In order to more precisely define the stage in cellular differentiation of the B-cell that gave rise to the tumor, we applied high-throughput sequencing techniques to profile the IGH repertoire in marrow at the time of diagnosis in a set of nine B-ALL patients who ultimately received a bone marrow transplant. These pediatric patients were enrolled on the Children’s Oncology Group ASCT0431 trial, a phase 3, randomized trial stem cell transplantation for high risk ALL. In eight of these patients we identified a dominant IGH VDJ clone accounting for 45%-99% of all sequences, consistent with the oncogenic transformation event occurring in a cell no longer capable of further IGH rearrangement. In the ninth patient, no single VDJ clone dominated, but we observed a highly recurrent rearrangement of a specific pair of IGH diversity-joining segments (D7–27 and J4) which also conserved a four base pair non-templated sequence at the N2 junction between D and J. We observed at least 143 rearrangements sharing this D-N2-J sequence, accounting for 22% of total sequences from the patient. These sequences recombined a variety of V-N1 sequences with the common D-N2-J sequence, consistent with a primary tumor that had initially rearranged the D-J segments but continued to express the RAG recombinases, allowing for full for V-DJ recombination. Of note, 140 out of 143 of these sequences were in frame, significantly more frequently than the one in three expected. This suggests that in this specific patient, the primary tumor bearing the common D-J rearrangement was relatively indolent, and signaling through a subsequently, successfully rearranged and expressed IGH protein was an important event contributing to to the malignancy. The rearranged IGH sequences show little evidence of somatic hypermutation, so the expansion signal being received through the IGH protein appears to be coinsistent with normal positive selection during pro-B cell development, rather than an antigen-specific signal. This is consistent with the signal delivered by the pre-B receptor at the pro to pre-B stage of B cell development. We are investigating additional samples to assess the frequency of this subtype of B-ALL (a single clonal rearrangement of D-J, followed by many subclones expressing different, in frame full V-DJ rearrangements), by sequencing patients at diagnosis, relapse pre-transplant, and relapse post-transplant. Our ongoing analysis of patients enrolled on this trial at various stages of relapse will allow us to determine the prevalence of this subtype among high-risk cases, and whether differential outcomes are observed in similar cases.

Rieder, M., et al. A normalization procedure for removal of residual multiplex PCR amplification bias from ultra-deep sequencing of the TCR repertoire; (Program #530W). Presented at the 62nd Annual Meeting of the American Society of Human Genetics, November 7, 2012 in San Francisco, California.

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The human adaptive immune system is a primary defense against the vast world of potential pathogens. To counter this enormous pathogenic diversity, B and T cells constituting the adaptive immune system rearrange surface receptors such that each rearrangement has the potential to code for a different amino acid sequence. For the T cell receptor (TCR), the CDR3 regions are formed by rearrangements of Variable and Joining (VJ) segments for the α chain and Variable, Diversity, and Joining (VDJ) segments for the β chain, in addition to nucleotide insertions/deletions at these junctions, creating the vast diversity in the TCR repertoire. As the adaptive immune system functions in part by clonal expansion, accurately measuring the changes in total abundance of each clone is vital for understanding the dynamics of an adaptive immune response. We developed an optimized multiplex PCR assay for gDNA rearrangements at the TCRβ locus that allows for quantitative assessment of each clonotype using ultra-deep DNA sequencing. This multiplex PCR consisted of 54 V segment and 13 J segment primers designed against all V-J gene segments of TCRβ. To produce an accurate quantitative assay, we need our assay to faithfully output the correct abundance of each clone from an arbitrary sample of T cells. We created a controlled, gold standard input template by synthesizing a single set of 702 oligonucleotides spanning all V-J gene pairs and mixing these at equal ratios. Using this standard as the template input, we first assessed the amplification bias using a PCR reaction containing equimolar ratios of TCRβ V-J primers. We performed additional experiments to assess 1) the independence of each specific V and J primer in the presence of a pools of complimentary primers (i.e. pools of all V or J primers) 2) cross-amplification due to variation in primer concentration (10-fold range) and 3) detection of specific spike-in oligonucleotides. Our results showed that V and J primers showed near complete independence with little interaction. We iterated through different primer rebalancing formulations producing nearly equal amplification across a large dynamic range as assessed by Illumina sequencing. Residual amplification bias following PCR optimization was computationally corrected to further improve the quantitation of the assay. Quantification of clones using this assay allows for accurate and robust tracking of TCRβ repertoire dynamics over time and between tissue types.

