Cell Repertoire Research Articles

The Evolving Landscape in Multiple Myeloma: From Risk Stratification to T Cell-Directed Advanced Therapies

Multiple myeloma is biologically and clinically a complex and heterogeneous disease which develops late in life, with the median age at the time of initial diagnosis being 66 years. In 1975, Durie and Salmon developed the first broadly adopted staging system in multiple myeloma, and in the ensuing decades, the risk stratification tools have improved and now incorporate different parameters to better predict the prognosis and to guide the treatment decisions. The International Staging System (ISS) was initially developed in 2005, revised in 2015 (R-ISS), and again in 2022 (R2-ISS). Tremendous progress has been achieved in multiple myeloma therapy over the past 25 years with the approval of immunomodulatory drugs, proteasome inhibitors, and anti-CD38 monoclonal antibodies, resulting in a major paradigm shift. The dysfunction of the innate and adaptive immune system, especially in the T cell repertoire, represents a hallmark of multiple myeloma evolution over time, supporting the need for additional therapeutic approaches to activate the host’s immune system and to overcome the immunosuppressive tumor microenvironment. Novel T cell-directed therapies include chimeric antigen receptor (CAR) T cell therapies and bispecific antibodies that leverage the immune system’s T cells to recognize and attack the tumor cells. Second-generation anti-BCMA CAR T cell therapies and bispecific antibodies that bind the tumor antigen BCMA or GPRC5D onto myeloma cells and CD3 on the T cell’s surface are currently available for the treatment of relapsed/refractory multiple myeloma. Despite impressive results obtained with currently approved treatments, multiple myeloma remains incurable, and almost all patients eventually relapse. Moreover, patients with extramedullary disease and plasma cell leukemia represent an unmet medical need that require additional strategies to improve the outcome. In this review, we provide an overview of the evolution of risk stratification and the treatment of multiple myeloma.

The Importance of Monitoring Antigen-Specific Memory B Cells, and How ImmunoSpot Assays Are Suitable for This Task

Determining an individual’s humoral immune reactivity to a pathogen, autoantigen, or environmental agent is traditionally accomplished through the assessment of specific antibody levels in blood. However, in many instances, titers of specific antibodies decline over time and thus do not faithfully reveal prior antigen exposure or establishment of immunological memory. To estimate an individual’s humoral immune competence, it is therefore necessary to assess functional B cell memory. Here, we describe novel B cell ELISPOT and FluoroSpot assays (collectively referred to as ImmunoSpot) that can be rapidly developed and validated to characterize the memory B cell (Bmem) repertoire specific for any desired antigen ex vivo and at single-cell resolution. Moreover, multiplexed variants of the B cell FluoroSpot assay enable high-throughput testing of antigen-specific B cells secreting distinct antibody classes and/or IgG subclasses, with minimal cell material requirements. B cell ImmunoSpot assays also enable measurement of affinity distributions within the antigen-specific Bmem compartment and permit cross-reactivity measurements that can provide insights into Bmem established against future pathogen variants. Collectively, the ImmunoSpot® system presented here is highly reproducible, and can be readily validated for regulated tests. The newly gained ability to monitor the antigen-specific Bmem compartment should catalyze a more comprehensive understanding of humoral immunity in health and disease.

Complete primer set for amplification and expression of full-length recombinant human monoclonal antibodies from single human B cells.

Human monoclonal antibodies (mAbs) are an important segment in precision therapeutics. Various methodologies are available for generating them. Recombinant human mAbs expression from sorted single B cells is preferred for its rapid expression using mammalian vectors while maintaining in vivo immunoglobulin (Ig) pairing. The success rate of generating recombinant mAbs from single sorted human B cells directly relies on Ig heavy (IgH) and light (IgL) gene coverage of the PCR primers. Existing primer sets fail to cover all functional human Ig gene rearrangements, exhibit high degeneracy leading to non-specific amplifications and mutations arising from primer mismatch/degeneracy, and require high amplification cycles. Some existing primer sets have high coverage but are not designed for expression as recombinant mAbs. Here, we have designed a primer set to amplify all functional V(D)J transcripts in human B cell repertoire using a nested RT-PCR approach. The resultant amplicons can be cloned into mammalian vectors for expression of recombinant mAb. Non-specific amplifications were minimized using isotype-specific primers for cDNA synthesis and limiting primer degeneracy. We validated the designed primers on single sorted B cells, bulk sorted B cells and peripheral blood mononuclear cells. We were successfully able to amplify paired heavy and light chain transcripts in 38.46 % (80/208) from naive, memory and B1 B cell subsets sorted as single B cells. Paired Ig transcripts from five single B cells were cloned into expression vectors and purified from mammalian cells as recombinant mAbs. Thus, our new primer set offers significant advantages over existing primers as it allows amplification of all functional V(D)J rearrangements, facilitating rapid generation of antigen-specific recombinant antibodies from diverse human B cell repertoires following vaccinations and infections previously inaccessible due to primer limitations.

