Human complete NFAT1 deficiency causes a triad of joint contractures, osteochondromas, and B-cell malignancy.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell-omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

Investigating microstructure evolution in block copolymer membranes.

Block copolymer self-assembly in conjunction with nonsolvent-induced phase separation (SNIPS) has been increasingly leveraged to fabricate integral-asymmetric membranes. The large number of formulation and processing parameters associated with SNIPS, however, has prevented the reliable construction of high performance membranes. In this study, we apply dynamical self-consistent field theory to model the SNIPS process and investigate the effect of various parameters on the membrane morphology: solvent selectivity, nonsolvent selectivity, initial film composition, and glass transition composition. We examine how solvent selectivity and concentration of polymers in the film impact the structure of micelles that connect to form the membrane matrix. In particular, we find that preserving the order in the surface layer and forming a connection between the supporting and surface layer are nontrivial and sensitive to each parameter studied. The effect of each parameter is discussed, and suggestions are made for successfully fabricating viable block copolymer membranes.

Slipped capital femoral epiphysis: emphasis on early recognition and potential pitfalls.

Slipped capital femoral epiphysis is a shearing injury through the growth plate of the proximal femur and is the most common hip disorder in adolescence. Delays in diagnosis persist across practice settings despite ongoing innovations in imaging. Recent insights into pathomechanics highlight the importance of femoral head surface morphology and rotational microinstability centered at the epiphyseal tubercle in causing early physeal changes, which can be detected on imaging prior to frank slip. Scrutiny of physeal morphology and comparison to the contralateral hip is critical at all stages of disease progression, and improper technique may result in undue diagnostic delay. Selective use of cross-sectional imaging can be helpful for troubleshooting equivocal early slips and can inform operative technique and adjuvant therapy candidacy in more severe cases. This review provides a comprehensive approach to imaging suspected slipped capital femoral epiphysis, with an emphasis on early detection and potential pitfalls.

Fully Automated Analysis of the Anatomic and Mechanical Axes From Pediatric Standing Lower Limb Radiographs Using Convolutional Neural Networks.

BACKGROUND: Lower limb alignment is the quantification of a set of parameters that are commonly measured radiographically to test for and track a wide range of skeletal pathologies. Determining limb alignment is a commonly performed yet laborious task in the pediatric orthopaedic setting and is therefore an interesting goal for automation. METHODS: We employ a machine learning approach using convolutional neural networks (CNNs) to segment pediatric weight-bearing lower limb radiographs. The results are then used with custom Matlab code to extract anatomic landmarks and to determine lower limb alignment parameters. RESULTS: Measurements obtained from the automated workflow proposed here were compared with manual measurements performed by orthopaedic surgery fellows. Mechanical axis deviation was determined within a mean of 2.02 mm. Lateral distal femoral angle and medial proximal tibial angle were determined with a mean deviation of 1.73 and 2.90 degrees, respectively. The calculation speed for the full set of mechanical and anatomic axis parameters was found to be ~2 seconds per radiograph. CONCLUSIONS: The CNN-based approach proposed in this work was shown to produce results comparable to orthopaedic surgery fellows at fast calculation speed. Although further work is needed to validate these results against radiographs and measurements from other centers, we see this as a promising start and a functional path that can be employed in further research. CLINICAL RELEVANCE: CNNs are a promising approach to automating commonly performed, repetitive tasks, especially those pertaining to image processing. The time savings are particularly important in clinical research applications where large sets of radiographs are routinely available and require analysis. With further development of these algorithms, we anticipate significantly improved agreement with expert-measured results and the calculation speed.

Synchronization and Clustering in Complex Quadratic Networks.

Synchronization and clustering are well studied in the context of networks of oscillators, such as neuronal networks. However, this relationship is notoriously difficult to approach mathematically in natural, complex networks. Here, we aim to understand it in a canonical framework, using complex quadratic node dynamics, coupled in networks that we call complex quadratic networks (CQNs). We review previously defined extensions of the Mandelbrot and Julia sets for networks, focusing on the behavior of the node-wise projections of these sets and on describing the phenomena of node clustering and synchronization. One aspect of our work consists of exploring ties between a network's connectivity and its ensemble dynamics by identifying mechanisms that lead to clusters of nodes exhibiting identical or different Mandelbrot sets. Based on our preliminary analytical results (obtained primarily in two-dimensional networks), we propose that clustering is strongly determined by the network connectivity patterns, with the geometry of these clusters further controlled by the connection weights. Here, we first explore this relationship further, using examples of synthetic networks, increasing in size (from 3, to 5, to 20 nodes). We then illustrate the potential practical implications of synchronization in an existing set of whole brain, tractography-based networks obtained from 197 human subjects using diffusion tensor imaging. Understanding the similarities to how these concepts apply to CQNs contributes to our understanding of universal principles in dynamic networks and may help extend theoretical results to natural, complex systems.

