Oligoclonal Expansion Research Articles

Immunotherapy of CT26 murine tumors is characterized by an oligoclonal response of tissue-resident memory T cells against the AH1 rejection antigen.

Mice bearing CT26 tumors can be cured by administration of L19-mIL12 or F8-mTNF, two antibody fusion proteins which selectively deliver their cytokine payload to the tumor. In both settings, cancer cures crucially depended on CD8+ T cells and the AH1 peptide (derived from the gp70 protein of the murine leukemia virus) acted as the main tumor-rejection antigen, with ∼50% of CD8+ T cells in the neoplastic mass being AH1-specific after therapy. In order to characterize the clonality of the Tcell response, its phenotype, and activation status, we isolated CD8+ T cells from tumors and secondary lymphoid organs and submitted them to Tcell receptor (TCR) and total mRNA sequencing. We found an extremely diverse repertoire of more than 40000 unique TCR sequences, but the ten most abundant TCRs accounted for >60% of CD8+ T-cell clones in the tumor. AH1-specific TCRs were consistently found among the most abundant sequences. AH1-specific T cells in the tumor had a tissue-resident memory phenotype. Treatment with L19-mIL12 led to overexpression of IL-12 receptor and of markers of cell activation and proliferation. These data suggest that the antitumor response driven by antibody-cytokine fusions proceeds through an oligoclonal expansion and activation of tumor-infiltrating CD8+ T cells.

Unraveling tumor-immune heterogeneity in advanced ovarian cancer uncovers immunogenic effect of chemotherapy.

In metastatic cancer, the degree of heterogeneity of the tumor microenvironment (TME) and its molecular underpinnings remain largely unstudied. To characterize the tumor-immune interface at baseline and during neoadjuvant chemotherapy (NACT) in high-grade serous ovarian cancer (HGSOC), we performed immunogenomic analysis of treatment-naive and paired samples from before and after treatment with chemotherapy. In treatment-naive HGSOC, we found that immune-cell-excluded and inflammatory microenvironments coexist within the same individuals and within the same tumor sites, indicating ubiquitous variability in immune cell infiltration. Analysis of TME cell composition, DNA copy number, mutations and gene expression showed that immune cell exclusion was associated with amplification of Myc target genes and increased expression of canonical Wnt signaling in treatment-naive HGSOC. Following NACT, increased natural killer (NK) cell infiltration and oligoclonal expansion of T cells were detected. We demonstrate that the tumor-immune microenvironment of advanced HGSOC is intrinsically heterogeneous and that chemotherapy induces local immune activation, suggesting that chemotherapy can potentiate the immunogenicity of immune-excluded HGSOC tumors.

B-cell oligoclonal expansions in renal tissue of patients with immune-mediated glomerular disease

B-cell clonal expansion has been sporadically described in the blood and/or renal tissue of patients with glomerulonephritides, albeit with unclear pathogenetic role. Herein, using spectratyping analysis, we observed oligoclonal intrarenal B-cell populations in 59% of glomerulonephritis patients with podocyte injury (6/7 with focal segmental glomerulosclerosis, 1/3 minimal change disease, 1/3 idiopathic membranous nephropathy, 3/4 IgA nephropathy, 2/5 membranous lupus nephritis), 20% of glomerulonephritis patients without podocyte involvement (4/13 with mesangial or proliferative lupus nephritis, 0/3 idiopathic membranoproliferative glomerulonephritis, 0/4 pauci-immune vasculitis) and 17% of control patients with renal cancer. In multivariate analysis, oligoclonal B-cells were associated with podocyte injury and the grade of glomerulosclerosis (both p = .009). B-cell oligoclonal expansions were not found in the paired peripheral blood samples. We postulate that B-cell expansion in the kidney results from local stimuli, including antigens expressed on podocytes. Further studies to unravel the role of oligoclonal B-cells in (auto)immune-mediated kidney disease are warranted.