Sherwood, A., et al. New Technologies for Measurements of Tumor Infiltrating Lymphocytes. J Immunother. 35:9.787.

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The presence, abundance, population, and diversity of Tumor Infiltrating Lymphocytes (TILs) have been identified as prognostic indicators in several immunogenic cancers. However, current methods to study TILs are largely limited to flow technologies to count and identify T cell type. Emerging cancer therapeutics including immunomodulators and adoptive T-cell therapy highlight the need to better understand and track this population of T cells. We’ve developed an amplification based high-throughput methods to characterize the adaptive immune response to a tumor. Our method utilizes a multiplex PCR to amplify rearranged T-cell receptor Beta (TCRB) chains; and uses high-throughput sequencing (HTS) to characterize the immune repertoire.

The majority of TCR diversity resides in the β chain, and each T cell clone expresses a single TCRB allele that has been rearranged from the germ-line TCRB locus to form one of many billions of possible TCRB genes. This immense diversity is generated by combining noncontiguous variable, diversity, and joining region gene segments, which collectively encode the CDR3 region and determine antigen specificity. This occurs after T cell lineage commitment and rearranged TCRB CDR3 chains are unique to T cells, so the number of rearranged chains is directly correlated with the number of αβ T cells. In our ddPCR assay, we use multiplex PCR primers and fluorescently labeled probes that specifically anneal to V genes to count T cells using the QuantaLife Droplet Reader. Our sequencing assay uses the Illumina system to sequence across the CDR3 region along with our previously developed bioinformatics tools to identify the diversity of T cell clones based on CDR3 sequence. In concert, these two assays can both count the number and characterize the repertoire of αβ T cells in a given tumor sample.

To show utility, we use our assay to characterize the heterogeneity of ovarian tumor TIL populations. We apply our assays to 10 primary and metastatic ovarian tumors collected from 5 patients. Each tumor is divided into a grid pattern with 8-10 sections and two samples are collected from each grid. DNA is isolated from each sample and used for HTS. For each tumor section we collect data on the number, diversity, and the unique CDR3 sequences carried by the TILs. We use these data to characterize the intra- tumor heterogeneity of TIL count, diversity, and T cell clone overlap. We find that within a tumor, adjacent samples show high similarity to each other suggesting that the TCR repertoire of the tumor environment is homogeneous.


Delaney, C., et al. Evolution and Clinical Implications of the T cell Repertoire Following Cord Blood Transplant. Biology of Blood and Marrow Transplantation. 19:2.S201.

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Cord blood transplant (CBT) recipients are at increased risk of transplant related mortality, in part due to delayed hematopoietic and immune system recovery resulting in increased infectious complications. However, without direct clinical measures of immunocompetence, the specific role of delayed immunity on CBT outcomes is not easily determined and intervention studies not possible.

Blood samples were collected (pre-transplant and at days 28, 56, 100, 180 and 365 post-transplant) in 34 consecutive patients undergoing myeloablative CBT for treatment of hematologic malignancies. All samples were subjected to Immunoseq, a novel T cell receptor (TCR) sequencing assay that delivers an unprecedented depth of sequencing data. From these data, clonal expansion and contraction of hundreds of thousands of clones were tracked over time and TCR diversity was directly measured. Basic clinical outcomes for all patients were also determined, including GvHD, overall survival, disease free survival, regimen related toxicities and infectious complications.