Multiscale modeling of solute diffusion in triblock copolymer membranes.

We develop a multiscale simulation model for diffusion of solutes through porous triblock copolymer membranes. The approach combines two techniques: self-consistent field theory (SCFT) to predict the structure of the self-assembled, solvated membrane and on-lattice kinetic Monte Carlo (kMC) simulations to model diffusion of solutes. Solvation is simulated in SCFT by constraining the glassy membrane matrix while relaxing the brush-like membrane pore coating against the solvent. The kMC simulations capture the resulting solute spatial distribution and concentration-dependent local diffusivity in the polymer-coated pores; we parameterize the latter using particle-based simulations. We apply our approach to simulate solute diffusion through nonequilibrium morphologies of a model triblock copolymer, and we correlate diffusivity with structural descriptors of the morphologies. We also compare the model's predictions to alternative approaches based on simple lattice random walks and find our multiscale model to be more robust and systematic to parameterize. Our multiscale modeling approach is general and can be readily extended in the future to other chemistries, morphologies, and models for the local solute diffusivity and interactions with the membrane.

Early Investigation and Bracing in DDH Impacts Maternal Wellbeing and Breastfeeding.

BACKGROUND: The outcomes of Pavlik Harness (PH) management for Developmental Dysplasia of the Hip (DDH) are equivalent regardless of the initiation timing, if it is within the first 6 weeks of life. A PH may be a physical barrier to breastfeeding, which is important for nutrition, immunity, and normal child development. The diagnosis of DDH and early management with a PH may also negatively affect the maternal psychosocial wellbeing and the infant-maternal relationship. The purpose of this study is to investigate the impact of the diagnosis of DDH and the management with a PH has on maternal wellbeing and maintenance of breastfeeding, compared with being screened for but not diagnosed with DDH. METHODS: A retrospective cohort of the mothers of infants who were diagnosed with DDH and treated with a PH brace was compared with the mothers of infants who were screened for DDH only. The Hip Worries Inventory and Edinburgh Postnatal Depression Scale were completed by the mothers in both groups. The PH group also completed an in-house questionnaire specific to PH and breastfeeding. RESULTS: Eighty completed surveys were included, 50 from the treatment group. The mean age of the PH initiation was 6.2 weeks. The modified Hip Worries Inventory score was higher in the treatment group, with a mean difference (MD) of 9.7 out of 50 (95% confidence interval, CI, 6.8, 12.5). The MD of the Edinburgh Postnatal Depression Scale was 2.0 out of 30 (CI -0.5, 4.5). Although there was no difference in the breastfeeding ease before and after the PH initiation (MD-0.2, CI-0.7, 0.2), 83% of mothers found breastfeeding more difficult with a PH and 11% of mothers stopped breastfeeding earlier than planned because of the PH. CONCLUSIONS: Mothers of infants with DDH worry more about their child's hips and the PH. Screening alone may contribute to maternal psychological dejection and negative thoughts. The presence of a PH makes breastfeeding more difficult. LEVEL OF EVIDENCE: Retrospective comparative study, level III.

Establishing content validity of LIMB-Q Kids: a new patient-reported outcome measure for lower limb deformities.