Clone‐Resolved Chemical Depletion of T cells via Cellular Proximity Chemistry

T cells play a pivotal role in the development of autoimmune diseases. To mitigate autoimmune inflammation without inducing global immunosuppression, it is crucial to selectively eliminate autoreactive T cell clones while preserving the normal T cell repertoire. In this study, we applied cellular proximity chemistry to develop a T‐cell depletion method with clonal precision. Using engineered dendritic cells (DCs) with surface‐bound photosensitizers, we generated reactive oxygen species (ROS) at immune synapses, leading to the targeted death of antigen‐specific T cells in close proximity. This process induces lipid oxidation in T cell membranes, triggering ferroptosis‐like cell death. The method enables the selective elimination of specific T cell clones without affecting others, in which the clonal resolution was demonstrated by TCR sequencing. Finally, we demonstrated the efficacy of this approach in a type 1 diabetes model by selectively depleting the pathogenic 8.3 T cell clone, thereby protecting islet β cells and preserving overall T cell function. This strategy offers a promising avenue for immunosuppressive therapy that targets pathogenic T cells while maintaining overall immune integrity.

Clone-Resolved Chemical Depletion of T cells via Cellular Proximity Chemistry.

T cells play a pivotal role in the development of autoimmune diseases. To mitigate autoimmune inflammation without inducing global immunosuppression, it is crucial to selectively eliminate autoreactive T cell clones while preserving the normal T cell repertoire. In this study, we applied cellular proximity chemistry to develop a T-cell depletion method with clonal precision. Using engineered dendritic cells (DCs) with surface-bound photosensitizers, we generated reactive oxygen species (ROS) at immune synapses, leading to the targeted death of antigen-specific T cells in close proximity. This process induces lipid oxidation in T cell membranes, triggering ferroptosis-like cell death. The method enables the selective elimination of specific T cell clones without affecting others, in which the clonal resolution was demonstrated by TCR sequencing. Finally, we demonstrated the efficacy of this approach in a type 1 diabetes model by selectively depleting the pathogenic 8.3 T cell clone, thereby protecting islet β cells and preserving overall T cell function. This strategy offers a promising avenue for immunosuppressive therapy that targets pathogenic T cells while maintaining overall immune integrity.

Abstract P004: Mapping the tumor-sentinel node immune migratome demonstrates a key role for CCR7+ dendritic cells in the successful response to immunoradiotherapy

