Quantifying T- and B-cell immune receptor distribution diversity to uncover their clinical relevance in clear cell renal cell carcinoma

Immune-modulating systemic therapies are often used to treat metastatic clear cell renal cell carcinoma (ccRCC). Used alone, sequence-based biomarkers neither accurately capture patient dynamics nor the tumor-immune microenvironment. To better understand the tumor ecology of this immune microenvironment, we used complementarity determining region-3 (CDR3) sequence recovery counts of the tumor infiltrating lymphocytes, and quantified tumor infiltration by sequences recovered from patient tumors by applying a generalized diversity index (GDI) to CDR3 sequence distributions across two distinct ccRCC cohorts. GDI can be understood as a curve over a continuum of diversity scales and allows sensitive characterization of distributions to capture richness, evenness, and subsampling uncertainty, along with other important metrics. For example, richness quantifies the total unique sequence count, while evenness quantifies similarities across sequence frequencies. We observed significant differences in receptor sequence diversity across gender and race. Further, our analysis revealed that patients with larger and more clinically aggressive tumors had increased richness of recovered tumoral CDR3 sequences, specifically in those from T-cell receptor alpha and B-cell immunoglobulin lambda light chain. We identified a novel and robust measure of distribution evenness, using GDI's inflection point (IP). High IP values associated with improved overall survival, suggesting that normal-like sequence distributions lead to better outcomes. Our results propose a new quantitative tool that can be used to better characterize patient-level differences related to immune cell infiltration and, can be used to identify unique characteristics of tumor-infiltrating lymphocyte heterogeneity in ccRCC and other malignancies.

Characterization of Plasmacytoid Dendritic Cells, Microbial Sequences, and Identification of a Candidate Public T-Cell Clone in Kikuchi-Fujimoto Disease

Objectives Kikuchi-Fujimoto disease (KFD) is a self-limited lymphadenitis of unclear etiology. We aimed to further characterize this disease in pediatric patients, including evaluation of the CD123 immunohistochemical (IHC) staining and investigation of potential immunologic and infectious causes. Methods Seventeen KFD cases and 12 controls were retrospectively identified, and the histologic and clinical features were evaluated. CD123 IHC staining was quantified by digital image analysis. Next generation sequencing was employed for comparative microbial analysis via RNAseq (5 KFD cases) and to evaluate the immune repertoire (9 KFD cases). Results In cases of lymphadenitis with necrosis, >0.85% CD123+ cells by IHC was found to be six times more likely in cases with a final diagnosis of KFD (sensitivity 75%, specificity 87.5%). RNAseq based comparative microbial analysis did not detect novel or known pathogen sequences in KFD. A shared complementarity determining region 3 (CDR3) sequence and use of the same T-cell receptor beta variable region family was identified in KFD LNs but not controls, and was not identified in available databases. Conclusions: Digital quantification of CD123 IHC can distinguish KFD from other necrotizing lymphadenitides. The presence of a unique shared CDR3 sequence suggests that a shared antigen underlies KFD pathogenesis.

Higher Order Restrictions of the Immunoglobulin Repertoire in CLL: The Illustrative Case of Stereotyped Subsets #2 and #169