PURPOSE: To establish the content validity of LIMB-Q Kids, a new patient-reported outcome measure (PROM) for children with lower limb deformities. This study focused on three key components of content validity, i.e., comprehension, comprehensiveness, and relevance. METHODS: Cognitive debriefing interviews (CDIs) with children with lower limb deformities were performed, and expert input from parents and clinicians directly involved in the care of children with lower limb deformities was obtained. CDIs were conducted with children from sites in Australia, Canada, and the USA. All interviews were recorded and transcribed verbatim. Analyses were conducted using the reparative approach, which involved close examination of the transcribed interviews and summarising edits after each interview. RESULTS: Forty patients, experts, and parents reviewed LIMB-Q Kids and provided feedback. At the start, LIMB-Q Kids had 10 scales and 124 total items. Five rounds of input was obtained from children, parents, and experts. Overall, 37 new items were added. Thirty-three of the new items were included to measure symptoms experienced in different parts of the legs. Final version of LIMB-Q Kids had 11 scales and 159 items. CONCLUSION: This study established content validity of the LIMB-Q Kids, a new PROM for children with lower limb deformities. An international field-test study is underway. Translation and cultural adaptation are underway for sites where English is not the first language. Scoring algorithms will be developed, following which the scale could be used to inform clinical practice and research.

Efficacy and Safety of "Sleeper Plate" in Temporary Hemiepiphysiodesis and the Observation of "Tethering".

BACKGROUND: Guided growth is commonly performed by placing an extraperiosteal 2-hole plate across the growth plate with one epiphyseal and one metaphyseal screw. Recent studies investigated the efficacy of the removal of the metaphyseal screw only (sleeper plate) after correction. They concluded the practice to be unnecessary as only 19% of patients showed recurrence of deformity. This study aims to examine the incidence of rebound and undesired bony in-growth of the plate (tethering) after metaphyseal screw removal only. METHODS: In this retrospective case series, patient data on 144 plates inserted around the knee were obtained. Plates still in situ (n=69) at the time of study and full hardware removal (n=50) were excluded. The remaining 25 plates had only the metaphyseal screw removed after completed deformity correction. We analyzed the rate of tethering, rebound, and maintenance of correction in 2 age groups at latest follow (mean of 3.5 y). The Fisher exact test with Freeman-Halton extension was used to analyze categorical data and the Student t test for descriptive variables. RESULTS: Twenty-five plates were identified as "sleeper plates" in our series. Thirteen plates (52%) maintained the achieved correction after a mean of 21 months (range: 4 to 39 mo), 9 plates (36%) required screw reinsertion due to rebound after a mean of 22 months (range: 12 to 48 mo) from screw removal, and 4 plates (16%) showed tethering with undesired continuation of guided growth after a mean of 14 months (range: 7 to 22 mo) from screw removal. Younger patients (<8 y at time of plate insertion) had higher rates of rebound and tethering ( P =0.0112, Fisher exact test). All tethering occurred in titanium plates, none occurred in steel plates. CONCLUSIONS: The sleeper plate is an acceptable treatment strategy for coronal deformities around the knee, however, tethering and rebound may occur, especially in younger patients. Titanium plates may increase the risk of tethering, however, further long-term follow-up is needed as there were only 6 steel plates versus 19 titanium in this study. We stress the importance of close postoperative follow-up to identify signs of tethering and rebound early to prevent over-correction. LEVEL OF EVIDENCE: Level IV-retrospective case study.

Survey of the Impact of COVID-19 on Pediatric Orthopaedic Surgeons Globally.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic required rapid, global health care shifts to prioritize urgent or pandemic-related care and minimize transmission. Little is known about impacts on pediatric orthopaedic surgeons during this time. We aimed to investigate COVID-19-related changes in practice, training, and research among pediatric orthopaedic surgeons globally. METHODS: An online survey was administered to orthopaedic surgeons with interest in pediatrics in April 2020 and a follow-up was administered in February 2021. The surveys captured demographics and surgeons' self-reported experiences during the pandemic. Participants were recruited from web media and available email lists of orthopaedic societies over a 2-month period. Descriptive statistics were used to analyze results, stratified by the severity of local COVID-19-related measures. RESULTS: A total of 460 responses from 45 countries were collected for initial survey. Of these, 358 (78.5%) respondents reported lockdown measures in their region at time of survey. Most (n=337, 94.4%) reported pausing all elective procedures. Surgeons reported a reduction in the average number of surgeries per week, from 6.89 (SD=4.61) prepandemic to 1.25 (SD=2.26) at time of survey (mean difference=5.64; 95% confidence interval=5.19, 6.10). Average number of elective outpatient appointments per week decreased from 67.89 (SD=45.78) prepandemic to 11.79 (SD=15.83) at time of survey (mean difference=56.10, 95% confidence interval=5.61, 60.58). In total, 177 (39.4%) surgeons reported using virtual modes of outpatient appointments for the first time. Of 290 surgeons with trainees, 223 (84.5%) reported implementation of systems to continue training such as webinars or virtual rounds. Of 192 respondents with research, 149 (82.8%) reported continuing research activities during the pandemic with most reporting either cessation (n=75, 64.15%), or reduction (n=25, 29.9%) in participant recruitment. A total of 111 responses from 28 countries were collected during follow-up. Surgeons described policy and circumstantial changes that facilitated resumption of clinical work. CONCLUSIONS: The COVID-19 pandemic and its related counter measures have had significant impacts on pediatric orthopaedic practice and increased uptake of technology to provide care continuity. Rigorous epidemiological studies are needed to assess impacts of delayed and virtual care on patient outcomes.