Abstract Immune checkpoint inhibition has demonstrated no benefit when combined with chemoradiotherapy for locally advanced HNSCC, raising the possibility that standard therapies compromise the response to immunotherapy. To address this hypothesis, we previously demonstrated that ablating tumor draining lymphatics abolishes the response to immune-oncology therapy. Specifically, we defined that the response to immunotherapy is coordinated by interferon type-I signaling through the cDC1 population in tumor-draining lymph nodes. These findings support the premise that successful tumor responses to immuno-oncology therapies is predicated upon intact and functional, locoregional anatomy. Accordingly, we hypothesize that lymphatic-preserving, tumor-directed immune oncology therapy will promote antitumor immunity by enhancing surveillance along the tumor-immune-lymphatic axis. To explore this, we employ a novel mouse reporter model that allows precise spatiotemporal labeling of immune effector cells. Combined with sentinel lymph node mapping, our reporter model enables selective labeling of immune effectors within the tumor microenvironment and subsequent tracking and characterization of these cells as they migrate to the sentinel lymph node. This approach led us to define the “cancer immune migratome” - the repertoire of immune cells actively migrating from tumors to lymph nodes during an antitumor response. For precise cellular identification, we employed CITE-sequencing, which integrates single-cell RNA expression with surface protein markers, allowing us to comprehensively define the characteristics of migrating immune cells. This work represents the first characterization of immune cell populations migrating from the tumor to the sentinel lymph node, revealing the diversity and dynamics of these populations in antitumor immunity. We then develop a tumor-directed, lymphatic-sparing radiotherapy model to modulate the tumor immune microenvironment without inducing tumor regression. Sequential administration of tumor-directed radiotherapy followed by PD-1 checkpoint inhibition achieves complete and durable responses. Analysis of the cancer immune migratome during immunoradiotherapy revealed a crucial role for an activated population of CCR7+ dendritic cells. These migratory dendritic cells are reprogrammed, via rational sequencing of radiation and immunotherapy, from their canonical role in immune tolerance to actively drive antitumor immunity. This reprogramming enhances T cell priming, clonotypic expansion, and, ultimately, the successful tumor response to immunoradiotherapy. Disruption of sentinel lymphatic channels or selective blockade of CCR7+ DC entry into the SLN abrogates the response to immunoradiotherapy. Overall, this work supports rationally sequencing immune-sensitizing, lymphatic-preserving, tumor-directed radiotherapy followed by immune checkpoint inhibition to optimize tumor response. Our findings support a therapeutic strategy that enhances host response to immunotherapy by directing activated dendritic cells to sentinel lymph nodes. Citation Format: Robert Saddawi-Konefka, Riyam Al Msari, Shiqi Tang, Lauren M. Clubb, Santiago Fassardi, Riley Jones, Farhoud Faraji, Shiruyeh Schokrpur, Bryan S. Yung, Michael M. Allevato, Shawn M. Jensen, Bernard A. Fox, Richard Bell, J. Silvio Gutkind, Andrew Sharabi, Joseph A. Califano. Mapping the tumor-sentinel node immune migratome demonstrates a key role for CCR7+ dendritic cells in the successful response to immunoradiotherapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P004

Human naïve B cells recognize prepandemic influenza virus hemagglutinins.

Understanding the naïve B cell repertoire and its specificity for potential zoonotic threats, such as the highly pathogenic avian influenza (HPAI) H5Nx viruses, may allow prediction of infection- or vaccine-specific responses. However, this naïve repertoire and the possibility to respond to emerging, prepandemic viruses are largely undetermined. Here, we profiled naïve B cell reactivity against a prototypical HPAI H5 hemagglutinin (HA), the major target of antibody responses. We found that the frequency of H5-specific human naïve B cells targeting the HA "head" domain was increased relative to cross-reactive B cells to a circulating seasonal H1N1 strain. We classified the isolated monoclonal antibodies (mAbs) by the HA epitopes engaged and found that selected mAbs neutralized H5N1 at germline. We determined a cryo-electron microscopic structure of one mAb in complex with H5 HA to define its epitope. Our study defines the naïve human B cell repertoire recognizing a potentially zoonotic HPAI.

P0125 Crohn’s-associated invariant T Cells are associated with disease severity and location and are not affected by medication intake