The ability to track clones demonstrated tremendous oscillation, with an almost entirely new T cell repertoire appearing at least monthly in CBT recipients. Furthermore, in contrast to healthy controls whose blood was sampled on a similar time-course, where the most frequent T cell clone at one time point remains the top clone at subsequent time points, the largest clones observed early post-transplant in CBT recipients subsequently dropped below detection within weeks of direct measurement. Of the 34 patients studied, six died of infectious complications between day 100 and one year. Notably, TCR diversity values for these six patients were far lower than those of the remaining patients (P-value = 0.015, see figure).

The TCR repertoire is exceptionally dynamic following CBT, with many T cell clones rising to high frequency and then receding to an undetectably low level in a matter of weeks. By two months after transplant, TCR diversity accurately predicted risk of death due to infection in this patient cohort, suggesting that diversity of the TCR is a direct measure of immunocompetence and may be useful as a test to guide clinical decision making in patients with acquired or congenital immunosuppression.


Carlson, C., et al. 2012. Immune Profiling Suggests an IGH Signaling-Dependent Subtype of Aggressive B-ALL. Blood. 120:1428 (Abstr). (Oral Presentation).


Desmarais, C.L., and H. Robins. 2012. High-throughput sequencing of memory and naïve T cell receptor repertoires at the RNA and DNA levels reveals differences in relative expression of expanded TCR clones. J Immunol. 182: 178.12 (Abstr).

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Recent advances in high throughput sequencing technologies have enabled the profiling of TCR repertoires at an unprecedented level of resolution. Adaptive Biotechnologies has developed sequencing methods that are able to sequence TCR rearrangements by sequencing either using cDNA isolated from RNA, or using genomic DNA directly. We used Adaptive’s immunoSEQ technology to profile CD8+ Memory, CD8+ Naïve, and unsorted PBMC repertoires from 2 individuals using cDNA and genomic DNA isolated from the same starting population of cells. Using this method, it is possible to identify instances of either differential over- or under-expression of particular rearrangements, and to study the overall impact of starting material on sequencing results. Understanding the implications of assaying TCR repertoires at the genomic versus RNA level is important for the interpretation of experimental results, as well as for informing the design of immune profiling experiments that use high-throughput sequencing methodologies.

Robins, H. and Cindy Desmarais. 2012. Effects of aging on the human adaptive immune system revealed by high-throughput DNA sequencing of T cell receptors. J Immunol. 182: 47.16 (Abstr).

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As we age, our adaptive immune system undergoes changes that affect vaccine efficacy as well as susceptibility to disease and infection. Identifying the factors that determine immune repertoire diversity and studying the impact of changes in diversity through the life span is therefore an important public health concern. The objective of our study was to characterize alterations that occur in the T cell receptor repertoire as we age. To address these questions we utilized a high-throughput sequencing (HTS) approach developed by Adaptive Biotechnologies. This technology (immunoSEQ) amplifies rearranged T-cell receptor CDR3 chains and uses HTS to capture millions of CDR3 sequences. CD4+ Memory, CD4+ Naïve, CD8+ Memory and CD8+ Naïve T cell populations from peripheral blood were isolated from a total of 15 healthy volunteers, ranging in age from 25 to 80. The CDR3 region of each TCRB chain was then sequenced, generating a comprehensive profile of the T cell receptor repertoire for each cell population. We observed a reduction in the relative contribution of CD8+ naïve T cells with advancing age. This effect was exacerbated by CMV infection. These results provide a basis to further characterize the effects of immunosenescence on the adaptive immune system.

Robins, H., R.O. Emerson, and C.L. Desmarais. 2012. CD4+ and CD8+ T cell β antigen receptors have different and predictable V and J gene usage and CDR3 lengths. J Immunol. 182: 115.10 (Abstr).

