T-cell Receptor Gene Research Articles

Abstract 14: Identification of tumor-reactive T cell receptors through functional single cell interaction analyses for personalized T cell therapy

Abstract Innovative immunotherapy approaches such as adoptive transfer of chimeric antigen receptor (CAR) T cells or tumor infiltrating lymphocytes (TILs) have shown great success in the treatment of solid tumors and hematological malignancies. Although treatment of multiple myeloma with CAR T cells can induce deep responses, relapses frequently occur due to antigen escape and limited CAR T cell persistence. TCR-engineered T cells may show prolonged persistence in vivo and could mediate sustained antitumor effects. A further benefit of TCR transgenic T cells is the ability to target intracellular antigens that are inaccessible to CAR T cells, expanding the range of potential targets for immunotherapy. In our project, we propose to identify T cell receptors (TCRs) specifically targeting autologous myeloma cells. Tumor-reactive T cells were identified using the Bruker Cellular Analysis Lightning® platform, allowing simultaneous functional analysis of up to 1500 individual T cell/target cell interactions on a chip. Reactive T cells were identified upon detection of secreted cytokines (IFNγ, TNFα, IL2) and measurement of 4-1BB (CD137) surface expression. Tumor-reactive T cells showing various cytokine secretion patterns and 4-1BB expression profiles were detected in each myeloma patient (on average 11.9 T cells out of 1243 cells tested per assay run). Individual tumor-reactive T cells have been isolated and their TCRs were sequenced. TCR sequences of tumor-reactive T cells were mapped to single-cell RNA sequencing data of T cells from the same patiens to reveal a gene expression signature of myeloma-reactive T cells. TCR genes of reactive T cells were cloned and overexpressed in autologous T cells for functional validation and analysis of tumor derived neoepitope specificity. In summary, we present a pipeline allowing identification of myeloma-recognizing T cells and recovery of bona fide tumor-reactive TCRs eligible for patient-individualized T cell therapy. Citation Format: Tim Robin Wagner, Niklas Kehl, Simon Steiger, Michael Kilian, Bruno Schönfelder, Tamara Boschert, Katharina Lindner, Patrick Schmidt, Karsten Rippe, Hartmut Goldschmidt, Marc-Steffen Raab, Michael Platten, Mirco Friedrich, Stefan B. Eichmüller. Identification of tumor-reactive T cell receptors through functional single cell interaction analyses for personalized T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 14.

Abstract 7011: The DNA methylation landscape across the TCR loci genes in patients with acute myeloid leukemia

Abstract The T-cell receptor (TCR) repertoire is partly shaped by epigenetic modifications, including DNA methylation. Hypomethylating agents like decitabine are used to treat patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The precise DNA methylation patterns within the TCR loci and their dynamics associated with HMAs treatments remain underexplored. Here, we analyzed methylation patterns across TCR alpha and beta gene loci in T cells from GSE175758 and GSE67170 datasets, comparing patterns between patients with AML and healthy controls. We also investigated the impact of decitabine on the methylation patterns of TCR gene loci in patients with AML and MDS (GSE175758 and GSE80762).The overall levels of methylation between T cells from healthy donors and patients with AML were significantly different within the TRAV (0.55 vs 0.42, P < 0.001) and TRBV regions (0.70 vs 0.59, P < 0.001) with 46 TRAV and 33 TRBV sites differentially methylated positions, respectively. However, methylation patterns in HSCs (GSE63409), T cells from healthy donors and patients with AML and those in AML cells were strongly correlated (R > 0.67, range 0.67-0.99, P < 0.001).Because decitabine preferentially acts on leukemic cells and given the high correlation levels between AML cells and T cells across datasets we analyzed the effects of decitabine on TCR DNA methylation patterns in AML cells. In the leukemic cells decitabine treatment led to significant demethylation across 33 and 30 CpG sites within the TRAV and TRBV genes, respectively. In the pre-treatment AML samples higher methylation beta values were observed in differentially methylated positions (DMPs) compared with non-DMPs (TRAV: 0.847 vs 0.407; P < 0.001; TRBV: 0.771 vs 0.538; P = 0.049). In samples largely enriched of myeloid leukemic blasts, we found that TRAV and TRBV loci methylation patterns differ significantly between AML patients with good, moderate, and poor risk patients (P < 0.001). We found TRBV6-1 position 1 and 2 methylation was lower in patients with good risk, compared with intermediate risk (pos1: 0.07 vs 0.13, P=0.04; pos2: 0.1 vs 0.2, P = 0.002) and poor risk (pos 1: 0.07 vs 0.21, P < 0.001; pos 2: 0.1 vs 0.32, P < 0.001) and was lower in patients with intermediate risk compared with poor risk (pos1: 0.13 vs 0.21, p=0.007; pos 2: 0.21 vs 0.32, P = 0.001). The high correlation of TCR DNA methylation patterns between the T cells and the myeloid cells, suggests the need for similar analyses in the context of AML T cells. The study highlights the presence of a conserved TCR loci methylated signatures which is highly correlated between myeloid leukemic cells and T cells and is associated with clinical outcomes, offering a potential avenue for therapeutic intervention. Hypomethylating agents, such as decitabine, can modulate TCR loci methylation patterns. Citation Format: Mateusz Pospiech, John Beckford, Advaith M. Kumar, Mukund Tamizharasan, Jaqueline Brito, Gangning Liang, Serghei Mangul, Houda Alachkar. The DNA methylation landscape across the TCR loci genes in patients with acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7011.

