Functional antibodies exhibit light chain coherence.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

High-Complexity cellular barcoding and clonal tracing reveals stochastic and deterministic parameters of radiation resistance.

It is elusive whether clonal selection of tumor cells in response to ionizing radiation (IR) is a deterministic or stochastic process. With high resolution clonal barcoding and tracking of over 400 000 HNSCC patient-derived tumor cells the clonal dynamics of tumor cells in response to IR was analyzed. Fractionated IR induced a strong selective pressure for clonal reduction which significantly exceeded uniform clonal survival probabilities indicative for a strong clone-to-clone difference within tumor cell lines. IR induced clonal reduction affected the majority of tumor cells ranging between 96% and 75% and correlated to the degree of radiation sensitivity. Survival to IR is driven by a deterministic clonal selection of a smaller population which commonly survives radiation, while increased clonogenic capacity is a result of clonal competition of cells which have been selected stochastically. A 2-fold increase in radiation resistance results in a 4-fold (P < .05) higher deterministic clonal selection showing that the ratio of these parameters is amenable to radiation sensitivity which correlates to prognostic biomarkers of HNSCC. Evidence for the existence of a rare subpopulation with an intrinsically radiation resistant phenotype commonly surviving IR was found at a frequency of 0.6% to 3.3% (P < .001, FDR 3%). With cellular barcoding we introduce a novel functional heterogeneity associated qualitative readout for tracking dynamics of clonogenic survival in response to radiation. This enables the quantification of intrinsically radiation resistant tumor cells from patient samples and reveals the contribution of stochastic and deterministic clonal selection processes in response to IR.

Profiling the specificity of clonally expanded plasma cells during chronic viral infection by single-cell analysis.

Plasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine BM plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single-cell sequencing approach recovered full-length and paired heavy- and light-chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class-switching, and somatic variants. Furthermore, antibodies from the highly expanded and class-switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross-reactivity to nonviral and autoantigens. Integrating single-cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross-reactive antibodies.

CXCL10 chemokine regulates heterogeneity of the CD8+ T cell response and viral set point during chronic infection.

CD8+ T cells responding to chronic infection adapt an altered differentiation program that provides some restraint on pathogen replication yet limits immunopathology. This adaptation is imprinted in stem-like cells and propagated to their progeny. Understanding the molecular control of CD8+ T cell differentiation in chronic infection has important therapeutic implications. Here, we find that the chemokine receptor CXCR3 is highly expressed on viral-specific stem-like CD8+ T cells and that one of its ligands, CXCL10, regulates the persistence and heterogeneity of responding CD8+ T cells in spleens of mice chronically infected with lymphocytic choriomeningitis virus. CXCL10 is produced by inflammatory monocytes and fibroblasts of the splenic red pulp, where it grants stem-like cells access to signals promoting differentiation and limits their exposure to pro-survival niches in the white pulp. Consequently, functional CD8+ T cell responses are greater in Cxcl10-/- mice and are associated with a lower viral set point.

Increased B Cell Selection Stringency In Germinal Centers Can Explain Improved COVID-19 Vaccine Efficacies With Low Dose Prime or Delayed Boost.

The efficacy of COVID-19 vaccines appears to depend in complex ways on the vaccine dosage and the interval between the prime and boost doses. Unexpectedly, lower dose prime and longer prime-boost intervals have yielded higher efficacies in clinical trials. To elucidate the origins of these effects, we developed a stochastic simulation model of the germinal center (GC) reaction and predicted the antibody responses elicited by different vaccination protocols. The simulations predicted that a lower dose prime could increase the selection stringency in GCs due to reduced antigen availability, resulting in the selection of GC B cells with higher affinities for the target antigen. The boost could relax this selection stringency and allow the expansion of the higher affinity GC B cells selected, improving the overall response. With a longer dosing interval, the decay in the antigen with time following the prime could further increase the selection stringency, amplifying this effect. The effect remained in our simulations even when new GCs following the boost had to be seeded by memory B cells formed following the prime. These predictions offer a plausible explanation of the observed paradoxical effects of dosage and dosing interval on vaccine efficacy. Tuning the selection stringency in the GCs using prime-boost dosages and dosing intervals as handles may help improve vaccine efficacies.

