Helper T cell immunity in humans with inherited CD4 deficiency.

CD4+ T cells are vital for host defense and immune regulation. However, the fundamental role of CD4 itself remains enigmatic. We report seven patients aged 5-61 years from five families of four ancestries with autosomal recessive CD4 deficiency and a range of infections, including recalcitrant warts and Whipple's disease. All patients are homozygous for rare deleterious CD4 variants impacting expression of the canonical CD4 isoform. A shorter expressed isoform that interacts with LCK, but not HLA class II, is affected by only one variant. All patients lack CD4+ T cells and have increased numbers of TCRαß+CD4-CD8- T cells, which phenotypically and transcriptionally resemble conventional Th cells. Finally, patient CD4-CD8- αß T cells exhibit intact responses to HLA class II-restricted antigens and promote B cell differentiation in vitro. Thus, compensatory development of Th cells enables patients with inherited CD4 deficiency to acquire effective cellular and humoral immunity against an unexpectedly large range of pathogens. Nevertheless, CD4 is indispensable for protective immunity against at least human papillomaviruses and Trophyrema whipplei.

DOCK2-deficiency causes defects in anti-viral T cell responses and impaired control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, analysis of these in patients is complicated by their treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. We also uncovered a critical, cell intrinsic role of DOCK2 in the priming of anti-viral CD8+ T cells and in particular their initial expansion, despite apparently normal early activation of these cells. When this defect was overcome by priming in vitro, DOCK2-deficient CD8+ T cells were surprisingly protective against HSV-1-disease, albeit not as effectively as wild type cells. These results shed light on a cellular deficiency that is likely to impact anti-viral immunity in DOCK2-deficient patients.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome.

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

Isolation and characterisation of PR3-specific B cells and their immunoglobulin sequences.

BACKGROUND: PR3 autoantibodies are essential to the diagnosis and monitoring of granulomatosus with polyangiitis, but to date no PR3 autoantibody sequences have been published. OBJECTIVES: To identify and characterise PR3-specific B cells from the peripheral blood of patients with PR3 autoantibodies. METHODS: Peripheral blood mononuclear cells from seven patients with PR3 autoantibodies were stained with PR3. B cells that bound PR3 underwent single cell sorting, transcriptome sequencing, and their immunoglobulin sequences expressed as antibodies and tested for PR3-specificity by ELISA. RESULTS: We identified 19 PR3-specific B cells from only one PR3-seropositive patient at a low frequency (0.0075 % of B cells) in the peripheral blood. These were polyclonal, IgG+ and enriched for IgG4, lambda pairing, IGHJ6 gene usage, CDRH3 length, IGHE and CD71 expression. They demonstrated relatively low levels of somatic hypermutation and variably reduced PR3 binding when reverted to germline. CONCLUSIONS: Identifying PR3-specific B cells in the peripheral blood is possible but challenging and those we did identify exhibited features suggesting that PR3-self reactivity may occur early in B-cell development.

DOCK2-deficiency causes defects in anti-viral T cell responses and poor control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, patients with these diseases are by definition rare. In addition, any analysis is complicated by treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. Further, we found that they have a critical, cell intrinsic role of DOCK2 in the clonal expansion of anti-viral CD8+ T cells despite normal early activation of these cells. Finally, while the major deficiency is in clonal expansion, the ability of primed and expanded DOCK2-deficient CD8+ T cells to protect against HSV-1-infection is also compromised. These results provide a contributing cause for the frequent and devastating viral infections seen in DOCK2-deficient patients and improve our understanding of anti-viral CD8+ T cell immunity.

Network analysis reveals a major role for 14q32 cluster miRNAs in determining transcriptional differences between IGHV-mutated and unmutated CLL.

Chronic lymphocytic leukaemia (CLL) cells can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear how these differences reflect the tumour cells' innate pre/post germinal centre origin or their BCR signalling competence, we applied mRNA/miRNA sequencing to 38 CLL cases categorised into three subsets by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. Of the IGHV-associated miRNAs, (14/38 (37%)) derived from chr14q32 clusters where all miRNAs were co-expressed with the MEG3 lncRNA from a cancer associated imprinted locus. Integrative analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was potentially regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3'UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.