Abstract Background Multiple alterations in the immune repertoire of IBD patients have been identified including, an expansion of a subset of type II natural killer T cells in CD patients, termed Crohn’s-associated invariant T cells (CAIT)1. Previously, we showed that CAIT cells respond to small molecules that resemble multiple microbial and drug metabolites2. This begs the question of whether CAIT cells are expanded due to the ongoing disease or the medications administrated to control the disease. Methods We profiled the T cell receptor alpha chain repertoire of 246 symptomatic controls, 228 treatment-naïve CD and 357 UC patients in addition to 176 and 329 treated CD and UC patients, respectively, from the Norwegian inception cohort IBSEN III3. Results Given that our cohort contained adult and paediatric IBD patients, we initially analysed the expansion of CAIT cells independently. Among adults, CAIT cells were significantly expanded in treatment-naïve CD patients (Fig. 1A) with a similar trend in paediatric patients (Fig. 1B) which arguably due to the small sample size was not significant. By combing the two groups, we observed a higher expansion of CAIT cells in treatment-naïve CD patients (Fig. 1C). The expansion of CAIT cells was also higher in treated-CD patients relative to controls and treated UC patients (Fig. 1D). By comparing the T cell repertoire of the UC and CD patients before and after treatment, we did not observe any significant difference in the expansion of CAIT cells due to medications (Fig. 1E, 1F). CAIT cells were significantly expanded in ileocolonic and ileal CD patients relative to colonic CD patients and controls (Fig. 2A). This effect persisted in treated patients (Fig. 2B), additionally, the expansion of CAIT cells did not differ significantly in the same patient before treatment and after treatment (Fig. 2C). The expansion also correlated with the disease-behavior4, with patients suffering from a stricturing CD showing higher levels of CAIT expansion relative to controls and CD patients with non-stricturing, non-penetrating (NSNP) disease (Fig. 2D). After treatment, patients with a stricturing disease had a higher CAIT expansion relative to controls and also to NSNP patients (Fig. 2E). By comparing the expansion of CAIT cells in CD patients with NSNP disease before and after treatment, we did not observe any significant difference (Fig. 2F). Conclusion Our results indicate that CAIT cells are not induced by treatment and that disease severity and location strongly correlates with their expansion, future efforts shall focus on identifying the spatial distribution of CAIT cells in the gut tissues as well as identifying potential antigens driving their expansion.

Glycan-reactive antibodies isolated from human HIV-1 vaccine trial participants show broad pathogen cross-reactivity.

HIV-1 continues to pose a significant global health challenge, requiring ongoing research into effective prevention and treatment strategies. Understanding the B cell repertoire that can be engaged upon vaccination in humans is crucial for the development of future preventive vaccines. In this study, PBMCs from HIV-negative participants in the multivalent HVTN124 human HIV-1 vaccine clinical trial were interrogated for HIV-reactive B cells using LIBRA-seq, a high-throughput B cell mapping technology. We report the discovery of glycan-reactive antibodies capable of neutralizing diverse heterologous HIV-1 virus strains. Further, isolated antibodies showed broad cross-reactivity against antigens from a variety of other pathogens, while remaining mostly negative on autoreactivity assays. The emerging class of glycan- reactive virus-neutralizing antibodies with exceptional breadth of pathogen cross- reactivity may present an effective target for vaccination at the population level.

Identification of novel KRASG12D neoantigen specific TCRs and a strategy to eliminate off-target recognition

BackgroundT cell receptor (TCR)–engineered T cells targeting neoantigens originated from mutations in KRAS gene have demonstrated promising outcomes in clinical trials against solid tumors. However, the challenge lies in developing tumor-specific TCRs that avoid cross-reactivity with self-antigens to minimize the possibility of severe clinical toxicities. Current research efforts have been put towards strategies to eliminate TCR off-target recognition.MethodsNaive T cell repertoire was used for screening KRASG12D-reactive TCRs. Specific TCRs were subsequently identified and their functionality was assessed using TCR Jurkat cells and TCR T cells. Peptide specificity was evaluated using the X-scan assay. To enhance TCR specificity for KRASG12D and reduce their reactivity to self-peptide SMC1A29-38, mammalian TCR display libraries were employed for the design of modification in the complementarity-determining region (CDR).ResultsHLA-A*11:01-restricted TCRs targeting the KRASG12D epitope were isolated, and TCR1 was characterized with superior functional avidity and specificity. Alongside a robust recognition of endogenous KRASG12D epitope, this TCR displayed cross-reactivity with the SMC1A29-38 epitope. With an approach utilizing structural-guided mutations in the CDR-1A region of TCR1, we obtained an engineered TCR variant (TCR1a7). Functional characterization of TCR1a7 showed that this TCR not only exhibited enhanced specificity towards KRASG12D, but also demonstrated successful elimination of the off-target recognition of SMC1A29-38.ConclusionsTCRs targeting the KRASG12D peptide could be isolated from naive T cell repertoires. Integrating the TCR-peptide-HLA complex structure with a mammalian TCR library system could serve as a functional strategy to reduce potential TCR cross-reactivity with self-antigens, such as SMC1A29-38. Our findings evidenced an operable method to enhance TCR peptide specificity, while maintaining advanced functional avidity and potent anti-tumor activity.

Combining T cell receptor sequencing and transcriptomics to characterize tissue-resident T cells from human gut biopsies.