Donor T-Cell Repertoire Profiling in Recipient Lymphoid and Parenchyma Organs Reveals GVHD Pathogenesis at Clonal Levels After Bone Marrow Transplantation in Mice.

The diversity and composition of T-cell receptor (TCR) repertoire, which is the result of V, (D), and J gene recombination in TCR gene locus, has been found to be implicated in T-cell responses in autoimmunity, cancer, and organ transplantation. The correlation of T-cell repertoire with the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation remains largely undefined. Here, by utilizing high-throughput sequencing of the genes encoding TCRß-chain, we comprehensively analyzed the profile of T-cell repertoire in recipient lymphoid and GVHD target organs after bone marrow transplantation (BMT) in mice. In lymphoid organs, TCR diversity was narrowed, accompanied with reduced numbers of unique clones while increased accumulation of dominant clones in allogeneic T cells compared to syngeneic T cells. In an individual allogeneic recipient, donor-derived TCR clones were highly overlapped among tissue sites, and the degree of overlapping was increasing from day 7 to 14 after allogeneic BMT. The top clones in peripheral blood, gut, liver, and lungs were highly mutually shared in an allogenic recipient, indicating that blood has the potential to predict dominant clones in these GVHD target organs. T cells in GVHD target organs from allogeneic recipients had fewer overlapped clones with pre-transplant donor T cells compared to those from syngeneic recipients. Importantly, the top 10 clones in allogeneic recipients were not detectable in pre-transplant donor T cells, indicating clonal expansion of rare rearrangements. Interestingly, even starting from the same pool of donor repertoires, T cells had very few overlapped clones between each allogeneic recipient who developed completely different dominant clones. We were only able to trace a single clone shared by three replicate allogeneic recipients within the top 500 clones. Although dominant clones were different among allogeneic recipients, V26 genes were consistently used more frequently by TCR clones in allogeneic than syngeneic recipients. This is the first study to extensively examine the feature of T-cell repertoire in multiple lymphoid and parenchyma organs, which establishes the association between T-cell activation and GVHD pathogenesis at the level of TCR clones. Immune repertoire sequencing-based methods may represent a novel personalized strategy to guide diagnosis and therapy in GVHD.

High Throughput Human T Cell Receptor Sequencing: A New Window Into Repertoire Establishment and Alloreactivity.

Recent advances in high throughput sequencing (HTS) of T cell receptors (TCRs) and in transcriptomic analysis, particularly at the single cell level, have opened the door to a new level of understanding of human immunology and immune-related diseases. In this article, we discuss the use of HTS of TCRs to discern the factors controlling human T cell repertoire development and how this approach can be used in combination with human immune system (HIS) mouse models to understand human repertoire selection in an unprecedented manner. An exceptionally high proportion of human T cells has alloreactive potential, which can best be understood as a consequence of the processes governing thymic selection. High throughput TCR sequencing has allowed assessment of the development, magnitude and nature of the human alloresponse at a new level and has provided a tool for tracking the fate of pre-transplant-defined donor- and host-reactive TCRs following transplantation. New insights into human allograft rejection and tolerance obtained with this method in combination with single cell transcriptional analyses are reviewed here.

SRSF1 plays a critical role in invariant natural killer T cell development and function.

Invariant natural killer T (iNKT) cells are highly conserved innate-like T lymphocytes that originate from CD4+CD8+ double-positive (DP) thymocytes. Here, we report that serine/arginine splicing factor 1 (SRSF1) intrinsically regulates iNKT cell development by directly targeting Myb and balancing the abundance of short and long isoforms. Conditional ablation of SRSF1 in DP cells led to a substantially diminished iNKT cell pool due to defects in proliferation, survival, and TCRα rearrangement. The transition from stage 0 to stage 1 of iNKT cells was substantially blocked, and the iNKT2 subset was notably diminished in SRSF1-deficient mice. SRSF1 deficiency resulted in aberrant expression of a series of regulators that are tightly correlated with iNKT cell development and iNKT2 differentiation, including Myb, PLZF, Gata3, ICOS, and CD5. In particular, we found that SRSF1 directly binds and regulates pre-mRNA alternative splicing of Myb and that the expression of the short isoform of Myb is substantially reduced in SRSF1-deficient DP and iNKT cells. Strikingly, ectopic expression of the Myb short isoform partially rectified the defects caused by ablation of SRSF1. Furthermore, we confirmed that the SRSF1-deficient mice exhibited resistance to acute liver injury upon α-GalCer and Con A induction. Our findings thus uncovered a previously unknown role of SRSF1 as an essential post-transcriptional regulator in iNKT cell development and functional differentiation, providing new clinical insights into iNKT-correlated disease.