Characterizing the TCR repertoire in Neuropsychiatric Lupus

Abstract Approximately 40% of lupus patients experience neurological and/or psychiatric disorders. However, the mechanisms contributing to the wide range of neuropsychiatric manifestations (NPSLE) have not yet been fully elucidated. We have demonstrated that the choroid plexus (CP) in the MRL/lpr NPSLE lupus mouse model is heavily infiltrated with effector T cells. We investigated T cell receptor (TCR) usage and repertoire diversity in CP infiltrating T cells, to enhance our understanding of this T cell population and its possible contribution to neuropsychiatric disease. The CP, salivary glands, and spleens were dissected from female MRL/lpr mice (n=9, n=9, n=4 respectively) and the spleens from female MRL/MpJ (n=5) at 17–22 weeks of age, at which time MRL/lpr mice display prominent neurobehavioral deficits. The TCR repertoire was evaluated by isolating genomic DNA, amplifying the CDR3 sequence by multiplex PCR (Adaptive Technologies), and analyzing the sequences with the Adaptive Technologies immunoSeq Analyzer and the R software, LymphoSeq. MRL/lpr CP tissues had increased numbers of T cell sequences, unique productive sequences, and clonality. The top 10 productive rearrangements were more abundant in MRL/lpr CP when compared to the MRL/lpr and MRL/MpJ splenic tissues (p = 0.0673 and p = 0.0003 respectively). The MRL/lpr CP had an increased frequency of TCRBV02, TCRBV04, TCRBV12, and TCRBV26 genes compared to MRL/lpr splenic and salivary gland tissues. Despite the lymphoproliferation found in this lupus strain, the increased oligoclonal expansion and limited TCR V beta gene usage in the MRL/lpr CP T cell repertoire, as compared to other MRL/lpr tissues, implies a distinct and specific infiltration of T cells into the CP.

P083 Characterisation of T-cell receptor repertoire patterns of circulating a4b7 positive memory T cells in ulcerative colitis

Abstract Background Memory T cells play an important role in mediating inflammatory responses in IBD. The integrin a4b7 is highly expressed on activated T cells, and is thought to direct homing of lymphocytes to the intestine, following its binding to MADCAM-1 expressed exclusively on intestinal endothelial cells. Since UC is characterised by oligoclonal expansion of specific T-cell clonotypes, we hypothesised that circulating memory T cells with gut-homing potential would exhibit unique T-cell receptor repertoire features. Methods Peripheral blood mononuclear cells were collected from 5 control subjects and 6 pediatric patients with active UC. Following CD3 MACS sorting cells were FACS sorted into a4b7 positive and a4b7 negative CD3+CD45RO+ memory T cells. DNA was Isolated from each subset and subjected to next-generation sequencing of the TCRB. This high-throughput platform employs massive parallel sequencing to process millions of rearranged T-cell receptor (TCR) products simultaneously, and permits an in-depth analysis of individual TCRs at the nucleotide level. Comparisons of different indices of diversity, CDR3 length and clonal biochemical characteristics were performed between a4b7 positive and a4b7 negative populations for each subject, and between controls and UC patients. Results PBMCs were isolated from active UC patients during endoscopic assessment. Four patients had a Mayo endoscopic score of 2, and two patients had a score of 1. Only one patient was treated with an immunosuppressive medication (azathioprine), and five out of six patients were treated with 5ASAs. Percentages of memory T cells (43.8 ± 12.3% vs. 32.2 ± 13.1%, p = 0.17) and a4b7 positive T cells (33.6 ± 15.7% vs. 36.0 ± 17.6%, p = 0.81) were comparable between controls and UC patients. Interestingly, a4b7 positive memory T cells displayed a polyclonal distribution, in both control subjects and in UC patients, without expansion of specific clones. Different indices of diversity, including shanon’s H, clonality index and entropy, were similar among controls and patients, both for a4b7 positive and a4b7 negative populations. Finally, clonal overlap between a4b7 positive and a4b7 negative memory T cells, for each subject was high, ranging between 30–50% for controls and 27–48% for UC patients. Conclusion a4b7 expressing memory T cells exhibited a polyclonal repertoire in both control subjects and patients with active UC, with high rates of overlap with a4b7 negative memory T cells. Our study, along with additional recent reports, challenge the dogma of the importance of a4b7 expression for T-cell migration to the gut, and may suggest that vedolizumab’s suppresses intestinal inflammation by blocking the trafficking of innate immune subsets.

Initial cerebrospinal fluid-restricted oligoclonal bands associate with anti-N-methyl-D-aspartate receptor encephalitis severity: a pilot study.

Intrathecal antibody production is thought to underly the pathogenesis and symptomatology of N-methyl-D-aspartate receptor encephalitis (NMDARE). In the present study, the clinical correlation of cerebrospinal fluid (CSF) restricted oligoclonal bands (OCBs), as a measure of intrathecal antibody synthesis, was examined in confirmed NMDARE cases. The present study included patients with a confirmed diagnosis of NMDARE who underwent initial CSF evaluation and were followed up for a minimum of 12 months. Disease severity was assessed at baseline and 1, 3, 6, 9, and 12 months. Data regarding duration of hospitalization and intensive care unit (ICU) stay, the presence of uncontrolled seizures, and antiepileptic drug requirement were obtained for each patient. Among the 14 confirmed NMDARE patients, seven had CSF-OCBs. The presence of CSF-OCBs was associated with a more severe disease at baseline (p = 0.004), worse final outcome (p = 0.005), and longer hospitalization (median, 19 vs. 173 days; p < 0.001) and ICU stay (median, 0 vs. 29 days; p = 0.006). CSF-OCB positivity was closely associated with treatment refractoriness within 4 weeks (p = 0.029). The presence of CSF-OCBs at the onset of disease in NMDARE patients was associated with initial treatment refractoriness and a more severe disease course leading to longer hospitalization, ICU admission, intractable seizures, and a poorer outcome. The results indicate that CSF-OCBs may be useful for prognostication. Furthermore, severe disease in NMDARE may be accompanied by oligoclonal expansion antibody-producing B cells.