Structural and functional characterization of capsid binding by anti-AAV9 monoclonal antibodies from infants after SMA gene therapy.

Success in the treatment of infants with spinal muscular atrophy (SMA) underscores the potential of vectors based on adeno-associated virus (AAV). However, a major obstacle to the full realization of this potential is pre-existing natural and therapy-induced anti-capsid humoral immunity. Structure-guided capsid engineering is one possible approach to surmounting this challenge but necessitates an understanding of capsid-antibody interactions at high molecular resolution. Currently, only mouse-derived monoclonal antibodies (mAbs) are available to structurally map these interactions, which presupposes that mouse and human-derived antibodies are functionally equivalent. In this study, we have characterized the polyclonal antibody responses of infants following AAV9-mediated gene therapy for SMA and recovered 35 anti-capsid mAbs from the abundance of switched-memory B (smB) cells present in these infants. For 21 of these mAbs, seven from each of three infants, we have undertaken functional and structural analysis measuring neutralization, affinities, and binding patterns by cryoelectron microscopy (cryo-EM). Four distinct patterns were observed akin to those reported for mouse-derived mAbs, but with early evidence of differing binding pattern preference and underlying molecular interactions. This is the first human and largest series of anti-capsid mAbs to have been comprehensively characterized and will prove to be powerful tools for basic discovery and applied purposes.

CARD11 gain-of-function mutation drives cell-autonomous accumulation of PD-1+ ICOShigh activated T cells, T-follicular, T-regulatory and T-follicular regulatory cells.

Introduction: Germline CARD11 gain-of-function (GOF) mutations cause B cell Expansion with NF-κB and T cell Anergy (BENTA) disease, whilst somatic GOF CARD11 mutations recur in diffuse large B cell lymphoma (DLBCL) and in up to 30% of the peripheral T cell lymphomas (PTCL) adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL) and Sezary Syndrome. Despite their frequent acquisition by PTCL, the T cell-intrinsic effects of CARD11 GOF mutations are poorly understood. Methods: Here, we studied B and T lymphocytes in mice with a germline Nethyl-N-nitrosourea (ENU)-induced Card11M365K mutation identical to a mutation identified in DLBCL and modifying a conserved region of the CARD11 coiled-coil domain recurrently mutated in DLBCL and PTCL. Results and discussion: Our results demonstrate that CARD11.M365K is a GOF protein that increases B and T lymphocyte activation and proliferation following antigen receptor stimulation. Germline Card11M365K mutation was insufficient alone to cause B or T-lymphoma, but increased accumulation of germinal center (GC) B cells in unimmunized and immunized mice. Card11M365K mutation caused cell-intrinsic over-accumulation of activated T cells, T regulatory (TREG), T follicular (TFH) and T follicular regulatory (TFR) cells expressing increased levels of ICOS, CTLA-4 and PD-1 checkpoint molecules. Our results reveal CARD11 as an important, cell-autonomous positive regulator of TFH, TREG and TFR cells. They highlight T cell-intrinsic effects of a GOF mutation in the CARD11 gene, which is recurrently mutated in T cell malignancies that are often aggressive and associated with variable clinical outcomes.

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients.

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

Tracking the clonal dynamics of SARS-CoV-2-specific T cells in children and adults with mild/asymptomatic COVID-19.

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. A novel population of naïve interferon-activated T cells is expanded in acute COVID-19 and is recruited into the memory compartment during convalescence in adults but not children. This was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection.

STAT3 gain-of-function mutations connect leukemia with autoimmune disease by pathological NKG2Dhi CD8+ T cell dysregulation and accumulation.

