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.

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.

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.

Factors that influence the thymic selection of CD8αα intraepithelial lymphocytes.

Thymocytes bearing αß T cell receptors (TCRαß) with high affinity for self-peptide-MHC complexes undergo negative selection or are diverted to alternate T cell lineages, a process termed agonist selection. Among thymocytes bearing TCRs restricted to MHC class I, agonist selection can lead to the development of precursors that can home to the gut and give rise to CD8αα-expressing intraepithelial lymphocytes (CD8αα IELs). The factors that influence the choice between negative selection versus CD8αα IEL development remain largely unknown. Using a synchronized thymic tissue slice model that supports both negative selection and CD8αα IEL development, we show that the affinity threshold for CD8αα IEL development is higher than for negative selection. We also investigate the impact of peptide presenting cells and cytokines, and the migration patterns associated with these alternative cell fates. Our data highlight the roles of TCR affinity and the thymic microenvironments on T cell fate.

Role, function and regulation of the thymocyte selection-associated high mobility group box protein in CD8+ T cell exhaustion.

Thymocyte selection-associated high mobility group box protein (TOX), a member of the high-motility group box (HMG) protein superfamily, is an evolutionarily conserved DNA-binding protein. It functions as a transcription factor that modulates transcriptional programs by binding to DNA in a structure-dependent manner. It has been well established that TOX is required for the development of CD4+ T cells, natural killer (NK) cells and innate lymphoid cells (ILCs), as well as the autoimmunity mediated by CD8+ T cells. Recently, emerging evidence supports an essential role for TOX in the induction of T cell exhaustion in the setting of tumor or chronic viral infection by mediating transcriptional and epigenetic changes, which are cardinal hallmarks of exhausted T cells. Moreover, TOX plays a key role in the persistence of antigen-specific T cells and in the mitigation of tissue damage caused by immunopathology over the course of tumorigenesis and chronic infection. Additionally, TOX contributes to the high level of programmed cell death protein 1 (PD-1) on the cell surface by participating in the process of endocytic recycling of PD-1. In this review, we summarize the most recent information about the role of TOX in the process of T cell exhaustion, which enriches our understanding of the molecular mechanisms of CD8+ T cell exhaustion upon chronic antigen stimulation and reveals promising therapeutic targets for persisting infection and cancer.

Comprehensive mapping of immune perturbations associated with severe COVID-19.

Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified extensive induction and activation of multiple immune lineages, including T cell activation, oligoclonal plasmablast expansion, and Fc and trafficking receptor modulation on innate lymphocytes and granulocytes, that distinguished severe COVID-19 cases from healthy donors or SARS-CoV-2-recovered or moderate severity patients. We found the neutrophil to lymphocyte ratio to be a prognostic biomarker of disease severity and organ failure. Our findings demonstrate broad innate and adaptive leukocyte perturbations that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation.

Clonal haematopoiesis and COVID-19: A possible deadly liaison.

We provide evidence for a linear correlation between the frequency of clonal haematopoiesis and COVID-19 mortality rate. We discuss the mechanistic explanations for this association mediated by a pathological inflammatory response. Our hypothesis can be tested in COVID-19-infected patients and eventually lead to new approaches to risk stratification and therapy.

Epitope-directed antibody selection by site-specific photocrosslinking.

Currently, there are no methods available offering solutions to select and identify antibodies binding to a specific conformational epitope of an antigen. Here, we developed a method to allow epitope-directed antibody selection from a phage display library by photocrosslinking bound antibodies to a site that specifically incorporates a noncanonical amino acid, p-benzoyl-l-phenylalanine (pBpa), on the target antigen epitope. By one or two rounds of panning against antibody phage display libraries, those hits that covalently bind to the proximity site of pBpa on specific epitopes of target antigens after ultraviolet irradiation are enriched and selected. This method was applied to specific epitopes on human interleukin-1ß and complement 5a. In both cases, more than one-third of hits identified bind to the target epitopes, demonstrating the feasibility and versatility of this method.

Eomes broadens the scope of CD8 T-cell memory by inhibiting apoptosis in cells of low affinity.

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.

Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy.

Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable anti-tumor responses in patients with B-cell malignancies. However, clonal kinetics and transcriptional programs that regulate the fate of CAR-T cells after infusion remain poorly understood. Here we perform TCRB sequencing, integration site analysis, and single-cell RNA sequencing (scRNA-seq) to profile CD8+ CAR-T cells from infusion products (IPs) and blood of patients undergoing CD19 CAR-T immunotherapy. TCRB sequencing shows that clonal diversity of CAR-T cells is highest in the IPs and declines following infusion. We observe clones that display distinct patterns of clonal kinetics, making variable contributions to the CAR-T cell pool after infusion. Although integration site does not appear to be a key driver of clonal kinetics, scRNA-seq demonstrates that clones that expand after infusion mainly originate from infused clusters with higher expression of cytotoxicity and proliferation genes. Thus, we uncover transcriptional programs associated with CAR-T cell behavior after infusion.

Autoreactive B Cell Differentiation in Diffuse Ectopic Lymphoid-Like Structures of Inflamed Pemphigus Lesions.