CD8+ T cell-mediated endotheliopathy is a targetable mechanism of neuro-inflammation in Susac syndrome

Neuroinflammation is often associated with blood-brain-barrier dysfunction, which contributes to neurological tissue damage. Here, we reveal the pathophysiology of Susac syndrome (SuS), an enigmatic neuroinflammatory disease with central nervous system (CNS) endotheliopathy. By investigating immune cells from the blood, cerebrospinal fluid, and CNS of SuS patients, we demonstrate oligoclonal expansion of terminally differentiated activated cytotoxic CD8+ T cells (CTLs). Neuropathological data derived from both SuS patients and a newly-developed transgenic mouse model recapitulating the disease indicate that CTLs adhere to CNS microvessels in distinct areas and polarize granzyme B, which most likely results in the observed endothelial cell injury and microhemorrhages. Blocking T-cell adhesion by anti-α4 integrin-intervention ameliorates the disease in the preclinical model. Similarly, disease severity decreases in four SuS patients treated with natalizumab along with other therapy. Our study identifies CD8+ T-cell-mediated endotheliopathy as a key disease mechanism in SuS and highlights therapeutic opportunities.

Deconstructing the Clonal Advantage and Clonal Stability of 5q- Candidate Genes in Del(5q) MDS on a Single Cell Level

Hematopoietic Stem/Progenitor cells (HSPCs) with 5q haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) acquire a clonal advantage in the bone marrow and out-compete normal hematopoiesis. A critical, yet unsolved question remains: how does genetic haploinsufficiency in del(5q) cells contribute to the clonal advantage of HSPCs? We investigated the role of haploinsufficiency for three candidate genes in the common deleted region on chromosome 5 (Csnk1a1, Egr1 and Apc) in direct competition with each other and wild-type (wt) cells on a single cell level by employing a novel lentiviral genetic barcoding strategy. We introduced genotype and cell-specific barcodes into HSPCs from murine models haploinsufficient for Csnk1a1, Egr1 or Apc. Barcoded HSPCs were sort-purified, genotypes mixed and subsequently competitively transplanted into lethally irradiated mice and re-transplanted after 16 weeks in a secondary transplant. The barcoded progeny was reliably recovered from peripheral blood and relative contribution of the barcoded clones to differentiated blood lineages was followed over 32 weeks. Despite heterogeneity in clonal evolution among the mice, all haploinsufficient clones had the potential to outcompete wt clones (3 of 5 mice in the primary transplant, 3 out of 4 mice in the secondary transplant). Csnk1a1 haploinsufficient clones showed the largest clonal abundance and clonal persistence. Expansion of oligoclonal Csnk1a1 haploinsufficient HSPCs was further enhanced in the secondary transplant in all mice. Egr1 haploinsufficient clones showed potential for prominent oligoclonal expansion in one mouse, but decreased in abundance in all secondary transplants. Apc haploinsufficient clones showed persistence but not expansion in 3 out of 5 mice. These results were validated by conventional competitive transplants, which demonstrated that Csnk1a1 and Egr1 haploinsufficient cells achieved the highest advantage over wt hematopoiesis in the primary transplant and more enhanced in the secondary transplant. Since Csnk1a1 regulates β-catenin protein stability, we hypothesized that the clonal expansion of Csnk1a1 haploinsufficient HSPCs is dependent on β-catenin levels. We performed a second genetic barcoding competitive transplant, comparing Csnk1a1-/+ HSPC directly to double haploinsufficient Csnk1a1-/+/Ctnnb1-/+ (β-catenin encoding) HSPCs. We included additional double haploinsufficient mutants Csnk1a1-/+/Apc-/+ and Csnk1a1-/+/Egr1-/+. Results showed pronounced expansion of Csnk1a1-/+ clones, while Csnk1a1-/+/Ctnnb1-/+ clones were outcompeted over time, suggesting that the advantage of Csnk1a1-/+ clones is β-catenin dependent. Csnk1a1-/+/Egr1-/+ and Csnk1a1-/+/Apc-/+ clones were less advantageous than Csnk1a1-/+ clones. To further investigate the mechanism of clonal fitness in Csnk1a1-/+ haploinsufficient HSPCs, we performed droplet based single cell RNA sequencing of Csnk1a1-/+ and wt Lin-Sca1+cKit+ (LSK) HSPCs. Csnk1a1 -/+ LSK were characterized by a higher fraction of cells expressing cell cycle genes compared to wt cells. In line, transcriptional alterations in the most primitive HSCs suggest that the clonal advantage is conveyed by canonical Wnt signaling activating downstream targets such E2F proteins. Csnk1a1-/+ haploinsufficient multipotent progenitors and myeloid/lymphoid primed progenitors expressed marked upregulation of metabolic pathways, mitochondrial respiration, cell cycle and differentiation, ubiquitination/proteasome system and deregulation of ribosome biogenesis. In conclusion, we demonstrate using a novel genetic barcoding approach in a competitive transplant setting that Csnk1a1-/+ haploinsufficient HSPCs have the potential for oligoclonal expansion and clonal persistence. Wnt/β-catenin signaling plays a central role in the clonal expansion. Interestingly, in Csnk1a1 haploinsufficiency the HSC state is preserved and the increased proliferation and metabolic activation are hallmark features of differentiating progenitor cells at MPP stage, increasing with cell cycle activation, thus ensuring clonal stability and preventing HSC exhaustion over time. Disclosures Brümmendorf: University Hospital of the RWTH Aachen: Employment; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; Merck: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Ebert:Celgene: Research Funding; Deerfield: Research Funding; Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute..