Modernization of bone age assessment: comparing the accuracy and reliability of an artificial intelligence algorithm and shorthand bone age to Greulich and Pyle.

Greulich and Pyle (GP) is one of the most common methods to determine bone age from hand radiographs. In recent years, new methods were developed to increase the efficiency in bone age analysis like the shorthand bone age (SBA) and automated artificial intelligence algorithms. OBJECTIVE: The aim of this study is to evaluate the accuracy and reliability of these two methods and examine if the reduction in analysis time compromises their efficacy. METHODS: Two hundred thirteen males and 213 females had their bone age determined by two separate raters using the SBA and GP methods. Three weeks later, the two raters repeated the analysis of the radiographs. The raters timed themselves using an online stopwatch. De-identified radiographs were securely uploaded to an automated algorithm developed by a group of radiologists in Toronto. The gold standard was determined to be the radiology report attached to each radiograph, written by experienced radiologists using GP. RESULTS: Intraclass correlation between each method and the gold standard fell within the range of 0.8-0.9, highlighting significant agreement. Most of the comparisons showed a statistically significant difference between the new methods and the gold standard; however, it may not be clinically significant as it ranges between 0.25 and 0.5 years. A bone age is considered clinically abnormal if it falls outside 2 standard deviations of the chronological age; standard deviations are calculated and provided in GP atlas. CONCLUSION: The shorthand bone age method and the automated algorithm produced values that are in agreement with the gold standard while reducing analysis time.

Support, information, and integration of genetics for children with congenital lower limb deficiencies in British Columbia, Canada.

OBJECTIVES: Children and families affected by congenital limb deficiencies (CLD) require a unique level of emotional support from diagnosis through to adolescence. The following study aims to collect data on Canadian paediatric patients affected by a CLD followed at BC Children's Hospital (BCCH), Department of Orthopaedics. METHODS: Parents of children with a CLD were asked to complete a written questionnaire examining their experiences. Qualitative and quantitative data were collected concerning parent satisfaction with patient referrals, emotional support, and knowledge of their child's diagnosis. RESULTS: Twenty-five completed questionnaires were returned. Fifty per cent of the parents reported they were either very satisfied, or satisfied, with the emotional support provided by health care providers (HCPs). Twenty-five per cent of the parents were unsatisfied with the emotional support received by HCPs. Forty-eight per cent of the parents could not recall the specific name of their child's diagnosis; 20% of the parents reported their child did not have diagnosis. All the patients in our study had received a clinical diagnosis. Twenty-eight per cent of the parents in this study were also seen in medical genetics. CONCLUSIONS: Families require additional resources for emotional support, peer support, and referrals to support organizations. Gaps in parent knowledge regarding their child's CLD suggest the need for formalized communication strategies for HCPs. Furthermore, patients with CLDs and their families may benefit from improved communication between orthopaedic and medical genetic services at the time of diagnosis. Integration of genetic counsellors may improve emotional supports and education for families with regards to testing and reproductive planning.

Southwick angle measurements and SCFE slip severity classifications are affected by frog-lateral positioning.

