Multiple myeloma (MM) is associated with skewed T cell activation and function which is present in asymptomatic myeloma precursor conditions, but underlying mechanisms of progression remain undefined. Here, we assemble a large single-cell RNA sequencing dataset of the bone marrow and blood from patients with MM, precursor conditions, and non-cancer controls. We demonstrate that, unlike solid cancers, MM is not characterized by T cell exhaustion, but by antigen-driven terminal memory differentiation. This is influenced by tumour-intrinsic features including tumour burden and expression of antigen-presentation genes. Expanded TCR clones accumulating in MM are not enriched with viral specificities but accumulate in effector states in highly-infiltrated marrows. Additionally, we identify a role for T cell dynamics in patients treated with autologous stem cell transplantation and demonstrate T cell features predict progression from precursor to symptomatic MM. Together, these results suggest that anti-tumour immunity drives a distinctive form of cancer-associated T cell differentiation in MM.

Notable large-scale immuno-oncology consortia formed in the recent era.

T cell-mediated immune surveillance is critical for cancer control, yet its role in bone marrow malignancies remains poorly understood. Here, we integrate TCR profiling, HLA immunopeptidomics, and functional screening to characterize tumor-reactive T cells in the bone marrow of patients with multiple myeloma (MM) and acute myeloid leukemia (AML). These cells are transcriptionally defined by a conserved effector program distinct from the exhausted phenotype of tumor-reactive T cells in solid cancers. Immunopeptidomic profiling reveals a partially shared antigen landscape enriched for noncanonical peptides driving convergent TCR responses. We develop TFiT (tumor-reactive features in T cells), a transcriptional classifier that identifies these cells and stratifies immunotherapy, but not chemotherapy, response across independent MM and AML cohorts, supporting its specificity for T cell-mediated tumor control. These findings reveal a latent but activatable anti-tumor T cell compartment in bone marrow malignancies and provide a framework for engaging endogenous immunity in MM and AML.

The adaptive immune system continuously encounters antigens from a wide range of sources, including pathogens, self-tissues, tumors, and environmental agents. While extensive studies have characterized how lymphocytes respond to antigen binding, most experimental frameworks consider the antigenic environment as static. In reality, antigen levels can fluctuate dramatically across a wide range of temporal and spatial scales. In this review, we examine how the dynamics of antigen presentation, ranging from molecular binding events to organism-level exposure, affect T cell activation and fate. We discuss the cellular and molecular mechanisms that allow T cells to detect and respond to changes in antigen concentration over timescales from seconds to days. These include kinetic proofreading of TCR signaling, frequency-dependent decoding in intracellular signaling networks, and population-level feedback circuits involving effector and regulatory T cells. Theoretical and experimental evidence suggests that T cells are tuned not only to antigen quantity but also to its rate of change, with implications for tolerance, immune activation, and memory formation. We highlight how manipulating the dynamics of antigen exposure, such as through controlled vaccine delivery, can modulate immune responses and suggest that incorporating temporal features into immunological models may improve our understanding of immune decision-making and inform therapeutic strategies.

MOTIVATION:The six complementarity determining regions (CDRs) of the T cell receptor (TCR) form multiple contacts with cognate peptide and major histocompatibility complex, thus determining antigen specificity. However, the contacts between the CDRs themselves are less understood. RESULTS:Our systematic study of all available TCR crystallographic structures identified consistent patterns of intra- and inter-chain CDR contacts in both free and antigen-bound TCRs. In addition, the protein sequences of TCRα and TCRβ from sets of TCRs which recognise a shared antigen shared mutual information and were not independent. As a result, sequence-based models can partially predict TCRα/TCRβ pairing de novo. The conserved patterns of CDR amino acid contacts, and the mutual sequence constraints between antigen-specific sets of TCR α and β chains represent an under-appreciated element of TCR structure, which may play an important role in T cell antigen recognition. AVAILABILITY AND IMPLEMENTATION:The code and data necessary to reproduce the analyses are available at https://github.com/mm523/TCRab-pairing.

Emerging evidence suggests that Parkinson's disease (PD) may have its origin in the enteric nervous system (ENS), from where α-synuclein (αS) pathology spreads to the brain1-4. Decades before the onset of motor symptoms, patients with PD suffer from constipation and present with circulating T cells responsive to αS, suggesting that peripheral immune responses initiated in the ENS may be involved in the early stages of PD1,5-7. However, cellular mechanisms that trigger αS pathology in the ENS and its spread along the gut-brain axis remain elusive. Here we demonstrate that muscularis macrophages (ME-Macs), housekeepers of ENS integrity and intestinal homeostasis, modulate αS pathology and neurodegeneration in models of PD8,9. ME-Macs contain misfolded αS, adopt a signature reflecting endolysosomal dysfunction and modulate the expansion of T cells that travel from the ENS to the brain through the dura mater as αS pathology progresses. Directed ME-Mac depletion leads to reduced αS pathology in the ENS and central nervous system, prevents T cell expansion and mitigates neurodegeneration and motor dysfunction, suggesting a role for ME-Macs as early cellular initiators of αS pathology along the gut-brain axis. Understanding these mechanisms could pave the way for early-stage biomarkers in PD.