The association between cancer and autoimmune disease is unexplained, exemplified by T cell large granular lymphocytic leukemia (T-LGL) where gain-of-function (GOF) somatic STAT3 mutations correlate with co-existing autoimmunity. To investigate whether these mutations are the cause or consequence of CD8+ T cell clonal expansions and autoimmunity, we analyzed patients and mice with germline STAT3 GOF mutations. STAT3 GOF mutations drove the accumulation of effector CD8+ T cell clones highly expressing NKG2D, the receptor for stress-induced MHC-class-I-related molecules. This subset also expressed genes for granzymes, perforin, interferon-γ, and Ccl5/Rantes and required NKG2D and the IL-15/IL-2 receptor IL2RB for maximal accumulation. Leukocyte-restricted STAT3 GOF was sufficient and CD8+ T cells were essential for lethal pathology in mice. These results demonstrate that STAT3 GOF mutations cause effector CD8+ T cell oligoclonal accumulation and that these rogue cells contribute to autoimmune pathology, supporting the hypothesis that somatic mutations in leukemia/lymphoma driver genes contribute to autoimmune disease.

Genetic and structural basis of the human anti-α-galactosyl antibody response.

Humans lack the capacity to produce the Galα1-3Galß1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.

Human T-bet governs the generation of a distinct subset of CD11chighCD21low B cells.

High-level expression of the transcription factor T-bet characterizes a phenotypically distinct murine B cell population known as "age-associated B cells" (ABCs). T-bet-deficient mice have reduced ABCs and impaired humoral immunity. We describe a patient with inherited T-bet deficiency and largely normal humoral immunity including intact somatic hypermutation, affinity maturation and memory B cell formation in vivo, and B cell differentiation into Ig-producing plasmablasts in vitro. Nevertheless, the patient exhibited skewed class switching to IgG1, IgG4, and IgE, along with reduced IgG2, both in vivo and in vitro. Moreover, T-bet was required for the in vivo and in vitro development of a distinct subset of human B cells characterized by reduced expression of CD21 and the concomitantly high expression of CD19, CD20, CD11c, FCRL5, and T-bet, a phenotype that shares many features with murine ABCs. Mechanistically, human T-bet governed CD21loCD11chi B cell differentiation by controlling the chromatin accessibility of lineage-defining genes in these cells: FAS, IL21R, SEC61B, DUSP4, DAPP1, SOX5, CD79B, and CXCR4. Thus, human T-bet is largely redundant for long-lived protective humoral immunity but is essential for the development of a distinct subset of human CD11chiCD21lo B cells.

SATB1 ensures appropriate transcriptional programs within naïve CD8+ T cells.

Special AT-binding protein 1 (SATB1) is a chromatin-binding protein that has been shown to be a key regulator of T-cell development and CD4+ T-cell fate decisions and function. The underlying function for SATB1 in peripheral CD8+ T-cell differentiation processes is largely unknown. To address this, we examined SATB1-binding patterns in naïve and effector CD8+ T cells demonstrating that SATB1 binds to noncoding regulatory elements linked to T-cell lineage-specific gene programs, particularly in naïve CD8+ T cells. We then assessed SATB1 function using N-ethyl-N-nitrosourea-mutant mice that exhibit a point mutation in the SATB1 DNA-binding domain (termed Satb1m1Anu/m1Anu ). Satb1m1Anu/m1Anu mice exhibit diminished SATB1-binding, naïve, Satb1m1Anu/m1Anu CD8+ T cells exhibiting transcriptional and phenotypic characteristics reminiscent of effector T cells. Upon activation, the transcriptional signatures of Satb1m1Anu/m1Anu and wild-type effector CD8+ T cells converged. While there were no overt differences, primary respiratory infection of Satb1m1Anu/m1Anu mice with influenza A virus (IAV) resulted in a decreased proportion and number of IAV-specific CD8+ effector T cells recruited to the infected lung when compared with wild-type mice. Together, these data suggest that SATB1 has a major role in an appropriate transcriptional state within naïve CD8+ T cells and ensures appropriate CD8+ T-cell effector gene expression upon activation.

Activation of the viral sensor oligoadenylate synthetase 2 (Oas2) prevents pregnancy-driven mammary cancer metastases.

BACKGROUND: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice. METHODS: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry. RESULTS: Oas2 mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when the Oas2 mutation was present. CONCLUSIONS: These data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy.

Platform for isolation and characterization of SARS-CoV-2 variants enables rapid characterization of Omicron in Australia.

Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.

STAT5B restrains human B-cell differentiation to maintain humoral immune homeostasis.