Deep Immunoprofiling By Flow Cytometry and NGS Reveals Distinct T Cell Profile in Pediatric Hepatitis-Associated Aplastic Anemia

Hepatitis-associated aplastic anemia (HAAA) is a rare variant of bone marrow (BM) failure that is typically diagnosed 2-3 months after an acute attack of hepatitis in patients that are seronegative for known hepatitis viruses. Although very little is known about its etiology, an autoimmune mechanism is presumed based on clinical response to immunosuppressive therapy (IST) and reports of oligoclonal T-lymphocyte expansion and skewing of CDR3 region length. However due to the rareness of this disease, the published evidence is still limited. We analyzed 18 pediatric patients consecutively diagnosed with HAAA in the Czech Republic between 2004-2019. Based on BM biopsy examination, 14 patients had aplastic anemia (AA) and interestingly 4 patients were histologically consistent with refractory cytopenia of childhood (RCC). Median age of these patients was 6.6 years (range 1-18) with a male to female ratio of 2:1. The prevalence of HAAA in our cohort of pediatric patients with BM failure (n=107; excluding patients with known genetic cause) was 17%. The presence of a paroxysmal nocturnal hemoglobinuria clone was excluded and cytogenetic evaluation showed no abnormalities. We examined both the diagnostic BM and peripheral blood by flow cytometry in all patients (with the exception of one peripheral blood sample) and performed deep immunophenotyping of T cells in 7 of them. Ten patients were selected for T cell receptor beta (TRB) repertoire and whole-exome sequencing (WES) based on the availability of DNA samples isolated from the BM before the start of the treatment. TRB sequencing was performed following the SOP developed by the EuroClonality-NGS working group with DNA input normalized to the equivalent of 20 000 CD3+ cells per sample based on flow cytometry data. Bioinformatic analysis was performed with ARResT/Interrogate. WES was performed on Illumina NextSeq 500 and libraries prepared using the Agilent SureSelectXT Human All Exon V6+UTRs kit. Vertebrate virus detection was performed using the VirCapSeq-VERT probe capture enrichment set and massive parallel sequencing from 8 BM DNA samples taken at the time of diagnosis. We found significant activation of CD8 positive T cells based on the expression of HLA-DR compared to non-HAAA RCC and AA patients (n=20 and n=21 respectively, Mann-Whitney p&lt;0.0001). Three out of 7 patients with deep T cell immunophenotype available showed a decrease of naive CD27pos45RAposCD8pos T cells. From TRB repertoire analysis we were able to identify a total of 5 private immunodominant clones in 3 patients with relative abundance of up to 11% from all productive TRB sequences. These clones were not found in any previous publications or clonotype databases based on their CDR3 sequence. We detected 5 clones shared by 3 or more patients; these were previously published in other cohorts and are not specific for HAAA. WES analysis did not reveal any previously published variant in genes associated with immune dysregulation or immunodeficiency, or any candidate variant for functional validation. No relevant vertebrate virus signal was detected in the 8 samples. Out of all 18 patients, 3 patients underwent MSD-HSCT without preceding immunotherapy. Fifteen patients were treated according to standard European Working Group on MDS/SAA protocols with anti-thymocyte globulin (horse n=6; rabbit n=9), corticosteroids and cyclosporin A. In total, 7 patients reached complete remission on IST by day 120 and 7 patients with insufficient response underwent MUD-HSCT. One patient died shortly after MUD-HSCT because of toxicity. We did not observe any correlation of T cell populations at diagnosis or day 120 of IST (activated and exhausted T cells) and clinical response. Similarly, the presence of an immunodominant clone or oligoclonal TRB repertoire with lower diversity at diagnosis had no significant impact on the outcome of these patients in our cohort. In conclusion, we present the biggest cohort of pediatric patients with HAAA analyzed by next-generation sequencing and flow cytometry so far. Even though our results show significantly increased activation of CD8 positive T-cells and presence of expanded clones, we did not confirm that these factors would be useful and significant for the prediction of treatment outcome. Large number of our patients did not fully respond to IST, requiring HSCT. Supported by AZV 18-07-00430, 16-32568A and PRIMUS/17/MED/11. Disclosures No relevant conflicts of interest to declare.