Gastrointestinal T cells (GI-T) play a critical role in mucosal immunity, balancing tolerance and pathogen defence. T cells recognize antigens via T cell receptors (TCRs). Next-generation sequencing (NGS) is utilized to analyse TCR repertoires in contexts such as health, haematological diseases, autoimmunity, and inflammation. While some studies have explored T cell involvement in GI conditions, the integration of different techniques and examination of diverse tissues remain underdeveloped. In our "proof of concept" study, for the first time, we combined flow cytometry, TCR sequencing and transcriptomics to analyse T cell repertoires from bulk sorted T cells and from single cells. This combination provides information about both, specificity and functionality of particular T cells. We focused on biopsy samples from the stomach, colon, and compared these to blood samples from patients with and without inflammation associated with erosive gastritis. This combined approach allows unique insights into T cell biology. Through TCR clonotype analysis, we identified oligoclonal expansion in inflamed biopsies, with minimal TCR clonotype overlap between individuals, highlighting personalized immune responses. Gene expression analysis revealed upregulation of T cell activation and signalling and chemokines in inflamed biopsies. Single-cell sequencing provided deeper insights into specific T cell populations, identifying dominant clonotypes with cytotoxic function. Our findings underscore the importance of studying affected sites to fully understand T cell responses and localized immune reactions. Our approach opens unique possibilities for studying TCR and gene expression from limited biopsy material, potentially leading to personalized therapies and biomarkers for gastrointestinal diseases.

Clonal analysis of SepSecS-specific B and T cells in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a rare chronic inflammatory liver disease characterized by the presence of autoantibodies, including those targeting O-phosphoseryl-tRNA:selenocysteine-tRNA synthase (SepSecS), also known as soluble liver antigen (SLA). Anti-SepSecS antibodies have been associated with a more severe phenotype, suggesting a key role for the SepSecS autoantigen in AIH. To analyze the immune response to SepSecS in patients with AIH at the clonal level, we combined sensitive high-throughput screening assays with the isolation of monoclonal antibodies (mAbs) and T cell clones. The anti-SepSecS mAbs isolated were primarily IgG1, affinity-matured compared with their germline versions, and recognized at least 3 nonoverlapping epitopes. SepSecS-specific CD4+ T cell clones were found in patients with AIH who were anti-SLA-positive and anti-SLA-negative,and, to a lesser extent, in patients with non-AIH liver diseases and in healthy individuals. SepSecS-specific T cell clones from patients with AIH produced IFN-γ, IL-4, and IL-10, targeted multiple SepSecS epitopes, and, in one patient, were clonally expanded in both blood and liver biopsy. Finally, SepSecS-specific B cell clones, but not those of unrelated specificities, were able to present soluble SepSecS to specific T cells. Collectively, our study provides the first detailed analysis of B and T cell repertoires targeting SepSecS in patients with AIH, offering a rationale for improved targeted therapies.

GZMK-expressing CD8+ T cells promote recurrent airway inflammatory diseases.

Inflammatory diseases are often chronic and recurrent, and current treatments do not typically remove underlying disease drivers1. T cells participate in a wide range of inflammatory diseases such as psoriasis2, Crohn's disease3, oesophagitis4 and multiple sclerosis5,6, and clonally expanded antigen-specific T cells may contribute to disease chronicity and recurrence, in part by forming persistent pathogenic memory. Chronic rhinosinusitis and asthma are inflammatory airway diseases that often present as comorbidities7. Chronic rhinosinusitis affects more than 10% of the general population8. Among these patients, 20-25% would develop nasal polyps, which often require repeated surgical resections owing to a high incidence of recurrence9. Whereas abundant T cells infiltrate the nasal polyps tissue10,11, T cell subsets that drive the disease pathology and promote recurrence are not fully understood. By comparing T cell repertoires in nasal polyp tissues obtained from consecutive surgeries, here we report that persistent CD8+ T cell clones carrying effector memory-like features colonize the mucosal tissue during disease recurrence, and these cells characteristically express the tryptase Granzyme K (GZMK). We find that GZMK cleaves many complement components, including C2, C3, C4 and C5, that collectively contribute to the activation of the complement cascade. GZMK-expressing CD8+ T cells participate in organized tertiary lymphoid structures, and tissue GZMK levels predict the disease severity and comorbidities better than well-established biomarkers such as eosinophilia and tissue interleukin-5. Using a mouse asthma model, we further show that GZMK-expressing CD8+ T cells exacerbate the disease in a manner dependent on the proteolytic activity of GZMK and complements. Genetic ablation or pharmacological inhibition of GZMK after the disease onset markedly alleviates tissue pathology and restores lung function. Our work identifies a pathogenic CD8+ memory T cell subset that promotes tissue inflammation and recurrent airway diseases by the effector molecule GZMK and suggests GZMK as a potential therapeutic target.