OBJECTIVE: Slipped capital femoral epiphysis (SCFE) is a hip disorder where the femoral head slips relative to the neck at the physis. Appropriate treatment of SCFE depends on the severity of the slip, commonly categorised using the Southwick (SW) angle. The SW angle is measured in the frog-lateral leg position, which can be painful and potentially unattainable for patients. The purpose of this study is to determine how errors in frog-lateral radiograph positioning affect measured SW angles and slip classifications. METHODS: Models of SCFE hips were produced from one CT scan of a normal hip; 360 deformities were created. SW angles were measured from a simulated frog-lateral position. Femoral lateral head-neck angles (LHNA; equivalent to SW in incorrect frog-lateral plane) were measured over a range of 837 incorrect frog-lateral leg positions with positioning errors in flexion and/or internal/external rotation. RESULTS: Seventy-six per cent of all imaging position-deformity combinations had error in the reported angle (>1° difference between LHNA and SW). Of those, 70% had <5°, 24% had 5° to 10°, and 6% had >10° of error from the actual SW angle. Three per cent of LHNAs that had >10° error resulted from <10° of positioning error. CONCLUSIONS: If the patient is limited in flexion or external rotation, more diagnostic testing should be considered if error in the reported slip measurement would affect treatment decisions or if accurate severity classification is needed for research. Small positioning errors in moderate and severe slips can cause a > 10° LHNA error; additional three-dimensional imaging should be considered.

PD-1 coinhibitory signals: the link between pathogenesis and protection.

In the majority of HIV-1 infected individuals, the adaptive immune response drives virus escape resulting in persistent viremia and a lack of immune-mediated control. The expression of negative regulatory molecules such as PD-1 during chronic HIV infection provides a useful marker to differentiate functional memory T cell subsets and the frequency of T cells with an exhausted phenotype. In addition, cell-based measurements of virus persistence equate with activation markers and the frequency of CD4 T cells expressing PD-1. High-level expression of PD-1 and its ligands PD-L1 and PD-L2 are found on hematopoietic and non-hematopoietic cells, and are upregulated by chronic antigen stimulation, Type 1 and Type II interferons (IFNs), and homeostatic cytokines. In HIV infected subjects, PD-1 levels on CD4 and CD8 T cells continue to remain high following combination anti-retroviral therapy (cART). System biology approaches have begun to elucidate signal transduction pathways regulated by PD-1 expression in CD4 and CD8 T cell subsets that become dysfunctional through chronic TCR activation and PD-1 signaling. In this review, we summarize our current understanding of transcriptional signatures and signal transduction pathways associated with immune exhaustion with a focus on recent work in our laboratory characterizing the role of PD-1 in T cell dysfunction and HIV pathogenesis. We also highlight the therapeutic potential of blocking PD-1-PD-L1 and other immune checkpoints for activating potent cellular immune responses against chronic viral infections and cancer.

Relationships Between Severity of Deformity and Impingement in Slipped Capital Femoral Epiphysis.

BACKGROUND: In situ pinning, a low-risk treatment for slipped capital femoral epiphysis (SCFE), leaves the slipped femoral head in place and may reduce range of motion (ROM) and cause impingement. It is unclear when a more complex surgery should be considered, because the relationships between severity, slip stability, remodeling, impingement, and ROM are unknown. RESEARCH QUESTIONS: (1) Do more severe acute SCFE deformities (no bony remodeling) result in a greater loss of flexion ROM?(2) Does the presence or location of impingement on the pelvis vary with severity of acute SCFE deformity? METHODS: We developed a 3D geometric model of acute SCFE deformity from 1 computed tomography scan of a normal adolescent hip. Ethics board approval was obtained from our institution. Bone models were created from the segmented pelvis, epiphysis, and subphyseal femur.In total, 3721 SCFE deformities were simulated by combining posterior and inferior slips in the axial and coronal planes, respectively. Southwick angles were estimated from a frog-leg lateral projection. Deformities were divided into mild (0 to 30 degrees), moderate (30 to 60 degrees), and severe (≥60 degrees) Southwick groups. Each joint was flexed in combination with internal/external rotation until contact occurred. A total of 121 ROM trials, with different degrees of internal/external rotation (0 to 90 degrees at 1.5-degree steps) were performed for each deformity. RESULTS: In total, 3355 simulated SCFE deformities (363 could not be rotated out of impingement) were analyzed.Increasing slip severity reduced flexion ROM across the range of internal/external rotation. Contact occurred for most mild deformities, and for all moderate and severe deformities in at least 1 ROM trial. Impingement was observed mainly on the anterosuperior aspect of the acetabulum. CONCLUSIONS: Increasing slip severity in acute SCFE reduced flexion and increased incidence of impingement, primarily occurring on the anterosuperior aspect of the acetabulum. The impingement patterns observed are consistent with damaged cartilage locations seen in clinical literature. CLINICAL RELEVANCE: In this experimental model, moderate and severe acute slips in SCFE lead to reduced ROM and impingement with the acetabulum. This suggests that in situ pinning may result in impingement of moderate and severe acute SCFE slips.