Stereotyped subset #2 (IGHV3-21/IGLV3-21) is the largest subset in CLL (~3% of all patients). Membership in subset #2 is clinically relevant since these patients experience an aggressive disease irrespective of the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene. Low-throughput evidence suggests that stereotyped subset #169, a minor CLL subset (~0.2% of all CLL), resembles subset #2 at the immunogenetic level. More specifically: (i) the clonotypic heavy chain (HC) of subset #169 is encoded by the IGHV3-48 gene which is closely related to the IGHV3-21 gene; (ii) both subsets carry VH CDR3s comprising 9-amino acids (aa) with a conserved aspartic acid (D) at VH CDR3 position 3; (iii) both subsets bear light chains (LC) encoded by the IGLV3-21 gene with a restricted VL CDR3; and, (iv) both subsets have borderline SHM status. Here we comprehensively assessed the ontogenetic relationship between CLL subsets #2 and #169 by analyzing their immunogenetic signatures. Utilizing next-generation sequencing (NGS) we studied the HC and LC gene rearrangements of 6 subset #169 patients and 20 subset #2 cases. In brief, IGHV-IGHD-IGHJ and IGLV-IGLJ gene rearrangements were RT-PCR amplified using subgroup-specific leader primers as well as IGHJ and IGLC primers, respectively. Libraries were sequenced on the MiSeq Illumina instrument. IG sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis conducted using an in-house, validated bioinformatics pipeline. Rearrangements with identical CDR3 aa sequences were herein defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. For the HC analysis of subset #169, we obtained 894,849 productive sequences (mean: 127,836, range: 87,509-208,019). On average, each analyzed sample carried 54 clonotypes (range: 44-68); the dominant clonotype had a mean frequency of 99.1% (range: 98.8-99.2%) and displayed considerable intraclonal heterogeneity with a mean of 2,641 subclones/sample (range: 1,566-6,533). For the LCs of subset #169, we obtained 2,096,728 productive sequences (mean: 299,533, range: 186,637-389,258). LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean: 148, range: 110-205); the dominant clonotype had a mean frequency of 98.1% (range: 97.2-98.6%). Intraclonal heterogeneity was also observed in the LCs, with a mean of 6,325 subclones/sample (range: 4,651-11,444), hence more pronounced than in their partner HCs. Viewing each of the cumulative VH and VL CDR3 sequence datasets as a single entity branching through diversification enabled the identification of common sequences. In particular, 2 VH clonotypes were present in 3/6 cases, while a single VL clonotype was present in all 6 cases, albeit at varying frequencies; interestingly, this VL CDR3 sequence was also detected in all subset #2 cases, underscoring the molecular similarities between the two subsets. Focusing on SHM, the following observations were made: (i) the frequent 3-nucleotide (AGT) deletion evidenced in the VH CDR2 of subset #2 (leading to the deletion of one of 5 consecutive serine residues) was also detected in all subset #169 cases at subclonal level (average: 6% per sample, range: 0.1-10.8%); of note, the 5-serine stretch is also present in the germline VH CDR2 of the IGHV3-48 gene; (ii) the R-to-G substitution at the VL-CL linker, a ubiquitous SHM in subset #2 and previously reported as critical for IG self-association leading to cell autonomous signaling in this subset, was present in all subset #169 samples as a clonal event with a mean frequency of 98.3%; and, finally, (iii) the S-to-G substitution at position 6 of the VL CDR3, present in all subset #2 cases (mean : 44.2% ,range: 6.3-87%), was also found in all #169 samples, representing a clonal event in 1 case (97.2% of all clonotypes) and a subclonal event in the remaining 5 cases (mean: 0.6%, range: 0.4-1.1%). In conclusion, the present high-throughput sequencing data cements the immunogenetic relatedness of CLL stereotyped subsets #2 and #169, further highlighting the role of antigen selection throughout their natural history. These findings also argue for a similar pathophysiology for these subsets that could also be reflected in a similar clonal behavior, with implications for risk stratification. Disclosures Sutton: Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.

Identification of Specificity Groups in Myeloma Patients T Cell Receptor (TCR) Repertoire through Single Cell TCR Sequencing