Negative feedback by NUR77/Nr4a1 restrains B cell clonal dominance during early T-dependent immune responses.

B cell clones compete for entry into and dominance within germinal centers (GCs), where the highest-affinity B cell receptors (BCRs) are selected. However, diverse and low-affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesize that a negative feedback loop may operate within B cells to preferentially restrain high-affinity clones from monopolizing the early GC niche. Here, we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We show that NUR77 expression scales with antigen stimulation and restrains B cell expansion. Although NUR77 is dispensable for regulating GC size when GCs are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. We propose that this is important to preserve early clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

A molecular theory of germinal center B cell selection and division.

The selection of B cells (BCs) in germinal centers (GCs) is pivotal to the generation of high-affinity antibodies and memory BCs, but it lacks global understanding. Based on the idea of a single Tfh-cell signal that controls BC selection and division, experiments appear contradictory. Here, we use the current knowledge on the molecular pathways of GC BCs to develop a theory of GC BC selection and division based on the dynamics of molecular factors. This theory explains the seemingly contradictory experiments by the separation of signals for BC fate decision from signals controlling the number of BC divisions. Three model variants are proposed and experiments are predicted that allow one to distinguish those. Understanding information processing in molecular BC states is critical for targeted immune interventions, and the proposed theory implies that selection and division can be controlled independently in GC reactions.

Infections in the monogenic autoimmune syndrome APECED.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by mutations in the Autoimmune Regulator (AIRE) gene, which impair the thymic negative selection of self-reactive T-cells and underlie the development of autoimmunity that targets multiple endocrine and non-endocrine tissues. Beyond autoimmunity, APECED features heightened susceptibility to certain specific infections, which is mediated by anti-cytokine autoantibodies and/or T-cell driven autoimmune tissue injury. These include the 'signature' APECED infection chronic mucocutaneous candidiasis (CMC), but also life-threatening coronavirus disease 2019 (COVID-19) pneumonia, bronchiectasis-associated bacterial pneumonia, and sepsis by encapsulated bacteria. Here we discuss the expanding understanding of the immunological mechanisms that contribute to infection susceptibility in this prototypic syndrome of impaired central tolerance, which provide the foundation for devising improved diagnostic and therapeutic strategies for affected patients.

Carbamazepine Induces Focused T Cell Responses in Resolved Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis Cases But Does Not Perturb the Immunopeptidome for T Cell Recognition.

Antiseizure medications (ASMs) are frequently implicated in T cell-mediated drug hypersensitivity reactions and cause skin tropic pathologies that range in severity from mild rashes to life-threatening systemic syndromes. During the acute stages of the more severe manifestations of these reactions, drug responsive proinflammatory CD8+ T cells display classical features of Th1 cytokine production (e.g. IFNγ) and cytolysis (e.g. granzyme B, perforin). These T cells may be found locally at the site of pathology (e.g. blister cells/fluid), as well as systemically (e.g. blood, organs). What is less understood are the long-lived immunological effects of the memory T cell pool following T cell-mediated drug hypersensitivity reactions. In this study, we examine the ASM carbamazepine (CBZ) and the CBZ-reactive memory T cell pool in patients who have a history of either Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) from 3-to-20 years following their initial adverse reaction. We show that in vitro drug restimulation of CBZ-reactive CD8+ T cells results in a proinflammatory profile and produces a mainly focused, yet private, T cell receptor (TCR) usage amongst human leukocyte antigen (HLA)-B*15:02-positive SJS or TEN patients. Additionally, we show that expression of these CBZ-reactive TCRs in a reporter cell line, lacking endogenous αßTCR, recapitulates the features of TCR activation reported for ASM-treated T cell lines/clones, providing a useful tool for further functional validations. Finally, we conduct a comprehensive evaluation of the HLA-B*15:02 immunopeptidome following ASM (or a metabolite) treatment of a HLA-B*15:02-positive B-lymphoblastoid cell line (C1R.B*15:02) and minor perturbation of the peptide repertoire. Collectively, this study shows that the CBZ-reactive T cells characterized require both the drug and HLA-B*15:02 for activation and that reactivation of memory T cells from blood results in a focused private TCR profile in patients with resolved disease.