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CD8+ and CD4+ T lymphocytes have different and complimentary roles: CD8+ (cytotoxic) T cells directly kill cells presenting non-self epitopes while CD4+ (helper) T cells regulate the immune response to a particular antigen. These T cells recognize antigens by binding to epitope MHC protein complexes, with CD8+ T cells binding class I and CD4+ T cells binding class II MHC protein complexes. While it’s known that the CD8 and CD4 proteins determine which MHC class the T cells bind, it is unknown if the antigen receptor sequences vary between the two populations. The diversity of possible receptors is huge and until recently this diversity precluded the possibility of capturing the antigen receptor repertoire. Adaptive Biotechnologies has developed a novel method that amplifies rearranged T cell receptor β (TCRB) CDR3 sequences and uses high throughput sequencing to sequence millions of TCRB CDR3 chains. To determine if the two populations of T cells have unique TCRB profiles, PBMC from 19 unrelated individuals were sorted 4 ways into CD4+ and CD8+ naïve and memory T cells. V and J gene usage and CDR3 sequence length could distinguish the TCRB CDR3 sequences amplified from CD4+ and CD8+ T cells. These sequence differences can be exploited and used to estimate the proportion of CD4+ to CD8+ T cells in a mixed population of T cells. These data indicate a biological difference between the antigen receptors that bind class I versus class II MHC protein complexes.

Wu, David et al. 2011. High-throughput sequencing of T-cell receptor gene loci for minimal residual disease monitoring in T Lymphooblastic Leukemia. Blood. 118:
2545 (Abstr).

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There is increasing evidence for the utility of minimal residual disease (MRD) assessment in predicting clinical outcomes of patients with T cell lymphoblastic leukemia (T-ALL). Evaluation of MRD by PCR-based analysis of T-cell receptor (TCR) genes has a sensitivity of 10–5, but requires the use of individualized patient-specific primers, which is laborious, expensive and difficult to implement for real-time, clinical decision-making. Multi-parametric flow cytometry is currently limited to a sensitivity of 10–4, requires viable cells, and is poorly standardized. High-throughput DNA sequencing offers the potential to equal or surpass the higher sensitivity of PCR-based MRD testing with reduced cost, improved turn-around time, and better standardization.

Paired samples of pediatric T-ALL from 14 patients enrolled on Children’s Oncology Group AALL0434 were obtained at diagnosis and at day 29 post-induction therapy. The complementarity determining regions (CDR3) regions of TCRB and TCRG were sequenced for all 28 specimens using an Illumina GA2 platform as previously described (see Blood, 114(19):4099–4107, 2009 and Sci Transl Med. 3(90):90ra61, 2011). Pre-treatment samples were used to obtain unique TCR sequences for the leukemic clone, and post-treatment samples were assessed for the frequency of each TCR sequence as a percentage of the total. The frequency of each sequence was also enumerated in post-treatment samples from all other patients to evaluate specificity. These results were compared to MRD results obtained by 9-color flow cytometry per trial protocol.

Eleven of 14 pre-treatment samples (78.6%) had a detectable clonal population based on TCRG sequence analysis, and 10 of these also had a clonal TCRB sequence. Five samples exhibited an additional unique TCRGsequence, consistent either with rearrangement of both TCRG loci or the presence of two clonal subpopulations. Two of 3 cases without a detectable clonal TCR gene sequence had the immunophenotype of early thymic precursor (ETP) T-ALL and would be expected to have germline TCRB and TCRG genes. No other cases were ETP. Clones were found in all 5 informative post-treatment samples positive for MRD by flow cytometry, as well as at a low level in 3 additional patients without MRD by flow cytometry, suggesting superior sensitivity for sequencing. The background sequence frequencies were very low (0–10–5) in other patient post-treatment samples, being slightly higher for TCRG than for TCRB, consistent with germline sequence diversity.

We demonstrate the potential of high-throughput sequencing for analysis of MRD in pediatric T-ALL. The number of cases in which the assay is informative (78.6%) is similar to that seen with standard PCR MRD methods, but evaluation of more cases is needed. MRD by sequencing appears to have a higher sensitivity than current flow cytometric methods, although direct comparison of MRD frequencies from the two techniques is problematic and will require normalization. The strong association of ETP status and lack of clonal TCR sequence identification at diagnosis suggests utility in identifying this poor outcome subset of T-ALL.


Carlson, C. et al. 2011. Profiling the repertoire of TCRB usage in induced and natural Treg cells. J Immunol. 186: 62.5 (Abstr).