Clonal Dynamics of Early Responder and Long-Term Surviving CAR-T Cells in Humans

CD19 CAR-T cells show unprecedented responses in relapsed/refractory Acute Lymphoblastic Leukaemia, but long-term persistence appears critical for their use as a stand-alone therapy. The origin of long-term persisting CAR-T cells has yet to be defined and will be critical in designing manufacturing protocols to optimise long-term persistence. Previous data are conflicting with groups showing prolonged persistence of CAR-T cells from cell products with a predominantly effector memory (TEM) phenotype, whereas others suggesting that the dominant clones originate instead from infused stem cell memory (TSCM) and central memory (TCM) T cells. To date, it has not been possible to isolate long-term (&amp;gt; 1 year) persisting CAR-T cells in patients. In the CARPALL Phase I study, the use of an improved low-affinity CD19 CAR resulted in enhanced expansion and persistence of CAR-T cells in vivo (Ghorashian et al, Nature Medicine, in press). Combining this unique experimental setting with our well-established clonal tracking platform based on high-resolution integration sites (IS) analysis has enabled us to track the fate of the infused CAR-T cells. We analysed 2 patients with long-term persistent CAR-T cells detectable by flow cytometry in peripheral blood. CAR-T cells comprised 13 and 53% circulating CD3+ cells respectively at day 14 post-infusion, 7.1 and 7.7% circulating at 1 month, 0.7 and 1.3% at 6 months and 0.1% for both at the latest follow-ups (24-28 months). Blood samples taken at early (14d, 30d) and later (6m to 28m) time points were flow-sorted for CAR+ TCM/TEM mixed population and TSCM T cells, while the corresponding infused gene-modified products were separated into three subpopulations: TSCM, TCM and TEM. The integration profile of each sorted cell populations was established using linear amplification mediated-PCR (LAM-PCR) combined with high throughput sequencing. We identified a total of 7,105 and 4,692 IS from 2 patients overtime before infusion and up to 28 months after infusion. The infused CAR-T cell population was highly polyclonal before infusion. Although the total number of CAR-T cell clones decreased substantially upon in vivo selection, we did not observe any sign of aberrant clonal drifts and diversity was preserved long-term. Early after infusion during the response peak, TSCM underwent two waves of transient oligoclonal expansion. In both patients two distinct sets of individual TSCM clones contributed to the 73% and 97% of the whole analysed TSCM population at day 14 and 74% and 99% at day 30. Conversely, the largest memory/effector clones detected at the same timepoints spanned from 4% to 21% of the total TCM/TEM population. These TSCM clones subsequently contracted and were not observed at 6-28 months after infusion suggesting that different clones are responsible for the early response and prolonged immune surveillance. After 6 months post-infusion, when the IS profile of circulating CAR-T cells was compared with selected populations from the infused product, only 1.8%-6.1% of long term clones were derived from the infused TCM population, despite this accounting for the majority of IS in the products (72.7/75.8% of clones). Conversely, in both patients the majority of IS associated with long term persistence (90.7%/55.5%) were derived from the TSCM compartment. Our preliminary results raise two hypotheses on the clonal dynamics of infused CAR T cells: 1) There is an early expansion of a defined group of clones during the first 30 days, which is more pronounced in the precursor TSCM compartment. These early waves do not seem to be originated from clones that have substantially expanded in vitro such that their clonal mark could not be retained in the batch of the infused cell product analysed. Further, these clones rapidly disappear after the early anti-tumour response. 2) The long-term population of CAR-T cells seem to have a higher relation with TSCM clones that have expanded in vitro before infusion, supporting the notion that such cells in the infused batch would be the one primarily responsible for the preservation of circulating CAR-T cells in the treated patients. This study suggests for the first time that anti-leukemic response occurs along rapid waves of clonal succession and that TSCM are primarily responsible for the long-term survival of CAR-T cells. Disclosures Ghorashian: novartis: Honoraria; UCLB: Patents &amp; Royalties: UCLB; Celgene: Honoraria. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Thrasher:4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Amrolia:UCLB: Patents &amp; Royalties.