Skewed HBV-specific CD8+T cell repertoire in chronic versus acute-resolving HBV infection

Skewed HBV-specific CD8+T cell repertoire in chronic versus acute-resolving HBV infection

Immunoregulatory programs in anti-N-methyl-D-aspartate receptor encephalitis identified by single-cell multi-omics analysis.

Anti-N-methyl-D-aspartate receptor encephalitis (anti-NMDARE) is a prevalent type of autoimmune encephalitis caused by antibodies targeting the NMDAR's GluN1 subunit. While significant progress has been made in elucidating the pathophysiology of autoimmune diseases, the immunological mechanisms underlying anti-NMDARE remain elusive. This study aimed to characterize immune cell interactionsand dysregulation in anti-NMDARE by leveraging single-cell multi-omics sequencing technologies. Peripheral blood mononuclear cells (PBMCs) from patients in the acute phase of anti-NMDARE and healthy controls were sequenced using single-cell joint profiling of transcriptome and chromatin accessibility. Differential gene expression analysis, transcription factor activity profiling, and cell-cell communication modeling were performed to elucidate the immune mechanisms underlying the disease. In parallel, single-cell B cell receptor sequencing (scBCR-seq) and repertoire analysis were conducted to assess antigen-driven clonal expansion within the B cell population. The study revealed a significant clonal expansion of B cells, particularly plasma cells, in anti-NMDARE patients. The novel finding of type I interferon (IFN-I) pathway activation suggests a regulatory mechanism that may drive this expansion and enhance antibody secretion. Additionally, activation of Toll-like receptor 2 (TLR2) in myeloid cells was noted, which may connect to tumor necrosis factor-alpha (TNF-α) secretion. This cytokine may contribute to the activation of B and T cells, thereby perpetuating immune dysregulation. This study presents a comprehensive single-cell multi-omics characterization of immune dysregulation in anti-NMDARE, highlighting the expansion of B cell and the activation of the IFN-I and TLR2 pathways. These findings provide deeper insights into the molecular mechanism driving the pathogenesis of anti-NMDARE and offer promising targets for future therapeutic intervention. Significant B cell clonal expansion, particularly in plasma cells, driven by antigen recognition. IFN-I pathway activation in plasma cells boosts their antibody production and potentially exacerbates immune dysregulation. TLR2 pathway activation in myeloid cells contributes to TNF-α secretion and could influence adaptive immune responses.

CAR-based cell therapies for systemic lupus erythematosus.

The remarkable efficacy of chimeric antigen receptor (CAR) T cell therapy in hematological malignancies has provided a solid basis for the therapeutic concept, wherein specific pathogenic cell populations can be eradicated by means of targeted recognition. During the past few years, CAR-based cell therapies have been extensively investigated in preclinical and clinical research across various non-tumor diseases, with particular emphasis in the treatment of autoimmune diseases (ADs), yielding significant advancements. The recent deployment of CD19-directed CAR T cells has induced long-lasting, drug-free remission in patients with systemic lupus erythematosus (SLE) and other systemic AD, alongside a more profound immune reconstruction of B cell repertoire compared with conventional immunosuppressive agents and B cell-targeting biologics. Despite the initial success achieved by CAR T cell therapy, it is critical to acknowledge the divergences in its application between cancer and AD. Through examining recent clinical studies and ongoing research, we highlight the transformative potential of this therapeutic approach in the treatment of SLE, while also addressing the challenges and future directions necessary to enhance the long-term efficacy and safety of CAR-based cell therapies in clinical practice.