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In our recent research, we have found that a subset of all possible T cell receptor beta (TCRB) rearrangements is produced more frequently than expected in unrelated individuals. This subset appears to be defined by rearrangements that are near to germline, with relatively few non-templated nucleotides inserted at the V-D and D-J junctions within complementarity determining region 3. The degree of overlap between TCRB repertoire between individuals does not appear to be dramatically HLA context-dependent. Other groups have described several subsets of regulatory CD4 T cells (Treg), including both thymus-derived natural Tregs (nTregs) and peripherally induced Tregs (iTregs). While both subtypes express the FOXP3 transcription factor that defines them as regulatory T cells, it has been asserted that the expression of a second transcription factor (Helios) can differentiate nTregs (Helios+) from induced Tregs (Helios-). We hypothesize that the nTreg population is likely to be enriched for near-germline TCRB rearrangments, as recurrently produced TCRB that predispose to the nTreg fate are likely to be shared between individuals. We will compare and contrast the TCRB repertoire in three cellular populations within several individuals: CD4+ FoxP3- Helios-, CD4+ FoxP3+ Helios-, and CD4+ FoxP3+ Helios+.

Robins, H. 2010. Overlap and effective size of the human CD8+ T cell repertoire. Keystone Symposia. Immunological Mechanisms of Vaccination (Oral Presentation).



Abstracts submitted by Customers of Adaptive Biotechnologies

Identification and Monitoring of Pathogenic T Cell Clones Using Deep TCR Sequencing in T-Large Granular Lymphocyte Leukemia and Aplastic Anemia
Result Type: Paper
Number: 3477
Program: Oral and Poster Abstracts
Session: 508. Bone Marrow Failure – Inherited Disorders: Poster III
Time and Location:
Monday, December 10, 2012: 6:00 PM-8:00 PM
Hall B1-B2 (Georgia World Congress Center)
Michael J. Clemente, Bartlomiej Przychodzen, Hideki Makishima, Manuel G. Afable II, Marcin W Wlodarski, Alan E. Lichtin and Jaroslaw P. Maciejewski


American Society of Hematology. 2012. “Improving Virus-Specific Immune Reconstitution after Cord Blood Transplantation Using Cord Blood-Derived Virus-Specific Cytotoxic T Lymphocytes: A Phase I Clinical Study” – abstract achievement award.

Authors: Patrick J Hanley, PhD, Caridad A Martinez, MD, Kathryn S Leung, MD, Conrad Russell Y Cruz, MD, PhD, Sharon Lam, Barbara Savoldo, MD, PhD, Gianpietro Dotti, MD, Malcolm K Brenner, MD, PhD, Cliona Rooney, PhD, Helen E Heslop, MD, Robert A Krance, MD, Elizabeth J Shpall, MD and Catherine Bollard, MD.


American Society of Gene and Cell Therapy. 2012. “Phase I Clinical Study to Improve Virus-Specific Immune Reconstitution after Cord Blood Transplantation Using Cord Blood-Derived Virus-Specific Cytotoxic T Lymphocytes.” – Excellence in Research Award for high scoring abstracts

Authors: Patrick J Hanley, PhD, Caridad A Martinez, MD, Kathryn S Leung, MD, Barbara Savoldo, MD, PhD, Gianpietro Dotti, MD, Malcolm K Brenner, MD, PhD, Cliona Rooney, PhD, Helen E Heslop, MD, Robert A Krance, MD, Elizabeth J Shpall, MD and Catherine Bollard, MD.


American Society of Hematology. 2011. “Phase I Clinical Study to Improve Virus-Specific Immune Reconstitution after Cord Blood Transplantation Using Cord Blood-Derived Virus-Specific Cytotoxic T Lymphocytes.” – Abstract achievement award

Authors: Patrick J Hanley, PhD, Caridad A Martinez, MD, Kathryn S Leung, MD, Barbara Savoldo, MD, PhD, Gianpietro Dotti, MD, Malcolm K Brenner, MD, PhD, Cliona Rooney, PhD, Helen E Heslop, MD, Robert A Krance, MD, Elizabeth J Shpall, MD and Catherine Bollard, MD.