Thymic atrophy creates holes in Treg-mediated immuno-regulation via impairment of an antigen-specific clone.

Age-related thymic atrophy results in reduced output of naïve conventional T (Tcon) cells. However, its impact on regulatory T (Treg) cells is insufficiently understood. Given evidence that thymic Treg (tTreg) cell generation is enhanced in the aged, atrophy thymus and that the aged periphery accumulates peripheral Treg (pTreg) cells, we asked why these Treg cells are unable to effectively attenuate increased autoreactivity-induced chronic inflammation in the elderly. We designed a mock-self-antigen chimera mouse model, in which membrane-bound ovalbumin (mOVA) transgenic mice, bearing a FoxN1-floxed gene for induction of conditional thymic atrophy, received OVA-specific (OT-II) T-cell receptor (TCR) transgenic progenitor cells. The chimeric mice with thymic atrophy exhibited a significant decrease in OVA-specific tTreg and pTreg cells but not polyclonal (pan)-Treg cells. These OVA-specific pTreg cells were significantly less able to suppress OVA-specific stimulation-induced proliferation in vitro and exhibited lower FoxP3 expression. Additionally, we conducted preliminary TCR repertoire diversity sequencing for Treg cells among recent thymic emigrants (RTEs) from RagGFP -FoxP3RFP dual-reporter mice and observed a trend for decreased diversity in mice with thymic atrophy compared to littermates with normal thymus. These data indicate that although the effects of age-related thymic atrophy do not affect pan-Treg generation, certain tissue-specific Treg clones may experience abnormal agonist selection. This, combined with enhanced pan-pTreg cells, may greatly contribute to age-related chronic inflammation, even in the absence of acute autoimmune disease in the elderly.

In Pursuit of Adult Progenitors of Thymic Epithelial Cells.

Peripheral T cells capable of discriminating between self and non-self antigens are major components of a robust adaptive immune system. The development of self-tolerant T cells is orchestrated by thymic epithelial cells (TECs), which are localized in the thymic cortex (cortical TECs, cTECs) and medulla (medullary TECs, mTECs). cTECs and mTECs are essential for differentiation, proliferation, and positive and negative selection of thymocytes. Recent advances in single-cell RNA-sequencing technology have revealed a previously unknown degree of TEC heterogeneity, but we still lack a clear picture of the identity of TEC progenitors in the adult thymus. In this review, we describe both earlier and recent findings that shed light on features of these elusive adult progenitors in the context of tissue homeostasis, as well as recovery from stress-induced thymic atrophy.

CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells.

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4+ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4+ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5lo and CD5hi naïve human CD4+ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4+ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies.

Eosinophils interact with thymocytes and proliferate in the human thymus.

Eosinophils differentiate and mature in the thymus, outside of the bone marrow, in healthy individuals. Locally developed thymic eosinophils may contribute to the maturation and selection of human thymocytes.

Generation of self-reactive, shared T-cell receptor α chains in the human thymus.

The T-cell receptor (TCR) repertoire is generated in a semistochastic process of gene recombination and pairing of TCRα to TCRß chains with the estimated total TCR diversity of >108. Despite this high diversity, similar or identical TCR chains are found to recur in immune responses. Here, we analyzed the thymic generation of TCR sequences previously associated with recognition of self- and nonself-antigens, represented by sequences associated with autoimmune diabetes and HIV, respectively. Unexpectedly, in the CD4+ compartment TCRα chains associated with the recognition of self-antigens were generated in significantly higher numbers than TCRα chains associated with the recognition of nonself-antigens. The analysis of the circulating repertoire further showed that these chains are not lost in negative selection nor predominantly converted to the regulatory T-cell lineage. The high abundance of self-reactive TCRα chains in multiple individuals suggests that the human thymus has a predilection to generate self-reactive TCRα chains independently of the HLA-type and that the individual risk of autoimmunity may be modulated by the TCRß repertoire associated with these chains.

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus.

TCRαß+ CD8α+ CD8ß- intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely ß2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86neg/low and MHCI+ . However, selective depletion of ß2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.

Role of the thymus in spontaneous development of a multi-organ autoimmune disease in human immune system mice.

We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.