The naïve T cell receptor (TCR) repertoire forms the immunological background from which adaptive cellular immune responses emerge. We examine the fundamental properties of the human naïve TCR repertoire through the perspective of Diversity, Equality, and Inclusion. We first consider the richness of the repertoire. A combination of experimental and computational approaches has been used to estimate that the human repertoire contains at least 100 million distinct naïve clonotypes. This estimate suggests an average size of each clonotype of 1000 T cells. However, evidence from both mathematical modeling and large-scale single-cell sequencing indicates that clonotype family sizes are very unequal, with a small subset of naïve TCRs present at substantially higher frequencies. Somatic recombination itself does not contribute significantly to this inequality, as the probability of generating an identical clonotype multiple times in an individual is very small. Instead, clonotype size is likely to be largely driven by thymic or post-thymic expansion, but the mechanisms driving heterogeneity remain poorly understood. Finally, despite clonal deletion being a cornerstone of immunological dogma, experimental evidence for functional "holes" in the naïve repertoire caused by negative thymic selection is surprisingly limited. Alternative tolerance mechanisms, including regulatory T cells and T cell quorum sensing, are likely to play important roles. This review highlights the need for further research to identify the mechanisms that shape the frequency distribution of naïve TCR clone sizes and to define its impact on primary immune responses. Further research is also needed to understand the role of quorum sensing in maintaining T cell tolerance, while avoiding potential vulnerabilities arising from extensive 'holes' in the TCR repertoire.

Controlled human influenza infection studies can uniquely interrogate the early immune factors associated with clinical outcome. In this study, 27 healthy volunteers with low strain-specific serum neutralizing antibody levels were challenged with influenza A/H3N2 virus. Twenty-two became infected, with 18 developing mild-to-moderate symptoms and four remaining asymptomatic. Local and systemic immune profiling revealed innate pathways that engaged more rapidly and to a higher level in symptomatic participants. Earlier monocyte and dendritic cell activation correlated with higher symptom scores but also enhanced natural killer and CD8+ T cell activation thereafter. At baseline, peripheral blood mononuclear cells from symptomatic participants were more responsive to in vitro challenge, indicating a predisposition to divergent immunological outcomes at the time of virus exposure that was subsequently modulated by infection. These results show that human innate cell responsiveness is a predeterminant of both symptomatic disease and cellular immune responses known to promote viral clearance, suggesting potential targets for therapeutic intervention if decoupled.

Ageing is associated with significant immune changes, with unhealthy ageing characterised by chronic inflammation and immune dysregulation. Here we identify a population of CD8⁺ TEMRA cells during unhealthy ageing, which exhibit features of premature senescence and are regulated in part by TGFβ. These cells show impaired cytotoxic function and altered migratory behaviour, including an increased presence in tissues. TGFβ plays a pivotal role in modulating their phenotype by inducing CD103 expression and downregulating KLRG1, causing these cells to resemble tissue-resident memory cells. This disruption to receptor recycling leads to defective degranulation potentially altering the capacity of these cells to mount an effective immune response. Overall, these findings suggest that TEMRA cells in the context of unhealthy ageing are a pathogenic T cell subset that accumulate in tissues where they are unable to exert an effector function. ### Competing Interest Statement LAC is currently employed by ADC Therapeutics. All work was undertaken while LAC was at QMUL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. British Heart Foundation, https://ror.org/02wdwnk04, FS/15/69/32043, FS/CRTF/20/24058, CH/15/2/32064, RG/20/8/34995, AA/18/5/34222 Academy of Medical Sciences, https://ror.org/00c489v88, SBF001\1013 Barts Charity, MGU0536, G-002143 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/X009610/1 Wellcome Trust, WT 212885/Z/18/Z, WT 084052/Z/07/Z, WT 203148/Z/16/Z Engineering and Physical Sciences Research Council, EP/S032789/1