Transcriptome Sequencing Demonstrates Unique Signature Associated with Durable Clinical Response to DC/AML Fusion Vaccine

Transcriptome Sequencing Demonstrates Unique Signature Associated with Durable Clinical Response to DC/AML Fusion Vaccine

Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib Show Expansion of T-Cell Clonotypes Composed of Antitumor Cytotoxic CD8+ T-Cells

We recently showed that ibrutinib induced a more oligoclonal T-cell receptor (TCR) repertoire in patients with chronic lymphocytic leukemia (CLL). Using next generation sequencing (NGS), we found that overrepresented TCRβ clonotypes were mostly patient-specific and that the degree of oligoclonal expansion correlated with CD8+ T-cell counts. Here, we tested the hypothesis that overrepresented clonotypes are CD8+ T-cells that respond to tumor antigens. Flow cytometric profiling of TCR Vβ (TRBV) chain usage, T-cell subset distribution, and granzyme B (GrB) expression was performed on cryopreserved peripheral blood mononuclear cells obtained at baseline and during response to single-agent ibrutinib (n = 5). Antibodies against TRBV were selected to investigate the ten most abundant TCRβ clonotypes identified by NGS. The cumulative frequency of these clonotypes evaluable by flow cytometry increased at the time of response (mean 31.2% ± SEM 3.9% of all T-cells) compared to baseline (mean 24.1% ± SEM 2.3% of all T-cells). Within each TRBV subpopulation, the proportion of CD4+ T-cells decreased (mean fold-change 0.78 ± SEM 0.04), while the proportion of CD8+ T-cells increased (mean fold-change 1.79 ± SEM 0.36). We further observed that the TRBV clonotypes expanding the most during treatment were comprised exclusively of CD8+ T-cells in every patient. These highly expanded CD8+ clonotypes expressed GrB, consistent with a cytotoxic phenotype. To evaluate whether expanding CD8+ clonotypes are tumor-specific, T-cells were expanded ex vivo in two conditions: (1) co-culture with autologous CLL cells, CD40L, IL-2, and IL-7 or (2) with anti-CD3/CD28/CD137 beads, IL-2, and IL-7. Overall, T-cells expanded with autologous CLL cells had a higher proportion of CD8+ GrB+ cells than T-cells expanded with anti-CD3/CD28/CD137 beads. The dominant CD8+ clonotypes detected in each patient at the time of response were preferentially expanded by co-culture with autologous CLL cells compared to expansion with beads. The biased expansion of cytotoxic T-cell clonotypes in the CLL-primed T-cell product supports a TCR mediated response to tumor antigens. Next, cytotoxicity assays were performed by mixing the expanded T-cell products described above with autologous CLL cells at an effector to target ratio of 3:1, 1:1 and 1:3. CLL-primed T-cells killed CLL cells in a dose-dependent fashion. In contrast, bead-expanded T-cells were ineffective at killing CLL cells or even protected tumor cells from spontaneous apoptosis. Thus, tumor-specific cytotoxic T-cells expand during treatment with ibrutinib and are further enriched ex vivo by co-culture with autologous CLL cells. In summary, increased TCR oligoclonality in patients responding to ibrutinib is driven by the expansion of specific CD8+ clonotypes. These clonotypes expand in co-culture with, and are cytotoxic against, autologous CLL cells. Tumor-specific CD8+ T-cell clonotypes may contribute to the overall treatment response with ibrutinib and supports the investigation of therapeutic strategies combining ibrutinib with T-cell directed immunotherapy. Disclosures Baskar: NIH: Patents &amp; Royalties: ROR1 mAb 2A2. Wiestner:Nurix: Research Funding; Acerta: Research Funding; Merck: Research Funding; Pharmayclics: Research Funding.

Immune and Circulating Tumor DNA Profiling After Radiation Treatment for Oligometastatic Non-Small Cell Lung Cancer: Translational Correlatives from a Mature Randomized Phase II Trial