Abstract 7425: UMI-nea: A fast and robust UMI analysis approach to accurately identify and quantify TCR repertoire from targeted RNA sequencing with wide range of input molecules

Abstract The T-cell receptor (TCR) population in humans is comprised of highly diversified heterodimers, regulating the recognition of antigen-major histocompatibility complex. Tremendous TCR sequence diversity is produced by somatic recombination of several TCR gene loci each consisting of multiple gene segments. Next-generation sequencing has enabled comprehensive profiling of the TCR repertoire from different physiological and disease conditions ushering in much interest in using TCR-seq to assess T-cell diversity. However, during NGS library construction and sequencing, errors and enzymatic inefficiencies can compromise the accuracy of the final data, particularly around calling of the VD and VDJ recombined regions and subsequent clonotype assignment. To increase the accuracy of NGS sequencing, Unique Molecular Identifiers (UMIs), consisting of short random nucleotide bases, can be used to mark original molecules in NGS library allowing for error and bias corrections. There are two well studied technical limitations to apply UMIs: 1.) UMI sequences tend to collide when input molecule number is large 2.) UMI sequences are not insulated from PCR and sequencing errors. To address these limitations, many computational approaches had been published. Among them, very few can be used to solve UMI colliding errors and over-simplified error models were implemented for UMI sequencing error handling. Here we report a novel strategy and UMI structure which uses more complex UMIs that is longer and of different length. This results in minimizing UMI collision while maximizing sequencing quality. Our UMI analysis pipeline, “UMI-nea” is able to handle not only substitution errors but also indel errors and UMIs with different lengths. We developed a novel computational framework to parallelly process sequence comparisons to mitigate the elevated computational burden. To account for the varied dispersion of PCR efficiency for different molecules and error bearing UMIs from libraries with different input and with different sequencing depth, we also developed a statistical framework leveraging negative binomial model and single-cell knee plot to set a dynamic threshold for original molecule estimate. We verified UMI-nea with several simulated data and demonstrated that UMI-nea can achieve >99% completeness and homogeneity to recover the original molecule count with various error rates and UMI lengths, outperforming existing tools and methods in comparison. We applied UMI-nea to profile TCR for 8 PBMC samples sequenced on different Illumina platforms with different sequencing depths. We observed >85% reproducibility of clonotype calls on all samples. To test the sensitivity and specificity of UMI-nea, we sequenced pure cell line samples and cell line spike-in samples with different ratios and discovered very high recall and precision rates. Citation Format: Jixin Deng, Jingxiao Zhang, Song Tian, Samuel J. Rulli, Hong Xu, John DiCarlo, Eric Lader. UMI-nea: A fast and robust UMI analysis approach to accurately identify and quantify TCR repertoire from targeted RNA sequencing with wide range of input molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7425.

Abstract 3976: Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach

Abstract Single-cell immune receptor profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000-5,000 cells) using microwell arrays or droplet microfluidics technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput when compared to bulk immune receptor profiling methods. Here, we describe a low-throughput, single-cell immune profiling strategy using sorted cells in 96-well plates. The plate is pre-aliquoted with either T-cell receptor (TCR) ɑ/β or TCR γ/δ primers along with 30 crucial T-cell markers. We perform multiplex RT-PCR amplification and sequencing of the CDR3 regions. The resulting data provides the abundant clonotype counts along with the chain pairing information for these ɑ/β and γ/δ chains, together with the T-cell subtype information using gene-expression profiles. By analyzing the TCR gene rearrangement at the single-cell level, researchers can better understand T cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. This method provides a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay make it a valuable tool for unraveling the immune dynamics in various diseases. Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Lester Kobzik, Khadija Ghias, Paul Diehl. Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3976.

Cutaneous lymphoproliferative disorders: Back to the future.

In the 1980s, immunohistochemistry and clonality analyses became instrumental in the recognition and definition of new types of cutaneous T-cell lymphoma (CTCL) and cutaneous B-cell lymphoma (CBCL) and the development of new classifications. By accepting loss of pan-T-cell antigens and clonal T-cell receptor gene rearrangements as important criteria to differentiate between benign and malignant T-cell proliferations, and monotypic immunoglobulin light-chain expression and clonal immunoglobulin gene rearrangements as crucial criteria to distinguish between benign and malignant B-cell proliferations, many cases, until then diagnosed as cutaneous lymphoid hyperplasia or pseudolymphoma, were reclassified as primary cutaneous CD4+ small/medium T-cell lymphoma (PCSM-TCL) or primary cutaneous marginal zone lymphoma (PCMZL), respectively. However, in recent years there is growing awareness that neither these immunohistochemical criteria nor demonstration of T-cell or B-cell clonality is specific for malignant lymphomas. In addition, many studies have reported that these low-grade malignant CTCL and CBCL have an indolent clinical behavior and an excellent prognosis with disease-specific survival rates of or close to 100%. As a result, recent classifications have downgraded several low-grade malignant cutaneous lymphomas to lymphoproliferative disorder (LPD). Both the 5th edition of the WHO classification (2022) and the 2022 International Consensus Classification (ICC) of mature lymphoid neoplasms reclassified PCSM-TCL as primary cutaneous CD4+ small/medium T-cell LPD and primary cutaneous acral CD8+ T-cell lymphoma as primary cutaneous acral CD8+ T cell LPD. While the 2022 ICC introduced the term "primary cutaneous marginal zone LPD," in the 5th edition of the WHO classification PCMZL is maintained. In this review we describe the background and rationale of the continually changing terminology of these conditions and discuss the clinical consequences of downgrading malignant lymphomas to LPDs.

Comparison of 2 T-Cell Receptor-γ Clonality Assays on Skin Biopsies Suspicious for Mycosis Fungoides.