The immunological synapse: the gateway to the HIV reservoir.

A major challenge in the development of a cure for human immunodeficiency virus (HIV) has been the incomplete understanding of the basic mechanisms underlying HIV persistence during antiretroviral therapy. It is now realized that the establishment of a latently infected reservoir refractory to immune system recognition has thus far hindered eradication efforts. Recent investigation into the innate immune response has shed light on signaling pathways downstream of the immunological synapse critical for T-cell activation and establishment of T-cell memory. This has led to the understanding that the cell-to-cell contacts observed in an immunological synapse that involve the CD4(+) T cell and antigen-presenting cell or T-cell-T-cell interactions enhance efficient viral spread and facilitate the induction and maintenance of latency in HIV-infected memory T cells. This review focuses on recent work characterizing the immunological synapse and the signaling pathways involved in T-cell activation and gene regulation in the context of HIV persistence.

Synthesis of dibenzylamino-1-methylcyclohexanol and dibenzylamino-1-trifluoromethylcyclohexanol isomers.

The isomers of dibenzylamino-1-methylcyclohexan-1-ol and dibenzylamino-1-trifluoromethylcyclohexan-1-ol have been prepared. The stereochemistry of these compounds was unequivocally assigned through a combination of NMR spectroscopy and single crystal X-ray analysis. The cis-isomer of 3-N,N-dibenzylamino-1-trifluoromethylcyclohexanol and its derivatives display an unusual conformational behaviour in both solution-phase and the solid-state, where the amino group usually adopts an axial conformation.

B cells from knock-in mice expressing broadly neutralizing HIV antibody b12 carry an innocuous B cell receptor responsive to HIV vaccine candidates.

Broadly neutralizing Abs against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic ("knock-in") mice expressing, in the physiological Ig H and L chain loci, two well-studied broadly neutralizing Abs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the companion article (Doyle-Cooper et al., J. Immunol. 191: 3186-3191). In this article, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV envelope gp140 trimers and to BCR ligands. Upon transfer to wild-type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, although generating abundant precursors and Abs with affinity for Env, were unable to rapidly generate neutralizing Abs, highlighting the importance of developing Ag forms that better focus responses to neutralizing epitopes. The b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding nonprotective specificities.

Immune tolerance negatively regulates B cells in knock-in mice expressing broadly neutralizing HIV antibody 4E10.

A major goal of HIV research is to develop vaccines reproducibly eliciting broadly neutralizing Abs (bNAbs); however, this has proved to be challenging. One suggested explanation for this difficulty is that epitopes seen by bNAbs mimic self, leading to immune tolerance. We generated knock-in mice expressing bNAb 4E10, which recognizes the membrane proximal external region of gp41. Unlike b12 knock-in mice, described in the companion article (Ota et al. 2013. J. Immunol. 191: 3179-3185), 4E10HL mice were found to undergo profound negative selection of B cells, indicating that 4E10 is, to a physiologically significant extent, autoreactive. Negative selection occurred by various mechanisms, including receptor editing, clonal deletion, and receptor downregulation. Despite significant deletion, small amounts of IgM and IgG anti-gp41 were found in the sera of 4E10HL mice. On a Rag1⁻/⁻ background, 4E10HL mice had virtually no serum Ig of any kind. These results are consistent with a model in which B cells with 4E10 specificity are counterselected, raising the question of how 4E10 was generated in the patient from whom it was isolated. This represents the second example of a membrane proximal external region-directed bNAb that is apparently autoreactive in a physiological setting. The relative conservation in HIV of the 4E10 epitope might reflect the fact that it is under less intense immunological selection as a result of B cell self-tolerance. The safety and desirability of targeting this epitope by a vaccine is discussed in light of the newly described bNAb 10E8.