Pemphigus is an organ-specific autoimmune disease that targets skin and/or mucous membranes. Our previous study showed that infiltrating lymphocytes in pemphigus vulgaris (PV) lesions produce anti-desmoglein (Dsg) 1/3 antibodies after in vitro culture. In this study, we found diffuse ectopic lymphoid-like structures (ELSs) commonly present in the lesions of both PV and pemphigus foliaceus. Notably, pemphigus lesions contained centroblasts, plasmablasts, and plasma cells, which recapitulated the different stages of B cell differentiation. Elevated mRNA expression levels of the differentiation-related transcription factors BLIMP-1, IRF4, and BCL-6 were observed in pemphigus lesions. Moreover, B cell receptor repertoire analysis revealed the clonal expansion of the lesional B cells. Lesional B cells might recirculate among lesions, lymph nodes, and peripheral blood. Increased mRNA expression levels of multiple chemokines in pemphigus lesions and elevated expression levels of chemokine receptors on lesional B cells were also observed. Collectively, these results show that the ELSs in pemphigus lesions might act as a niche, supporting in situ B cell differentiation and clonal expansion.

Stochastic Expansions Maintain the Clonal Stability of CD8+ T Cell Populations Undergoing Memory Inflation Driven by Murine Cytomegalovirus.

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV.

PKC signaling contributes to chromatin decondensation and is required for competence to respond to IL-2 during T cell activation.

The clonal proliferation of antigen-specific T cells during an immune response critically depends on the differential response to growth factors, such as IL-2. While activated T cells proliferate robustly in response to IL-2 stimulation, naïve (quiescent) T cells are able to ignore the potent effects of growth factors because they possess chromatin that is tightly condensed such that transcription factors, such as STAT5, cannot access DNA. Activation via the T cell receptor (TCR) induces a rapid decondensation of chromatin, permitting STAT5-DNA engagement and ultimately promoting proliferation of only antigen-specific T cells. Previous work demonstrated that the mobilization of intracellular calcium following TCR stimulation is a key event in the decondensation of chromatin. Here we examine PKC-dependent signaling mechanisms to determine their role in activation-induced chromatin decondensation and the subsequent acquisition of competence to respond to IL-2 stimulation. We found that a calcium-dependent PKC contributes to activation-induced chromatin decondensation and that the p38 MAPK and NFκB pathways downstream of PKC each contribute to regulating the proper decondensation of chromatin. Importantly, we found that p44/42 MAPK activity is required for peripheral T cells to gain competence to properly respond to IL-2 stimulation. Our findings shed light on the mechanisms that control the clonal proliferation of antigen-specific peripheral T cells during an immune response.

Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen.

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, "LES" with an affinity (∼15-25 µM) comparable to many αß TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined.

Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches.

Current advances in antibody engineering driving the strongest growth area in biotherapeutic agents development. Affinity improvement that is mainly important for biological activity and clinical efficacy of therapeutic antibodies, has still remained a challenging task. In the human body, during a course of immune response affinity maturation increase antibody activity by several rounds of somatic hypermutation and clonal selection in the germinal center. The final outputs are antibodies representing higher affinity and specificity against a particular antigen. In the realm of biotechnology, exploring of mutations which improve antibody affinity while preserving its specificity and stability is an extremely time-consuming and laborious process. Recent advances in computational algorithms and DNA sequencing technologies help researchers to redesign antibody structure to achieve desired properties such as improved binding affinity. In this review, we briefly described the principle of affinity maturation and different corresponding in vitro techniques. Also, we recapitulated the most recent advancements in the field of antibody affinity maturation including computational approaches and next-generation sequencing (NGS).

High Peptide Dose Vaccination Promotes the Early Selection of Tumor Antigen-Specific CD8 T-Cells of Enhanced Functional Competence.

CD8 T-cell response efficiency critically depends on the TCR binding strength to peptide-MHC, i.e., the TCR binding avidity. A current challenge in onco-immunology lies in the evaluation of vaccine protocols selecting for tumor-specific T-cells of highest avidity, offering maximal immune protection against tumor cells and clinical benefit. Here, we investigated the impact of peptide and CpG/adjuvant doses on the quality of vaccine-induced CD8 T-cells in relation to binding avidity and functional responses in treated melanoma patients. Using TCR-pMHC binding avidity measurements combined to phenotype and functional assays, we performed a comprehensive study on representative tumor antigen-specific CD8 T-cell clones (n = 454) from seven patients vaccinated with different doses of Melan-A/ELA peptide (0.1 mg vs. 0.5 mg) and CpG-B adjuvant (1-1.3 mg vs. 2.6 mg). Vaccination with high peptide dose favored the early and strong in vivo expansion and differentiation of Melan-A-specific CD8 T-cells. Consistently, T-cell clones generated from those patients showed increased TCR binding avidity (i.e., slow off-rates and CD8 binding independency) readily after 4 monthly vaccine injections (4v). In contrast, the use of low peptide or high CpG-B doses required 8 monthly vaccine injections (8v) for the enrichment of anti-tumor T-cells with high TCR binding avidity and low CD8 binding dependency. Importantly, the CD8 binding-independent vaccine-induced CD8 T-cells displayed enhanced functional avidity, reaching a plateau of maximal function. Thus, T-cell functional potency following peptide/CpG/IFA vaccination may not be further improved beyond a certain TCR binding avidity limit. Our results also indicate that while high peptide dose vaccination induced the early selection of Melan-A-specific CD8 T-cells of increased functional competence, continued serial vaccinations also promoted such high-avidity T-cells. Overall, the systematic assessment of T-cell binding avidity may contribute to optimize vaccine design for improving clinical efficacy.