PCR-based fragment analysis of the T-cell receptor (TCR) gene is used extensively in diagnostic labs to assess clonality in T-cell populations in multiple tissue sites. Of the numerous TCR assays that have been reported, studies assessing use on biopsies suspicious for mycosis fungoides specifically are lacking. We compared clonality findings from a previously run 2-tube/2-fluorochrome dye assay to a redesigned 1-tube/1-fluorochrome dye assay on formalin-fixed skin biopsies. Overall, the accuracy of the 2-tube assay was marginally better (75.7% vs. 71.4%), when using clinical history combined with histologic diagnosis as the gold standard. The 2-tube assay had better sensitivity (73.7% vs. 65.8%), while the 1-tube assay had superior specificity (93.8% vs. 87.5%). Clonality results were easier to interpret with the 1-tube assay. In nearly 19% of cases, a change of assays on the same biopsy resulted in a change of clonality interpretation. For laboratories that change TCR-γ clonality assays, follow-up biopsies for mycosis fungoides assessment may result in a change of diagnosis.

Interstitial mycosis fungoides: A rare presentation of mycosis fungoides with overlapping granulomatous and folliculotropic features.

Interstitial mycosis fungoides (IMF) is a rare subtype of mycosis fungoides (MF) characterized by atypical lymphocytes infiltrating the reticular dermis between collagen bundles with limited epidermotropism and variable granulomatous features. Retrospective single institution review of 31 cases of IMF including clinical characteristics, disease course and pathological features. Our cohort was predominately male (19; 61%, M:F 1.6:1) with a mean age at diagnosis of 43 years (range 11-85), mean signs/symptoms duration of 7 years prior to diagnosis, and 6 years mean follow-up duration. Clinically, patients often exhibited symmetric ill-defined patches/plaques involving intertriginous regions with tan-yellow hyperpigmentation and follicular-based papules, wrinkling, and alopecia. Lymphadenopathy was noted in seven patients. Fifteen (52%) patients were in near or complete clinical remission at the latest follow-up. T-cell receptor gene rearrangement was positive in 23/24 (96%) cases. Histopathologically, atypical cells were small-medium, CD4+ (29; 94%) or rarely CD4+/CD8+ (1; 3%) lymphocytes infiltrating the reticular dermis with thickened collagen bundles (27; 87%), multinucleated giant cells (12; 39%), and often tracing along adnexa with subtle folliculotropism (12/20; 60%). Our study demonstrates IMF is an indolent subtype of MF with distinct features, including frequent granulomatous and subtle follicular involvement resulting in alopecia.

Identification, expression and molecular polymorphism of T-cell receptors α and β from the glacial relict Hucho bleekeri

The T-cell receptor (TCR) is a specific molecule on the surface of all T cells that mediates cellular adaptive immune responses to antigens. Hucho bleekeri is a critically endangered species and is regarded as a glacial relict that has the lowest-latitude distribution compared with any Eurasian salmonid. In the present study, two TCR genes, namely, TCR α and β, were identified and characterized in H. bleekeri. Both TCR α and TCR β have typical TCR structures, including the IgV domain, IgC domain, connecting peptide, transmembrane and cytoplasmic domains. The two TCR genes were constitutionally expressed in various tissues, with the highest expression found in the spleen for TCR α and in the trunk kidney for TCR β. Challenge of H. bleekeri with LPS or poly(I:C) resulted in significant upregulation of both TCR α and β expression in headkidney and spleen primary cells, indicating their potential roles in the immune response. Molecular polymorphism analysis of the whole ORF regions of TCR α and β in different individuals revealed high diversity of IgV domains of these two genes, especially in complementarity-determining region (CDR) 3. The ratio of nonsynonymous substitution occurred at a significantly higher frequency than synonymous substitution in the CDR of TCR α and β, demonstrating the existence of positive selection. The results obtained in the present study enhance our understanding of TCR roles in regulating immune mechanisms and provide new information for the study of TCR lineage diversity in fish.

Micro-RNAs in the Diagnosis of Cutaneous T-Cell Lymphomas

Rationale. Early diagnosis of mycosis fungoides (MF), as the most common form of T-cell lymphoma, presents significant challenges. The diagnosis of MF is based on the following criteria: comprehensive assessment of the clinical picture of the disease, histological and immunohistochemical examination of the skin, and determination of rearrangement of the T-cell receptor gene, but even this does not always aid in diagnosis. The aim of the study is to investigate the expression of micro-RNAs (miR-223, -423, -663, -16, -326, -711) in the blood plasma and leukocytes of patients with a presumptive diagnosis of MF to improve the disease diagnosis. Methods. This study included 50 patients aged 24 to 79 years, of whom 30 patients had a preliminary diagnosis of MF and 20 patients with small plaque parapsoriasis, who formed the comparison group. All patients underwent histological, immunohistochemical examination of skin biopsies, and determination of micro-RNA (miR-223, -423, -663, -16, -326, -711) expression in blood plasma and leukocytes by real-time PCR. Results. Analyzing the results of clinical-anamnestic, histological, and immunohistochemical research methods, the diagnosis of MF was established in 22 of 30 (73.3%) patients, of which 9 of 14 (64.3%) were in the early stages of the disease. small plaque parapsoriasis. Conclusion. During our study, it was found that the studied micro-RNAs (miR-326, -663, -711, -223, -423, -16) in the blood plasma and leukocytes of patients with MF have statistically significant levels of expression compared to the low level of expression of these micro-RNAs in patients with small plaque parapsoriasis. The expression of micro-RNAs we studied in the skin contributes to the improvement of MF diagnosis with an accuracy of up to 90%.