Quantifying T cell response during primary infection in humans is crucial for understanding adaptive immunity. Leveraging a controlled human challenge to SARS-CoV-2, we characterized antigen-specific T cell response within and across individuals. Notably, individual clones reached similar maximum frequencies despite differences in the timing of their peak expansion. Mathematical modeling showed that this observation is consistent with precursor frequency, but not TCR signal strength, as the source of inter-clonal variability. Single-cell profiling revealed distinct temporal programs for CD4+ and CD8+ T cells, with CD4+ cells expanding earlier but contracting to a lower frequency. Clones with similar receptors, likely recognizing the same antigen, expanded at similar times. Together, these findings highlight how clone-intrinsic properties such as precursor frequency and lineage shape T cell clonal kinetics. These insights provide a quantitative framework for understanding T cell response in humans, with implications for vaccine design. ### Competing Interest Statement The authors have declared no competing interest.

Neoantigen vaccines are under investigation for various cancers, including epidermal growth factor receptor (EGFR)-driven lung cancers1,2. We tracked the phylogenetic history of an EGFR mutant lung cancer treated with erlotinib, osimertinib, radiotherapy and a personalized neopeptide vaccine (NPV) targeting ten somatic mutations, including EGFR exon 19 deletion (ex19del). The ex19del mutation was clonal, but is likely to have appeared after a whole-genome doubling (WGD) event. Following osimertinib and NPV treatment, loss of the ex19del mutation was identified in a progressing small-cell-transformed liver metastasis. Circulating tumour DNA analyses tracking 467 somatic variants revealed the presence of this EGFR wild-type clone before vaccination and its expansion during osimertinib/NPV therapy. Despite systemic T cell reactivity to the vaccine-targeted ex19del neoantigen, the NPV failed to halt disease progression. The liver metastasis lost vaccine-targeted neoantigens through chromosomal instability and exhibited a hostile microenvironment, characterized by limited immune infiltration, low CXCL9 and elevated M2 macrophage levels. Neoantigens arising post-WGD were more likely to be absent in the progressing liver metastasis than those occurring pre-WGD, suggesting that prioritizing pre-WGD neoantigens may improve vaccine design. Data from the TRACERx 421 cohort3 provide evidence that pre-WGD mutations better represent clonal variants, and owing to their presence at multiple copy numbers, are less likely to be lost in metastatic transition. These data highlight the power of phylogenetic disease tracking and functional T cell profiling to understand mechanisms of immune escape during combination therapies.

Locally advanced esophageal adenocarcinoma remains difficult to treat and the ecological and evolutionary dynamics responsible for resistance and recurrence are incompletely understood. Here, we performed longitudinal multiomic analysis of patients with esophageal adenocarcinoma in the MEMORI trial. Multi-region multi-timepoint whole-exome and paired transcriptome sequencing was performed on 27 patients before, during and after neoadjuvant treatment. We found major transcriptomic changes during treatment with upregulation of immune, stromal and oncogenic pathways. Genetic data revealed that clonal sweeps through treatment were rare. Imaging mass cytometry and T cell receptor sequencing revealed remodeling of the tumor microenvironment during treatment. The presence of genetic immune escape, a less-cytotoxic T cell phenotype and a lack of clonal T cell expansions were linked to poor treatment response. In summary, there were widespread transcriptional and environmental changes through treatment, with limited clonal replacement, suggestive of phenotypic plasticity.

Computational prediction of the interaction of T cell receptors (TCRs) and their ligands is a grand challenge in immunology. Despite advances in high-throughput assays, specificity-labeled TCR data remain sparse. In other domains, the pre-training of language models on unlabeled data has been successfully used to address data bottlenecks. However, it is unclear how to best pre-train protein language models for TCR specificity prediction. Here, we introduce a TCR language model called SCEPTR (simple contrastive embedding of the primary sequence of T cell receptors), which is capable of data-efficient transfer learning. Through our model, we introduce a pre-training strategy combining autocontrastive learning and masked-language modeling, which enables SCEPTR to achieve its state-of-the-art performance. In contrast, existing protein language models and a variant of SCEPTR pre-trained without autocontrastive learning are outperformed by sequence alignment-based methods. We anticipate that contrastive learning will be a useful paradigm to decode the rules of TCR specificity. A record of this paper's transparent peer review process is included in the supplemental information.