Blood immune profiling of Ethiopian patients with breast cancer highlights different forms of immune escape

ABSTRACT Breast cancer (BC) is a leading cause of death worldwide, particularly also among African woman. In order to better stratify patients for the most effective (immuno-) therapy, an in depth characterization of the immune status of BC patients is required. In this study, a cohort of 65 Ethiopian patients with primary BC underwent immune profiling by multicolor flow cytometry on peripheral blood samples collected prior to surgery and to any other therapy. Comparison with peripheral blood samples from healthy donors highlighted a general activation of the immune system, accompanied by the presence of exhausted CD4+ T cells and senescent CD8+ T cells with an inverted CD4/CD8 ratio in approximately 50% of BC cases. Enhanced frequencies of γδ T cells, myeloid-derived suppressor cells and regulatory T cells were also found. Correlation with clinical parameters demonstrated a progressive reduction in T cell frequencies with increasing histopathological grading of the tumor. Differences in CD8+ T cells and B cells were also noted among luminal and non-luminal BC subtypes. In conclusion, Ethiopian BC patients showed several alterations in the composition and activation status of the blood immune cell repertoire, which were phenotypically associated with immune suppression. The role of these immunological changes in the clinical outcome of patients with BC will have to be determined in follow-up studies and confirmed in additional patients’ cohorts.

Reduced thymic IL-4 impairs negative T cell selection in nonobese diabetic mice.

Type 1 diabetes (T1D) develops spontaneously despite functional antigen presentation machinery in the thymus and a perceptible central tolerance process. We found that intrathymic enrichment with IL-4 fine tunes signaling through the IL-4/IL-13 heteroreceptor (HR) in early thymic progenitors (ETPs), augments negative selection of self-reactive T cells, sustains a diverse T cell repertoire devoid of clones expressing disease-associated T cell receptor (TCR) genes, and protects the nonobese diabetic (NOD) mouse from T1D. Indeed, optimal IL-4 activates STAT transcription factors to program ETP fate decision toward CD11c+CD8α+ dendritic cells (DCs) agile in negative T cell selection and clonal deletion of diabetogenic T cells. However, due to diminished invariant natural killer T (iNKT) 2 cell frequency in the NOD thymus, IL-4 is as suboptimal level, metering STAT activation to program ETP fate decision toward the T cell lineage leading to diminished negative selection, a clonally restricted TCR repertoire, and manifestation of spontaneous T1D. These insights uncover yet another interplay by which IL-4 affects T1D.

T cell receptor-centric perspective to multimodal single-cell data analysis.

The T cell receptor (TCR), despite its importance, is underutilized in single-cell analysis, with gene expression features solely driving current strategies. Here, we argue for a TCR-first approach, more suited toward T cell repertoires. To this end, we curated a large T cell atlas from 12 prominent human studies, containing in total 500,000 T cells spanning multiple diseases, including melanoma, head and neck cancer, blood cancer, and lung transplantation. Here, we identified severe limitations in cell-type annotation using unsupervised approaches and propose a more robust standard using a semi-supervised method or the TCR arrangement. We showcase the utility of a TCR-first approach through application of the STEGO.R tool for the identification of treatment-related dynamics and previously unknown public T cell clusters with potential antigen-specific properties. Thus, the paradigm shift to a TCR-first can highlight overlooked key T cell features that have the potential for improvements in immunotherapy and diagnostics.

PD-1 and CD73 on naive CD4+ T cells synergistically limit responses to self.

Vaccination with self- and foreign peptides induces weak and strong expansion of antigen-specific CD4+ T cells, respectively, but the mechanism is not known. In the present study, we used computational analysis of the entire mouse major histocompatibility complex class II peptidome to test how much of the naive CD4+ T cell repertoire specific for self-antigens was shaped by negative selection in the thymus and found that negative selection only partially explained the difference between responses to self and foreign. In naive uninfected and unimmunized mice, we identified higher expression of programmed cell death protein 1 (PD-1) and CD73 mRNA and protein on self-specific CD4+ T cells compared with foreign-specific CD4+ T cells. Pharmacological or genetic blockade of PD-1 and CD73 significantly increased the vaccine-induced expansion of self-specific CD4+ T cells and their transcriptomes were similar to those of foreign-specific CD4+ T cells. We concluded that PD-1 and CD73 synergistically limited CD4+ T cell responses to self. These observations have implications for the development of tolerogenic vaccines and cancer immunotherapy.