Juvenile-Onset Non-Poikilodermatous CD8+CD56+ Mycosis Fungoides

The most frequent primary cutaneous lymphomas observed in childhood and adolescence are mycosis fungoides (MF) and CD30-positive lymphoproliferative diseases. We report a 22-year-old female who presented with a 6-year history of multiple well-demarcated large roundish-oval scaly and reddish-brownish patches and plaques on the trunk and extremities. Histopathology revealed the focal parakeratosis and prominent epidermotropism of atypical lymphocytes, which were positive for CD8, CD56, and TIA-1 and showed a loss of CD7 and CD5 expression. T-cell receptor (TCR) gene rearrangement analysis (multiplex-PCR, BIOMED-2) of the lesional skin demonstrated the rearrangement of the gamma chain (tube A: 162 nt). Based on clinicopathological findings and a complete work-up, she was diagnosed with juvenile non-poikilodermatous C8+/CD56+ MF in stage IA. Resolution of the skin lesions was achieved by 16-week narrowband UVB phototherapy and clobetasol propionate 0.05% ointment. Juvenile-onset non-poikilodermatous CD8+CD56+ MF represents a very rare MF subtype and is associated with an indolent course. In order to avoid too aggressive diagnostics and treatments, clinicians should be aware of this rare and indolent MF variant in childhood and adolescence.

Application of Blood Lymphocyte Immunophenotype and TCR Gene Rearrangement in the Diagnosis of CTCL.

The immunophenotype of peripheral blood lymphocytes and T-cell receptor (TCR) gene rearrangement of cutaneous T cell lymphoma (CTCL) patients were retrospectively analyzed to explore their value in the diagnosis of CTCL. A total of fifty patients' results were enrolled from 2013 to 2021, including 29 malignant skin disorders and 21 benign skin disorders. The immunophenotype of peripheral blood lymphocytes were analyzed by flow cytometry and TCR gene rearrangement was detected by capillary electrophoresis. Lymphocyte subsets, CD4/CD8 ratio, the percentage of CD3+CD4+CD7- cells and CD45RA/CD45RO ratio was calculated between malignant and benign skin disorders. Peripheral blood lymphocyte immunophenotype and TCR gene rearrangement was compared with skin biopsy to evaluate their sensitivity and specificity. Lymphocyte subsets between malignant and benign groups have no significant difference in percentage of T cell (p > 0.05). The CD4/CD8 ratio is higher in patients with malignant lymphoma than the healthy range. The percentage of CD3+CD4+CD7- cells in malignant groups is higher than that in benign groups and CD45RA/ CD45RO ratio has significant difference between malignant and benign groups (p < 0.05). The sensitivity and specificity of TCR rearrangement for CTCL were 51.7% and 42.9%. The sensitivity and specificity of peripheral blood lymphocyte immunophenotype for CTCL were 44.8% and 33.3%. Combining the two methods, the sensitivity and specificity reached 69.0% and 38.1%, respectively. CD4/CD8 ratio of lymphocyte subsets, the proportion of CD4+CD7-T cells and CD45RA/CD45RO ratio can effectively distinguish benign and malignant dermatosis. TCR rearrangement method combined with lymphocyte immunophenotype can improve the sensitivity and specificity of CTCL diagnosis.

TCL1-family negative T-cell prolymphocytic leukemia with rapid progression of extranodal disease despite a normal white blood cell count

Here we describe the case of a 69-year-old man who was found to have moderate thrombocytopenia and severe splenomegaly during a medical checkup at the age of 67. At the first visit, his white blood cell (WBC) count was 7,400/µl with 80% lymphocytes, and bone marrow aspiration showed 24% atypical lymphocytes. Flow cytometry of atypical lymphocytes was positive for mature T-cell markers, and T-cell clonality was revealed by T-cell receptor gene rearrangement. TCL1 was negative on immunohistochemistry. We diagnosed TCL1-family negative T-cell prolymphocytic leukemia (T-PLL) and employed watchful waiting. Thirty months after diagnosis, the patient developed urinary retention and right lower-limb paresis despite a normal WBC count, and an extradural tumor around the thoracic vertebrae and spinal cord compression were detected. The tumor was diagnosed as extranodal involvement of TCL1-family negative T-PLL, but the patient's general condition deteriorated rapidly, and no treatment was possible. T-PLL is a rare disease characterized by leukocytosis, and the WBC count generally increases with disease progression. Although blood counts are recommended for observation, it is important to keep in mind that the disease may worsen even if blood counts do not change.

Routine Whole Genome Sequencing Offers a More Comprehensive Assessment of IG/TCR Gene Rearrangements for MRD Targets in Acute Lymphoblastic Leukemia - Data from the First 62 Cases from the NHS England Genomic Medicine Service