BACKGROUND Thymic involution with age leads to reduced T cell output and impaired adaptive immunity. However, the extent to which thymic activity persists later in life and how this contributes to immunological aging remains unclear. This study aimed to assess the presence and function of thymic tissue in older adults and identify factors influencing residual thymopoiesis.METHODS Patients aged 50 or older undergoing cardiothoracic surgery were recruited. Thymic structures within mediastinal adipose tissue were evaluated using histology, immunofluorescence, flow cytometry, T cell receptor (TCR) sequencing, and RNA sequencing. Recent thymic emigrants (RTEs) were quantified in peripheral blood and correlated with transcriptomic, epigenetic, and TCR repertoire data. Primary outcomes included thymic tissue identification, RTE frequency, and immune correlates.RESULTS Functional thymic tissue was identified in mediastinal adipose tissue of older individuals. The frequency of CD31+CD4+ T cells (RTEs) positively correlated with the presence of thymic tissue. Thymic output showed substantial heterogeneity and was influenced by sex and smoking history. Thymic activity was associated with increased TCR repertoire diversity, improved immune protection against infections, and reduced epigenetic aging. Detailed profiling uncovered functional and phenotypic heterogeneity within naive CD4+ T cell subsets shaped by thymic activity.CONCLUSION This study demonstrates that thymic function can persist into later life and is modulated by factors such as sex and smoking. These findings suggest that thymic activity during aging is heterogeneous and influenced by more than chronological age alone, with potential implications for immune competence in older adults.

Abstract Genomic analysis of the T-cell receptor (TCR) reveals the strength, breadth, and clonal dynamics of the adaptive immune response to pathogens or cancer. The diversity of the TCR repertoire, however, means that sequencing is technically challenging, particularly for samples with low-quality, degraded nucleic acids. Here, we developed and validated FUME-TCRseq, a robust and sensitive RNA-based TCR sequencing methodology that is suitable for formalin-fixed paraffin-embedded samples and low amounts of input material. FUME-TCRseq incorporates unique molecular identifiers into each molecule of cDNA, allowing correction for sequencing errors and PCR bias. Using RNA extracted from colorectal and head and neck cancers to benchmark the accuracy and sensitivity of FUME-TCRseq against existing methods demonstrated excellent concordance between the datasets. Furthermore, FUME-TCRseq detected more clonotypes than a commercial RNA-based alternative, with shorter library preparation time and significantly lower cost. The high sensitivity and the ability to sequence RNA of poor quality and limited amount enabled quantitative analysis of small numbers of cells from archival tissue sections, which is not possible with other methods. Spatially resolved FUME-TCRseq analysis of colorectal cancers using macrodissected archival samples revealed the shifting T-cell landscapes at the transition to an invasive phenotype and between tumor subclones containing distinct driver alterations. In summary, FUME-TCRseq represents an accurate, sensitive, and low-cost tool for the characterization of T-cell repertoires, particularly in samples with low-quality RNA that have not been accessible using existing methodology. Significance: FUME-TCRseq is a TCR sequencing methodology that supports sensitive and spatially resolved detection of TCR clones in archival clinical specimens, which can facilitate longitudinal tracking of immune responses through disease course and treatment.

The biophysical interactions between the T cell receptor (TCR) and its ligands determine the specificity of the cellular immune response. However, the immense diversity of receptors and ligands has made it challenging to discover generalizable rules across the distinct binding affinity landscapes created by different ligands. Here, we present an optimization framework for discovering biophysical rules that predict whether TCRs share specificity to a ligand. Applying this framework to TCRs associated with a collection of SARS-CoV-2 peptides, we systematically characterize how cospecificity depends on the type and position of amino-acid differences between receptors. We also demonstrate that the inferred rules generalize to ligands highly dissimilar to any seen during training. Our analysis reveals that the matching of steric properties between substituted amino acids is more important for receptor cospecificity than the hydrophobic properties that prominently determine evolutionary substitutability. Our analysis also quantifies the substantial importance of positions not in direct contact with the peptide for specificity. These findings highlight the potential for data-driven approaches to uncover the molecular mechanisms underpinning the specificity of adaptive immune responses.

Antigen-specific antibody infusion is known to enhance or suppress germinal center (GC) responses depending on the affinity of the infusion. We hypothesized that infusing monoclonal antibodies (mAbs) of escalating affinity during an immunization regimen may progressively escalate selection pressure on competing B cells, increasing their affinity. To test this, we immunized mice with HIV envelope gp120 and infused CD4 binding-site (CD4bs)-specific mAbs. While mAb infusion reduced somatic hypermutation (SHM) and affinity in most CD4bs-specific B cells, a sub-population was identified with greater SHM and affinity than control. High-throughput sequencing of plasma cells revealed that CD4bs-specific plasma cells possessed elevated SHM after mAb infusion, with phylogenetic tree topology that suggested more rapid differentiation. We therefore conclude, in accordance with other studies, that high-affinity mAb infusion primarily suppresses recruitment of most competing B cells but can increase and expedite affinity maturation of certain epitope-specific B cells.