Background: A renewed interest in immune and cellular based therapeutics in multiple myeloma was recently fueled by the development of CD38 targeting monoclonal antibodies as well as the introduction of engineered CAR-T cells. Daratumumab treatment in myeloma patients was demonstrated to expand clonal CD8+T cells and T cell clonality was correlated with the depth of response consistent with a daratumumab mediated cytotoxic T cell effect. However the mechanisms behind this adaptive immune response and the identity of the T cell receptor (TCR) interacting with MHC presented tumoral peptide (pMHC) remains elusive. The large diversity of TCR combinations generated though somatic recombination of the VDJ gene sequences (~ 1015combinations) represents a major challenge for the accurate characterization of the antigen specific TCR. The aim of this study was to define the identity of the adaptive immune repertoire of the bone marrow infiltrating T lymphocytes (single cell TCR α/β paired sequencing) in myeloma patients treated to daratumumab and IMiDs based therapies. Methods and Results: BM aspirates from patients (n=24) treated with daratumumab single agent or in combination with pomalidomide or lenalidomide (MM014, MM3008 and MMY3012 trials) were collected post initiation of therapy (cycle 3, day 1) followed by magnetic beads sorting of CD3pos T cells from Ficoll generated mononuclear cell fractions. Using the 10x Genomics Single cell VDJ solution which combines single cell droplet microfluidics with 5' molecular barcoding, T cells from each patient were partitioned into droplets containing individual cells with primers specific for the constant region of the V(D)J locus allowing the PCR amplification and enrichment of α and β TCR individual cell barcoded cDNA. Paired-end sequencing was performed on Illumina NEXTseq platform. Cell Ranger VDJ pipeline was used for sample de-multiplexing, barcode processing and grouping of T cells into clonotypes with shared TCR α/β sequences. Of note, generated sequences span the full length of V-J genes (including CDR3) allowing faithful reconstruction of TCR transcripts. Consistent with the known high TCR diverse repertoire, we identified 32322 individual clonotypes corresponding to an average of 1346 clonotypes with paired α/β TCR sequences per patient. Clonotypes proportion (> 2%) and number of individual clonotypes did correlate with the depth of response (≥VGPR vs PR vs PD). Analysis of clonotype TCRs and CDR3 sequences identified 11 clonotypes with the exact paired CDR3 αβ sequences that were shared by at least 2 patients. Of interest the CDR3 sequence of one shared clonotype is predicted (https://vdjdb.cdr3.net) to bind an epitope derived for CD317 (also know as BST2 or HM1.24) previously demonstrated to be highly expressed on myeloma cells (Jalili A et al. Blood 2005). Both patients harboring this CD317 reactive T cell clone are in sCR for more than 2 years. Of note, TCRs that recognize the same peptide-MHC complexes do not always share the exact CDR3 sequences but rather have conserved CDR3 sequence features, rendering possible to predictively model epitope specificity. Indeed, recent studies demonstrated that similarity in CDR3 sequences (CDR3 differing by up to one amino acid) or shared CDR3 motifs of 2-4 amino acids in length, define the TCR clusters that are often contact points with the antigenic peptides. Such features in CDR3 sequences facilitates T cell target antigen discovery. Therefore, we applied the GLIPH algorithm (https://github.com/immunoengineer/gliph) to cluster the sequenced TCRs based on their high probability of sharing pMHC specificity owing to both conserved CDR3 motifs and global similarity in their CDR3 sequences. GLIPH grouped the TCRs sequences identified in our study into 171 unique clusters (with a minimum of 3 clones each) that are predicted to recognize the same pMHC ligands. Furthermore, it identified 26 CDR3 motifs that are elevated at least 10-fold over expected frequency in a naïve TCR reference pool (p <0.001). Functional validation of this myeloma-targeting TCR clusters through pMHC tetramer binding is ongoing. Conclusion: Single cell TCR profiling identified unique clonotypes that are highly enriched in marrow infiltrating T cells and are predicted to be reactive with myeloma peptides. This work facilitates the future development of TCR engineered T cells targeting myeloma neoepitopes. Disclosures Neri: Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. McCulloch:Takeda: Other: Travel expenses; Celgene: Honoraria. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Characteristics of the Vδ2 CDR3 Sequence of Peripheral γδ T Cells in Patients with Pulmonary Tuberculosis and Identification of a New Tuberculosis-Related Antigen Peptide

ABSTRACTAntigen-specific γδ T cells may play an important role in the immune response toMycobacterium tuberculosis. However, little is known about the characteristics of the length distribution of the δ2-chain complementarity determining region 3 (δ2 CDR3) of the γδ T-cell receptor (TCR) in patients with active pulmonary tuberculosis (TB) on a large scale. In addition,M. tuberculosis-activated γδ T cells potentially inhibit intracellular mycobacterial growth, but phosphoantigen-activated γδ T cells do not. Only a fewM. tuberculosis-related antigen peptides or proteins that are recognized by γδ TCR have been identified. Twenty-four healthy donors (HDs) and 27 TB patients were included in the present study. The gene-scanning technique found that the δ2 CDR3 length distribution patterns of γδ TCR in TB patients were perturbed, and each pattern included different predominant CDR3 sequences. The predominant δ2 CDR3 sequences of γδ TCRs, which originated from TB patients and HD γδ T cells that were stimulated byM. tuberculosisheat resistance antigen (Mtb-HAg), were used as probes to screen peptides recognized by γδ TCR using a phage display library. We identified four peptides that bound to the predominant δ2 CDR3 fragments and showed homology toM. tuberculosisgenes in a BLAST search. Notably, one peptide was related toM. tuberculosisH37Rv (QHIPKPP), and this fragment was confirmed as a ligand for the γδ TCR. Two fragments, Ag1 and Ag2, activated γδ T cells from HD or TB patients. In summary, the δ2 CDR3 lineage of TB patients apparently drifts, and the predominant δ2 CDR3 sequence that recognizesM. tuberculosismay exhibit specificity. The identifiedM. tuberculosis-related antigen peptides may be used as vaccines or adjuvants for protective immunity againstM. tuberculosis.