Introduction Minimal residual disease (MRD) monitoring is fundamental for risk stratification and treatment in acute lymphoblastic leukemia (ALL). Sensitive molecular assay requires presentation samples to be screened for immunoglobulin and T-cell receptor (IG/TCR) gene rearrangements. Previously samples were screened by PCR, heteroduplex analysis and Sanger sequencing to identify clone defining IG/TCR gene rearrangements. Subsequently these sequences were used to develop qPCR assays for MRD follow-up monitoring. More recently, targeted IG/TCR high-throughput sequencing (HTS) has been used with similar panels to identify gene rearrangements. Both these methods have limitations, in that only rearrangements covered by the panels are detected, and the techniques are labour intensive and expensive. Whole genome sequencing (WGS) has potential to discover all IG/TCR rearrangements in a single assay giving greater potential to detect markers for follow up MRD monitoring. In England all patients with ALL are eligible for WGS at diagnosis via the National Health Service, Genomic Medicine Service (NHS GMS). Methods Sixty-two patients, presenting with ALL at either Great Ormond Street Hospital or Bristol Children's Hospital, had short-read WGS via the NHS GMS performed concurrently with standard of care (SOC) targeted HTS to identify IG/TCR rearrangements. A bespoke bioinformatic pipeline was developed to identify IG/TCR rearrangements in the WGS samples. SOC targeted HTS analysis was performed using the open source Vidjil bioinformatic software. A cut-off of 5% (clone proportion of total clonotypes) was used to define an acceptable disease-specific clone for monitoring. Results All 62 samples had a greater number of rearrangements detected by WGS than targeted HTS. The mean number of clones &amp;gt;5% detected was 2.7 by targeted HTS and 3.1 by WGS. For rearrangements identified by targeted HTS at a 5% cut-off, 97% (185 out of 190) were identified by WGS. All targets were identified by WGS using a 10% cut-off (Table 1).Despite the lower depth of coverage of the WGS assay, it retrieved 89% of sequences seen in SOC down to a level of 1%, as well as additional re-arrangements not seen by the SOC assay (Figure 1). As well as additional rearrangements being discovered by WGS, we observed novel rearrangements (dd3/ja rearrangements) not previously identified using a targeted approach. To establish if the additional sequences identified by WGS were clinically applicable as MRD targets, we designed allele specific oligonucleotides for a representative 32 rearrangements for testing by qPCR. Over half (n=16) were able to be used to track MRD in follow-up samples. New primer/probe sets were designed for the novel dd3/ja rearrangements identified by WGS, these were also tested by qPCR and performed well enough to be clinically applicable for MRD monitoring. Conclusions WGS approach outperformed targeted HTS, identifying more IG/TCR rearrangements suitable for molecular MRD monitoring in all patients. This is of particularly benefit in cases where targeted HTS only identifies 1 or 2 potential MRD targets. WGS also has the advantage of identifying other key gene rearrangements (BCR-ABL, KMT2A, SIL-TAL, IKZF1) that make excellent targets for MRD monitoring. Beyond this, WGS delivers all diagnostic information, germline and additional somatic findings that aid diagnosis. For ALL, in one test WGS can now replace all molecular diagnostics. In England all patients with leukemia are eligible for centralised WGS. With continued improvements in turnaround times, and reducing costs, we propose that WGS should be the new diagnostic standard for pediatric ALL

Antigen Receptor Gene Repertoire Profiling in Kidney Transplant Recipients Post-Sars-Cov-2 Vaccination

Kidney transplant recipients (KTRs) are highly susceptible to viral infections, while also exhibiting a higher risk of developing severe COVID-19. Recent studies have demonstrated a suboptimal humoral response to current COVID-19 vaccines in this cohort, necessitating multiple vaccine doses. Nevertheless, low rates of seroconversion have been observed even after booster vaccinations, highlighting the need for the in-depth characterization of both humoral and cellular immune responses towards the development of innovative and effective immunoprophylactic strategies tailored for KTRs. Here, we investigated the immunogenetic features of the T cell receptor (TR) and B cell receptor immunoglobulin (BcR IG) gene repertoires in KTRs using targeted Next Generation Sequencing (NGS). In more detail, we studied 19 KTRs at two consecutive timepoints, after the 2 nd (T1) and multiple vaccination doses (T2), respectively. We included as a control group 19 healthy individuals (HIs) sampled either after two mRNA vaccination doses or after a documented SARS-CoV-2 infection. Seroconversion (&amp;gt;1300 AU/ml of anti-RBD IgG) was measured by the Abbott SARS-CoV-2 IgG assay: all HIs tested positive, in sharp contrast to KTRs who failed to exhibit humoral responses after the 2 nd dose (T1). Immunogenetic characterization of TR/BcR IG gene repertoires was performed by paired-end NGS; resultant data were processed through the TRIP software. Only productive V(D)J gene rearrangements were considered for the computation of clonotypes (i.e. TR/BcR IG gene rearrangements with identical V gene usage and complementarity-determining region 3 amino acid sequence-CDR3 aa). Starting with the TR gene repertoire, diversity significantly increased at T2 (average Shannon diversity: KTR-T1=638 vs KTR-T2=3212, p&amp;lt;0.0005| KTR-T2=3212 vs HI=2146, p&amp;lt;0.05). TR clonality (mean cumulative frequency of the 10 dominant TR clonotypes/sample) was significantly increased at T1 in KTRs vs HIs (KTR-T1=37% vs HI=25%, p&amp;lt;0.05), eventually decreasing at T2, reaching comparable levels to those measured in HIs (KTR-T2=27%). At T1, a median of 7.6% of TR clonotypes/sample in KTRs were SARS-specific (high-affinity TR clonotypes to SARS-specific peptide-MHC complexes, &amp;gt;0.9 binding score determined by the ERGO-II algorithm), almost identical to what we found for HIs (7.7%). Although the TR gene repertoire was renewed in T2, the percentage of SARS-specific clonotypes remained essentially identical (median of 7.8%). Turning to the BcR IG gene repertoire, despite the absence of a serological response at T1, the percentage of the predicted SARS-specific BcR IG clonotypes (high-homology with SARS-specific CDR3 aa sequences published at CoV-AbDab, &amp;gt;0.9 identity score) did not differ between KTRs (0.72% at T1 and 0.65% at T2) vs HIs (0.63%), raising the intriguing possibility of B cell anergy in the former. Overtime assessment revealed that BcR IG clonotype diversity significantly increased at T2 vs T1 in KTRs (average Shannon diversity: KTR-T1=12006 vs KTR-T2=38576 p&amp;lt;0.05) and strongly correlated with the presence of SARS-specific BcR IG clonotypes (r=0.65, p&amp;lt;0.001). Nevertheless, the cumulative fraction of SARS-specific BcR IG clonotypes at KTR-T2 decreased (median cumulative frequency of SARS-specific clonotypes: KTR-T2=0.45% vs HI=0.67%, p&amp;lt;0.05). Of note, multiple immunizations resulted to SARS-specific BcR IG clonotypes with a significantly increased number of somatic hypermutations (SHM) compared to either HIs (p&amp;lt;0.0005) or T1 (p&amp;lt;0.05). In conclusion, our findings suggest that the lack of seroconversion at T1 in KTRs is potentially counteracted by cellular sensitization, likely mediated by SARS-specific T cells, that is sustained by booster vaccination. The latter also drives the accumulation of SHM in BcR IG clonotypes, suggesting distinct differentiation trajectories (germinal center-independent at T1 vs germinal center-dependent at T2) that could give rise to new B cell clones with increased neutralizing potency.