BACKGROUND: Lymphocyte differentiation is regulated by coordinated actions of cytokines and signaling pathways. IL-21 activates STAT1, STAT3, and STAT5 and is fundamental for the differentiation of human B cells into memory cells and antibody-secreting cells. While STAT1 is largely nonessential and STAT3 is critical for this process, the role of STAT5 is unknown. OBJECTIVES: This study sought to delineate unique roles of STAT5 in activation and differentiation of human naive and memory B cells. METHODS: STAT activation was assessed by phospho-flow cytometry cell sorting. Differential gene expression was determined by RNA-sequencing and quantitative PCR. The requirement for STAT5B in B-cell and CD4+ T-cell differentiation was assessed using CRISPR-mediated STAT5B deletion from B-cell lines and investigating primary lymphocytes from individuals with germline STAT5B mutations. RESULTS: IL-21 activated STAT5 and strongly induced SOCS3 in human naive, but not memory, B cells. Deletion of STAT5B in B-cell lines diminished IL-21-mediated SOCS3 induction. PBMCs from STAT5B-null individuals contained expanded populations of immunoglobulin class-switched B cells, CD21loTbet+ B cells, and follicular T helper cells. IL-21 induced greater differentiation of STAT5B-deficient B cells into plasmablasts in vitro than B cells from healthy donors, correlating with higher expression levels of transcription factors promoting plasma cell formation. CONCLUSIONS: These findings reveal novel roles for STAT5B in regulating IL-21-induced human B-cell differentiation. This is achieved by inducing SOCS3 to attenuate IL-21 signaling, and BCL6 to repress class switching and plasma cell generation. Thus, STAT5B is critical for restraining IL-21-mediated B-cell differentiation. These findings provide insights into mechanisms underpinning B-cell responses during primary and subsequent antigen encounter and explain autoimmunity and dysfunctional humoral immunity in STAT5B deficiency.

Augmented neutralization of SARS-CoV-2 Omicron variant by boost vaccination and monoclonal antibodies.

Effective vaccines and monoclonal antibodies have been developed against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the appearance of virus variants with higher transmissibility and pathogenicity is a major concern because of their potential to escape vaccines and clinically approved SARS-CoV-2- antibodies. Here, we use flow cytometry-based binding and pseudotyped SARS-CoV-2 neutralization assays to determine the efficacy of boost immunization and therapeutic antibodies to neutralize the dominant Omicron variant. We provide compelling evidence that the third vaccination with BNT162b2 increases the amount of neutralizing serum antibodies against Delta and Omicron variants, albeit to a lower degree when compared to the parental Wuhan strain. Therefore, a third vaccination is warranted to increase titers of protective serum antibodies, especially in the case of the Omicron variant. We also found that most clinically approved and otherwise potent therapeutic antibodies against the Delta variant failed to recognize and neutralize the Omicron variant. In contrast, some antibodies under preclinical development potentially neutralized the Omicron variant. Our studies also support using a flow cytometry-based antibody binding assay to rapidly monitor therapeutic candidates and serum titers against emerging SARS-CoV-2 variants.

Uncontrolled CD21low age-associated and B1 B cell accumulation caused by failure of an EGR2/3 tolerance checkpoint.

CD21low age-associated or atypical memory B cells are autoantibody enriched and poised for plasma cell differentiation. These cells overaccumulate in chronic infections, autoimmune disease, and immunodeficiency, posing the question of what checkpoints normally oppose their accumulation. Here, we reveal a critical role for paralogous calcium-NFAT-regulated transcription factors EGR2 and EGR3 that are induced in self-reactive B cells. CD21low and B1 B cells lacking EGR2 and EGR3 accumulate and circulate in young mice in numbers 10- to 20-fold greater than normal and overexpress a large set of EGR2 ChIP-seq target genes, including known drivers of plasma cell differentiation. Most follicular B cells constitutively express Egr2 proportionally to surface IgM downregulation by self-antigens, and EGR2/3 deficiency abolishes this cardinal feature of B cell anergy. These results explain the cardinal features of B cell anergy, define a key transcriptional checkpoint repressing CD21low B cell formation, and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.