Local Clonal Diversification and Dissemination of B Lymphocytes in the Human Bronchial Mucosa.

The efficacy of the adaptive humoral immune response likely requires diverse, yet focused regional B cell antibody production throughout the body. Here we address, in the first study of its kind, the B cell repertoire in the bronchial mucosa, an important barrier to antigens inhaled from the atmosphere. To accomplish this, we have applied high-throughput Adaptive Immune Receptor Repertoire Sequencing (AIRR-Seq) to 10 bronchial biopsies from altogether four different sites in the right lungs from an asthmatic patient and a healthy subject. While the majority of identified B cell clones were restricted to a single site, many were disseminated in multiple sites. Members of a clone were shared more between adjacent biopsies than between distal biopsies, suggesting local mucosal migration and/or a homing mechanism for B cells through the blood or lymph. A smaller fraction of clones spanned the bronchial mucosa and peripheral blood, suggesting ongoing trafficking between these compartments. The bronchial mucosal B cell repertoire in the asthmatic patient was geographically more variable but less diverse compared to that of the healthy subject, suggesting an ongoing, antigen-driven humoral immune response in atopic asthma. Whether this is a feature of atopy or disease status remains to be clarified in future studies. We observed a subset of highly mutated and antigen-selected IgD-only cells in the bronchial mucosa. These cells were found in relative high abundance in the asthmatic individual but also, albeit at lower abundance, in the healthy subject. This novel finding merits further exploration using a larger cohort of subjects.

Capturing change in clonal composition amongst single mouse germinal centers.

Understanding cellular processes occurring in vivo on time scales of days to weeks requires repeatedly interrogating the same tissue without perturbing homeostasis. We describe a novel setup for longitudinal intravital imaging of murine peripheral lymph nodes (LNs). The formation and evolution of single germinal centers (GCs) was visualized over days to weeks. Naïve B cells encounter antigen and form primary foci, which subsequently seed GCs. These experience widely varying rates of homogenizing selection, even within closely confined spatial proximity. The fluidity of GCs is greater than previously observed with large shifts in clonality over short time scales; and loss of GCs is a rare, observable event. The observation of contemporaneous, congruent shifts in clonal composition between GCs within the same animal suggests inter-GC trafficking of memory B cells. This tool refines approaches to resolving immune dynamics in peripheral LNs with high temporospatial resolution and minimal perturbation of homeostasis.

Immunoglobulin gene analysis as a tool for investigating human immune responses.

The human immunoglobulin repertoire is a hugely diverse set of sequences that are formed by processes of gene rearrangement, heavy and light chain gene assortment, class switching and somatic hypermutation. Early B cell development produces diverse IgM and IgD B cell receptors on the B cell surface, resulting in a repertoire that can bind many foreign antigens but which has had self-reactive B cells removed. Later antigen-dependent development processes adjust the antigen affinity of the receptor by somatic hypermutation. The effector mechanism of the antibody is also adjusted, by switching the class of the antibody from IgM to one of seven other classes depending on the required function. There are many instances in human biology where positive and negative selection forces can act to shape the immunoglobulin repertoire and therefore repertoire analysis can provide useful information on infection control, vaccination efficacy, autoimmune diseases, and cancer. It can also be used to identify antigen-specific sequences that may be of use in therapeutics. The juxtaposition of lymphocyte development and numerical evaluation of immune repertoires has resulted in the growth of a new sub-speciality in immunology where immunologists and computer scientists/physicists collaborate to assess immune repertoires and develop models of immune action.

Germinal center responses to complex antigens.

Germinal centers (GCs) are the primary sites of antibody affinity maturation, sites where B-cell antigen-receptor (BCR) genes rapidly acquire mutations and are selected for increasing affinity for antigen. This process of hypermutation and affinity-driven selection results in the clonal expansion of B cells expressing mutated BCRs and acts to hone the antibody repertoire for greater avidity and specificity. Remarkably, whereas the process of affinity maturation has been confirmed in a number of laboratories, models for how affinity maturation in GCs operates are largely from studies of genetically restricted B-cell populations competing for a single hapten epitope. Much less is known about GC responses to complex antigens, which involve both inter- and intraclonal competition for many epitopes. In this review, we (i) compare current methods for analysis of the GC B-cell repertoire, (ii) describe recent studies of GC population dynamics in response to complex antigens, discussing how the observed repertoire changes support or depart from the standard model of clonal selection, and (iii) speculate on the nature and potential importance of the large fraction of GC B cells that do not appear to interact with native antigen.