T Cell Dysfunction and Exhaustion in Patients with CLL: The Impact of Long Term Ibrutinib Treatment

Introduction: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with defects in the immune system and is dependent on the microenvironmental niche for survival. Ibrutinib, the first irreversible inhibitor of Bruton's tyrosine kinase targets B-cell receptor signaling to kill tumor cells. In addition, ibrutinib also appears to regulate the tumor microenvironment and T-cell immunity, but this mechanism unknown. Increased expression of inhibitory receptors, and functional defects such as reduced proliferative capacity and cytotoxicity are common in T cells from patients with CLL. The effect of ibrutinib on T-cell immunity has not been extensively studied in the setting of long term ibrutinib treatment. Therefore, we investigated the phenotype and function of CD4+ and CD8+ T-cells from patients with CLL treated with ibrutinib, treatment naïve CLL and age-matched healthy controls. Methods: CLL patient samples were obtained from treatment naïve patients at diagnosis, and at the 3- and 5-year timepoints. Samples from patients on continuous ibrutinib who had not relapsed at the 3- and 5-year timepoints; and pre-ibrutinib were obtained as well. Age matched healthy donor-derived peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll. PBMCs from CLL samples and healthy donors were labeled with CellTrace Violet and treated with anti-CD3 and anti-CD28 (ThermoFisher) for 7 days. T-cells were stimulated to identify proliferation capacity and to identify immune profiles. Results: Efficient T-cell activation and proliferation requires multiple signals such as T-cell receptor (TCR) stimulation, a costimulatory signal, and cytokine production. We analyzed the proliferation of T cells in-vitro upon stimulation with anti-CD3/CD28 beads using CellTrace Violet on PBMCs and found that this stimulation resulted in increased expansion of CD4+ T cells and CD8+ T cells in all the samples. We also found significant difference in the proliferation between untreated and treated CLL samples. Interestingly, proliferating T cells had different expression pattern of the exhaustion markers and inhibitory molecules such as TIM3, TIGIT, LAG3, CD160, KLRG1, CD244 and PD-1 at different phases of the disease. Increased expression of CD160, CD244, and PD1 are the key features of T-cell exhaustion. We found significant increase in the percentage of T cells from CLL patients expressing CD244, CD160 and PD1 compared to healthy donor controls but not between naïve and ibrutinib treated samples. Long term ibrutinib treatment in 3yr and 5yr samples does not change the exhaustion state in patients. We also found T-cells from ibrutinib treated samples express higher levels of CD57 which is known as a marker of senescence (Figure 1A). The phenotypes of untreated CLL samples are CCR7+CD45RA- central memory T-cells (35%) and CCR7+CD45RA+ naïve T cells (25%) (p&amp;lt;0.05) after stimulation in-vitro on day 3. In the same setting, ibrutinib treated samples had higher levels of effector memory T-cells (p&amp;lt;0.05) (80%) compared to untreated CLL samples (Figure 1B). These results suggest that the phenotype has changed to central memory phenotype in untreated samples. Functional T-cell exhaustion in CLL also leads to failure in cytokine production. Therefore, we measured different cytokine levels after stimulation in all the patient samples and in healthy controls. We found significant increase in the levels of inflammatory cytokines such as IFN-γ (p&amp;lt;0.05) and TNF-α (p&amp;lt;0.05) in long term ibrutinib treated samples compared to naïve CLL samples and healthy controls. Conclusion: Several studies have showed that T-cell counts are increased after treatment with ibrutinib in patients with CLL but not in extended therapy. Our findings suggest that long term ibrutinib treatment does increase T-cell number and proliferative capacity in CLL. The proliferating T cells express higher levels of exhaustion markers and inhibitory molecules in-vitro making them dysfunctional. However, these T-cells still maintained good proliferative potential, on par to healthy donors and better than untreated patients which can be applied in expanding improved T cell grafts for various types of adoptive T cell therapy, such as TCR gene therapy, and CAR therapy. Also better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells.