NCT01725165 was a phase II prospective trial in which patients with non-small cell lung cancer were randomized to local consolidative therapy (LCT) versus maintenance therapy or observation (MT/O). Peripheral blood from patients enrolled on NCT01725165 were labeled as (1) baseline, (2) early follow-up (FU) if obtained in the first or second FU evaluation (6-18 weeks), and (3) late FU if obtained in the third to sixth FU evaluations (22-50 weeks). All patients who underwent LCT and were included in this analysis received radiation. Among 49 randomized patients, 21 patients underwent T cell CDR3 variable region sequencing using immunoSEQ, 31 patients underwent circulating tumor DNA (ctDNA) analysis using next-generation sequencing with a 1021 cancer gene panel, and cytokine concentration was assayed in 19 patients using enzyme-linked immunosorbent assay. All analyses were exploratory and not corrected for multiple testing. No associations were identified between baseline T cell repertoire and ctDNA metrics with patient outcomes. Among baseline cytokines, interleukin 1α was the only cytokine associated with both overall survival (hazard ratio, 0.02; 95% confidence interval, 0.1-0.5; P = .0006) and progression-free survival (hazard ratio, 0.5; 95% confidence interval, 0.2-0.9; P = .03). At early FU, LCT was associated with decreased ctDNA burden, including lower number of detected mutations (median, 2 [interquartile range {IQR}, 1-6] vs 6 [IQR, 4-18]) and decreased average variable allele frequency (VAF; median, 0.006 [IQR, 0.003-0.010] vs 0.011 [IQR, 0.007-0.014]) compared with MT/O. Among 6 patients with serial ctDNA analysis, a rise in ctDNA detected mutation burden preceded clinical progression by 6.7 months. At early FU, LCT was associated with changes in T cell clonality that suggested oligoclonal expansion specifically increased T cell clonality (median, 0.15 [IQR, 0.12-0.24] vs 0.10 [IQR, 0.05-0.13]) and frequency of top 10 clones (median, 0.14 [IQR, 0.06-0.18] vs 0.21[IQR, 0.19-0.28]). LCT was associated with decreased ctDNA burden and oligoclonal expansion at early FU timepoints. Baseline interleukin 1α was associated with improved patient outcomes.

Impact of CMV Infection on Natural Killer Cell Clonal Repertoire in CMV-Naïve Rhesus Macaques.

Recent functional, gene expression, and epigenetic studies have suggested the presence of a subset of mature natural killer (NK) cells responsible for maintaining NK cell memory. The lack of endogenous clonal markers in NK cells impedes understanding the genesis of these cell populations. In humans, primates, and mice, this phenotype and memory or adaptive functions have been strongly linked to cytomegalovirus or related herpes virus infections. We have used transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPC) to track clonal hematopoiesis in rhesus macaques and previously reported striking oligoclonal expansions of NK-biased barcoded clones within the CD56−CD16+ NK cell subpopulation, clonally distinct from ongoing output of myeloid, B cell, T cell, and CD56+16− NK cells from HSPC. These CD56−CD16+ NK cell clones segregate by expression of specific KIR surface receptors, suggesting clonal expansion in reaction to specific environmental stimuli. We have now used this model to investigate the impact of rhesus CMV(RhCMV) infection on NK clonal dynamics. Following transplantation, RhCMVneg rhesus macaques display less dominant and oligoclonal CD16+ NK cells biased clones compared to RhCMVpos animals, however these populations of cells are still clearly present. Upon RhCMV infection, CD16+ NK cells proliferate, followed by appearance of new groups of expanded NK clones and disappearance of clones present prior to RhCMV infection. A second superinfection with RhCMV resulted in rapid viral clearance without major change in the mature NK cell clonal landscape. Our findings suggest that RhCMV is not the sole driver of clonal expansion and peripheral maintenance of mature NK cells; however, infection of macaques with this herpesvirus does result in selective expansion and persistence of specific NK cell clones, providing further information relevant to adaptive NK cells and the development of NK cell therapies.

Primary EBV Infection Induces an Acute Wave of Activated Antigen-Specific Cytotoxic CD4+ T Cells.

CD4+ T cells are essential for immune protection against viruses, yet their multiple roles remain ill-defined at the single-cell level in humans. Using HLA class II tetramers, we studied the functional properties and clonotypic architecture of EBV-specific CD4+ T cells in patients with infectious mononucleosis, a symptomatic manifestation of primary EBV infection, and in long-term healthy carriers of EBV. We found that primary infection elicited oligoclonal expansions of TH1-like EBV-specific CD4+ T cells armed with cytotoxic proteins that responded immediately ex vivo to challenge with EBV-infected B cells. Importantly, these acutely generated cytotoxic CD4+ T cells were highly activated and transcriptionally distinct from classically described cytotoxic CD4+ memory T cells that accumulate during other persistent viral infections, including CMV and HIV. In contrast, EBV-specific memory CD4+ T cells displayed increased cytokine polyfunctionality but lacked cytotoxic activity. These findings suggested an important effector role for acutely generated cytotoxic CD4+ T cells that could potentially be harnessed to improve the efficacy of vaccines against EBV.

TCR repertoire and CDR3 motif analyses depict the role of αβ T cells in Ankylosing spondylitis.