Endogenous Ligands Selectively Stimulate Highly Avid Autoreactive Human Invariant Natural Killer T Cells with Distinctive T-Cell Receptor Vβ11 CDR3 Sequence Motifs

Abstract 999 Human CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of innate T-cells that carry an invariant T-cell receptor (TCR) Vα24 chain paired with a TCR Vβ11 chain. They constitutively express a variety of activation and memory markers and possess the capacity to rapidly produce a variety of cytokines including IFN-γ and IL-4 upon TCR engagement. As an immune response launches, iNKT cells can induce innate immune responses and serve as a bridge between innate and adaptive immunity. In addition, it has also been shown that iNKT cells themselves have anti-tumor and anti-infectious effects in mice. However, to translate these findings and develop iNKT cell-mediated immunotherapy, iNKT cells must recognize target cells while sparing normal cells. Autoreactivity is primarily dictated by 1) the specificity and avidity of the TCR on iNKT cells; 2) the expression level of CD1d on target cells; and 3) the density and stability of endogenous ligand(s) presented by CD1d on target cells. In this study, we sought to determine the TCR Vβ11 CDR3 sequence motifs that distinguish high avidity and low avidity iNKT cells. We developed an artificial antigen-presenting cell (aAPC) to expand iNKT cells by transducing K562 with CD1d, CD80, and CD83. Using this CD1d+aAPC loaded with or without α-galactosylceramide (αGC), we expanded CD1d-restricted iNKT cells by stimulating primary CD3+ T cells expressing canonical iNKT TCR Vα24 chain. Expanded iNKT cells were stained with αGC-loaded CD1d tetramers and were found to express TCR Vβ11 chain in conjunction with transgenic canonical TCR Vα24 chain. Sequence analysis of cloned Vβ11 CDR3 revealed that the clonality of iNKT cells generated using unloaded aAPC was significantly lower than that of iNKT cells generated using loaded aAPC. Surprisingly, when cotransfected with canonical TCR Vα24 chain, some TCR Vβ11 chains isolated from iNKT cells generated using unloaded aAPC were stained with unloaded CD1d tetramers produced in HEK293 cells. This result suggests that these reconstituted iNKT TCR recognized endogenous ligand(s) derived from HEK293 cells in the context of CD1d. A comprehensive analysis of the structural avidity demonstrated that these TCR Vβ11 chains reconstituted TCR with significantly higher structural avidity than those cloned from NKT cells generated using loaded aAPC. However, this significant difference was only observed when the structural avidity was measured using unloaded tetramers but not αGC-loaded tetramers. A univariate analysis found that structural avidity was significantly higher when 1) Vβ11 CDR3 used J2-5; 2) Vβ11 CDR3 consisted of exactly 23 amino acids; and 3) Vβ11 CDR3 encoded 3 or more acidic amino acids (asparagic and glutamic acids). A multivariate analysis confirmed that all three variables were independent predictors of higher structural avidity. Furthermore, each variable is sufficient to increase the structural avidity of an iNKT TCR with low structural avidity. Intriguingly, unloaded mouse CD1d tetramers produced in HEK293 cells also stained a human iNKT TCR with high structural avidity reconstituted on both human and mouse T cells. This result suggests that the human iNKT TCR with high structural avidity possessed a cross-species reactivity to mouse CD1d in conjunction with endogenous ligand(s) derived from HEK293 cells. We next studied the autoreactivity of cloned human iNKT TCR with high structural avidity. A human T cell line, Jurkat, reconstituted with high avidity iNKT TCR, was able to recognize cells expressing CD1d endogenously (itself, SUP-T1, and primary human monocytes) and ectopically (K562, C1R, Hela). Furthermore, mouse T cells expressing human iNKT TCR with high structural avidity recognized mouse cell lines, B16, EL4, and 58, all endogenously expressing CD1d. Finally, human primary T cells transduced with the iNKT TCR with high structural avidity were autoreactive, secreting both IFN-γ and IL-4 in response to CD1d+ target cells. Using our CD1d+ aAPC-based system, we successfully isolated human iNKT cells with high structural avidity and identified the TCR Vβ11 CDR3 sequence motifs that dictate the structural avidity of iNKT cells. To develop clinically effective iNKT cell-mediated immunotherapy without unwanted autoimmunity, it will be critically important to define the range of iNKT TCR avidity that enables reactivity to pathologic but not normal cells. Disclosures: No relevant conflicts of interest to declare.