Unraveling Tumorigenicity in iPSC-Derived Immune Cells: The Impact of Chromosomal Abnormalities and Proliferative Intermediates

Response to tumor cells by the immune system can determine the course of a cancer. This guiding principle and the dynamic, adaptable nature of our immune system underlies the drive to generate immune cells for cancer immunotherapy. Induced pluripotent stem cells (iPSCs) provide an unlimited resource for the generation of immune cells such as T cells and dendritic cells (DCs) for cancer immunotherapy. However, the use of iPSC-derived immune cells in clinical applications has been limited due to concerns of safety. In vitro passage of iPSCs can introduce selective pressures that promote the accumulation of mutations that confer a growth advantage. Over several passages, this can lead to the acquisition of chromosomal abnormalities. Moreover, differentiation protocols of varying efficiency permit the presence of immature cells, contaminating the differentiated cell population. However, the impact of chromosomal abnormalities and intermediate products with proliferation potential on tumorigenicity of iPSC-derived immune cells remains unclear. Here, we investigated the tumorigenic potential of iPSC-derived T cells and DCs in immunocompetent and immunodeficient mice. Pmel-1 T-cell receptor (TCR) transgenic mouse T cells were reprogrammed using Sendai virus vectors carrying Oct3/4, Sox2, Klf4 and cMyc. While our iPSCs were phenotypically normal, even at early passage numbers, various chromosomal abnormalities were found in the resulting cell population. These abnormalities included trisomy 8 and 11 and loss of X chromosome with high frequency. Furthermore, within a population of iPSCs determined to be karyotypically normal by G-banding, unbalanced translocations were found by spectral karyotyping. These findings are consistent with those reported in the literature and highlight the prevalence of chromosomal abnormalities in iPSC populations. As iPSCs can harbor chromosomal abnormalities that are undetectable by standard methods, we assessed the tumorigenic potential of iPSC-derived cells, similar to what may be used in clinical applications. Mouse iPSCs were differentiated into CD8αβ T cells by co-culture with murine OP9 bone marrow stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) and were enriched by CD8 positive selection. Activated iPSC-derived T cells were adoptively transferred to immunocompetent C57BL/6 and immunodeficient NOD/SCID, IL-2 receptor γ chain knockout mice. Immunodeficient but not immunocompetent mice developed hepatic tumors. Reverse transcription (RT)-PCR identified Pme1-l TCR gene in tumor cells, confirming tumor origin. Moreover, histology of liver tumors is suggestive of lymphoma or lymphosarcoma. Within the population of CD8+ iPSC-derived cells, a small subpopulation co-expressing CD8 and SSEA-1 was present. Sorting the cells by SSEA-1 positivity eliminated tumor formation in vivo. Mouse iPSCs were also differentiated into DCs by co-culture with OP9-DL1 cells and granulocyte macrophage colony-stimulating factor. Despite similar use of iPSCs with chromosomal abnormalities and prolonged passaging in vitro, SSEA-1+ cells were not identified when they were differentiated to DCs, and iPSC-derived DCs did not form tumors in immunodeficient mice. Taken together, this study revealed the potential risk of tumorigenicity of iPSC-derived immune cells in our preclinical model. The presence of chromosomal abnormalities did not definitively determine risk of tumorigenesis upon transplantation. The relationship between SSEA-1 positivity and tumor formation suggests contaminating cells may carry tumorigenic characteristics. Further work is required to develop differentiation methods that generate pure populations, free of tumorigenic intermediates, to potentiate the clinical use of iPSC-derived immunotherapy.

Progressively transformed germinal center-like follicular T-cell lymphoma:a clinicopathological analysis of 14 cases

Objective: To investigate the clinicopathologic features of progressively transformed germinal center-like follicular T-cell lymphoma (PTGC-like FTCL). Methods: The clinicopathologic data of 14 PTGC-like FTCL cases that were diagnosed at the Beijing Friendship Hospital Affiliated to the Capital Medical University from January 2017 to January 2022 were retrospectively collected. Clinicopathological features, immunophenotype, and Epstein-Barr virus (EBV) infection status were analyzed in these cases. Polymerase chain reaction (PCR) was performed to detect the clonal gene rearrangements of T cell receptor (TCR) and the immunoglobulin (Ig) in 10 and 8 cases, respectively. Results: The male to female ratio was 5∶2. The median age was 61 years (range 32-70 years). All patients had lymphadenopathy at the time of diagnosis. By using the Ann Arbor system staging, seven cases were classified as stage Ⅰ-Ⅱ, and seven cases as stage Ⅲ-Ⅳ. Seven cases had B symptoms, four cases had splenomegaly, and two cases had skin rash and pruritus. Previously, three cases were diagnosed as classic Hodgkin's lymphoma, three cases as small B-cell lymphoma, two cases as atypical lymphoid hyperplasia unable to exclude angioimmunoblastic T-cell lymphoma (AITL), one case as EBV-associated lymphoproliferative disorder, and one case as peripheral T-cell lymphoma (PTCL) associated with the proliferation of B cells. All the 14 cases showed that the large nodules were composed of mature CD20+, IgD+B lymphocytes admixed with small aggregates of neoplastic cells with pale to clear cytoplasm. Moreover, hyperplastic germinal centers (GCs) and Hodgkin/Reed-Sternberg-like (HRS-like) cells were seen within these nodules in two and five cases, respectively. The neoplastic cells expressed CD3 (14/14), CD4 (14/14), PD1 (14/14), ICOS (14/14), CD10 (9/14), bcl-6 (12/14), CXCL13 (10/14), and CD30 (10/14). The HRS-like cells in five cases expressed CD20 (2/5), PAX5 (5/5), CD30 (5/5), CD15 (2/5), LCA (0/5), OCT2 (5/5) and BOB1 (2/5). Moreover, neoplastic T cells formed rosettes around HRS-like cells. EBV-encoded RNA (EBER) in situ hybridization showed scattered, small, positive bystander B lymphocytes in 8/14 cases, including 3/5 cases containing HRS-like cells. All tested cases (including five with HRS-like cells) showed monoclonal TCR gene rearrangement and polyclonal Ig gene rearrangement. Conclusions: PTGC-like FTCL is a rare tumor originated from T-follicular helper cells. It could be distinguished from angioimmunoblastic T-cell lymphoma by the formation of follicular structure, and lack of follicular dendritic cell proliferation outside the follicles and the polymorphous inflammatory background. In addition, it should be differentiated from lymphocyte-rich classical Hodgkin's lymphoma and low-grade B cell lymphoma.