BackgroundAnkylosing spondylitis (AS) is a chronic inflammatory disease with worldwide high prevalence. Although AS is strongly associated with HLA-B27 MHC-I antigen presentation, the role played by αβ T cells in AS remains elusive.MethodsUtilizing TCRβ repertoire sequencing and bioinformatics tools developed in house, we analyzed overall TCR repertoire structures and antigen-recognizing CDR3 motifs in AS patients with different disease activities.FindingsWe found that disease progression is associated with both CD4+ and CD8+ T cell oligo-clonal expansion, which suggests that αβ T cell activation may mediate AS disease progression. By developing a bioinformatics platform to dissect antigen-specific responses, we discovered a cell population consisting of both CD4+ and CD8+ T cells expressing identical TCRs, herein termed CD4/8 T cells. CD4/8 clonotypes were highly enriched in the spondyloarthritic joint fluid of patients, and their expansion correlated with the activity of disease.InterpretationThese results provide evidence on the T cell clone side to reveal the potential role of CD4/8 T cells in the etiology of AS development.

Adaptive and Innate Immunity in Psoriasis and Other Inflammatory Disorders.

Over the past three decades, a considerable body of evidence has highlighted T cells as pivotal culprits in the pathogenesis of psoriasis. This includes the association of psoriasis with certain MHC (HLA) alleles, oligoclonal expansion of T cells in some cases, therapeutic response to T cell-directed immunomodulation, the onset of psoriasis following bone marrow transplantation, or induction of psoriasis-like inflammation by T cells in experimental animals. There is accumulating clinical and experimental evidence suggesting that both autoimmune and autoinflammatory mechanisms lie at the core of the disease. Indeed, some studies suggested antigenic functions of structural proteins, and complexes of self-DNA with cathelicidin (LL37) or melanocytic ADAMTSL5 have been proposed more recently as actual auto-antigens in some cases of psoriasis. These findings are accompanied by various immunoregulatory mechanisms, which we increasingly understand and which connect innate and adaptive immunity. Specific adaptive autoimmune responses, together with our current view of psoriasis as a systemic inflammatory disorder, raise the question of whether psoriasis may have connections to autoimmune or autoinflammatory disorders elsewhere in the body. While such associations have been suspected for many years, compelling mechanistic evidence in support of this notion is still scant. This review sets into context the current knowledge about innate and adaptive immunological processes in psoriasis and other autoimmune or autoinflammatory diseases.

Smooth Muscle Cell Phenotypic Diversity.

Vascular smooth muscle cells (SMC) play a critical role in controlling blood pressure and blood distribution, as well as maintaining the structural integrity of the blood vessel. SMC also participate in physiological and pathological vascular remodeling due to their remarkable ability to dynamically modulate their phenotype. During the past decade, the development of in vivo fate mapping systems for unbiased identification and tracking of SMC and their progeny has led to major discoveries as well as the reevaluation of well-established concepts about the contribution of vascular SMC in major vascular diseases including atherosclerosis. Lineage tracing studies revealed that SMC undergoes multiple phenotypic transitions characterized by the expression of markers of alternative cell types (eg, macrophage-like and mesenchymal-stem cell-like) and populate injured or diseased vessels by oligoclonal expansion of a limited number of medial SMC. With the development of high-throughput transcriptomics and single-cell RNA sequencing (scRNAseq), the field is moving forward towards in-depth SMC phenotypic characterization. Herein, we review the major observations put forth by lineage and clonality tracing studies and the evidence in support for SMC phenotypic diversity in healthy and diseased vascular tissue. We will also discuss the opportunities and remaining challenges of combining lineage tracing and single-cell transcriptomics technologies, as well as studying the functional relevance of SMC phenotypic transitions and identifying the mechanisms controlling them.

High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4\u201328/IGHJ4 gene in primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP)IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4–28 in ITP patients. One hundred eighty-six distinct IGHV4–28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4–28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4–28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4–28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP.

TRAV1-2+ CD8+ T-cells including oligoconal expansions of MAIT cells are enriched in the airways in human tuberculosis

Mucosal-associated invariant T (MAIT) cells typically express a TRAV1-2+ semi-invariant TCRα that enables recognition of bacterial, mycobacterial, and fungal riboflavin metabolites presented by MR1. MAIT cells are associated with immune control of bacterial and mycobacterial infections in murine models. Here, we report that a population of pro-inflammatory TRAV1-2+ CD8+ T cells are present in the airways and lungs of healthy individuals and are enriched in bronchoalveolar fluid of patients with active pulmonary tuberculosis (TB). High-throughput T cell receptor analysis reveals oligoclonal expansions of canonical and donor-unique TRAV1-2+ MAIT-consistent TCRα sequences within this population. Some of these cells demonstrate MR1-restricted mycobacterial reactivity and phenotypes suggestive of MAIT cell identity. These findings demonstrate enrichment of TRAV1-2+ CD8+ T cells with MAIT or MAIT-like features in the airways during active TB and suggest a role for these cells in the human pulmonary immune response to Mycobacterium tuberculosis.