Colonic mucosal associated invariant T cells in Crohn's disease have a diverse and non-public T cell receptor beta chain repertoire.

Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn's disease (CD). The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+ T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood. MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin α4β7 in the colon but not in the blood, where they were enriched for α4β7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts. MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin α4β7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.

CAR-NK Specific Targeting of Clonal Tcrvβ Chain to Treat T-Cell Non-Hodgkin's Lymphoma

Background T-cell Non-Hodgkin Lymphoma (T-NHL) is a diverse group of aggressive malignancies which are minimally researched and have poorer patient outcomes than those with B-cell NHL (Stuver et. al., 2022, Foss et. al., 2011). Therapies are characterized by pan T cell cytotoxicity, poor response rates and the requirement for multiple levels of treatments resulting in toxicity and suppression of the immune system that can lead to life threatening infections (Bellei et. Al., 2018). Clinical trials are considered a first line option for advanced disease, speaking to the lack of viable treatment options. One possible target for T cells is the T-cell receptor (TCR). TCRs possess tremendous diversity owing to diverse variable β chain (Vβ) (n=48) and α chain (Vα) (n=46) gene segments, that recombine with D and J gene segments to form a functional TCR. T Multiplexed PCR of the TCR gene has been used to identify the presence of a distinct malignant clone and facilitate diagnosis of T-NHL (Savage et. al., 2008). Thus, the Vβ chains represent a therapeutic target that discriminates the clonally expanded malignant T cells from the vast majority of healthy T-cells. Lucero et. al. (2023), showed specific targeting of T cell receptor Vβ chains, for T-NHL patients with an expanded TCRVβ clone using a naked antibody approach. Similar modalities have used BiTE and CAR-T. One downside of the naked antibody and BiTE approach (Paul et. Al., 2021) is that they require patients to have functional effector NK and CD8 T cells, and these cells are often not present in advanced T-NHL diseases. The expanded Vβ chain could also be targeted by CAR-T cell therapy; however, this approach is hampered by concerns of toxicity and poor autologous T cell quality due to the patients' disease (Marofi et. al., 2021). Therefore, we propose CAR-NK cell therapy utilizing the TCRVβ chain (Fig 1A) for patients with advanced T-NHL against the clonally expanded TCRVβ which will allow for an off the shelf approach that bypasses the need for autologous effector cells. Methods To show proof of concept, TCRVβ13 CAR-NK cells were manufactured from an NK-92 cell line, HaNK, which expresses a high affinity CD16 V158 FcγRIIIa receptor (Jochems, et. al., 2016). CAR transduction was confirmed via flow cytometry. Along with the CAR, NK/T-cell lymphoma cell lines, EL4 (mouse) and Jurkat (human), were transduced through a lentiviral vector containing a plasmid to express our Vβ of interest, Vβ13 (EL4-V13 and Jurkat-V13, respectively) (Lucero, 2023). We performed In vitro cytotoxicity co-culture assays on NK/T-cell lymphoma cell lines, EL4-V13 and Jurkat-V13, to confirm functional activity in CAR-HaNK cells compared to wild type HaNK cells at three different effector to target ratios. From the in vitro cytotoxicity assay, assays on supernatants from each of the co-cultures for changes of the cytokines INF-gamma and TNF-alpha levels to assess functional NK activity are in progress. Results The in vitro co-culture cytotoxicity assays showed increased cytotoxicity of the TCRVβ CAR-HaNK cells in comparison to HaNK cells in the presence of the TCRVβ for both EL-4-V13 and Jurkat-V13 cell lines. Targeting of the EL-4 cell line without the transduced TCRVβ showed similar percent cytotoxicity in CAR-HaNK cells and HaNK cells, while EL-4-V13 cell line showed significant increase in cytotoxicity when co-cultured with CAR-NK cells in comparison to the HaNK cells. In the 1:1 co-culture of either CAR-HaNK or HaNK cells with EL-4-V13 there was a 27% increase in cytotoxicity in the CAR-NK in comparison to the HaNK cells (P&amp;lt;0.0001), the 5:1 showed 71% increase in CAR-NK (P&amp;lt;0.0001), and the 10:1 showed an 80.5% increase in CAR-HaNK (P&amp;lt;0.0001) (Fig 1B). Similarly, in the human cell line Jurkat-V13, co-cultures with CAR-HaNK cells showed an increase in cytotoxicity in the 1:1 of 30% (P=0.0169), the 5:1 of 37% (P=0.0074), and 10:1 of 57.7% (P&amp;lt;0.0001) when comparing to HaNK cells (Fig 1B). Conclusions The goal of these preclinical studies is to leverage advances in CAR development with the efficacy of targeting TCRVβ as a direct and safe therapeutic option for patients with T-NHL. If successful, this will propel both cell-directed therapies and a novel target to a disease characterized by few efficacious, durable and safe therapies.

Mycosis fungoides palmaris and plantaris.

Mycosis fungoides (MF) palmaris and plantaris is a rare form of MF. Only few cases are reported in the literature. Different forms are described: eczematous lesions, dyshidrosis lesions, verrucous lesions, dry pulpitis, ulcerated lesions, pustulosis, and hyperkeratotic lesions. Histology is typical for MF with a positive T-cell receptor gene rearrangement in majority of cases. Prognosis is good. Resistance to topical steroids is common, and classical treatment consist of chlormethine gel and radiotherapy.