WNT2B Deficiency Causes Enhanced Susceptibility to Colitis Due to Increased Inflammatory Cytokine Production.

BACKGROUND & AIMS: Humans with WNT2B deficiency have severe intestinal disease, including significant inflammatory injury, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. METHODS: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon, and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue. RESULTS: Mice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased, epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6, and biopsy tissue from humans showed increased neutrophils. CONCLUSIONS: WNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation, particularly in the colon.

Applying the bronchopulmonary dysplasia framework to necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of the neonatal intestine, causing widespread intestinal necrosis as well systemic illness that frequently results in death. Because the clinical onset of NEC is sudden and difficult to predict, NEC is considered an acute event. However, NEC does not occur in utero, meaning that postnatal exposures are required, and it does not typically occur right after birth, suggesting that longitudinal changes may be occurring before NEC can develop. In this perspective, the author considers whether NEC should be re-considered as a problem of disordered intestinal epithelial development, with required maladaptation over time prior to the onset of the necrotic event. This framework is similar to how bronchopulmonary dysplasia is currently conceptualized. They also advocate that NEC researchers incorporate this possibility into future studies on NEC susceptibility and pathogenesis.

M1 and M2 Macrophages Differentially Regulate Colonic Crypt Renewal.

BACKGROUND: The colonic epithelium is the most rapidly renewing tissue in the body and is organized into a single cell layer of invaginations called crypts. Crypt renewal occurs through Lgr5 + gut stem cells situated at the crypt base, which divide, produce daughter cells that proliferate, migrate, differentiate into all the cells required for normal gut function, and are finally shed into the crypt lumen. In health, this rapid renewal helps maintain barrier function next to the hostile gut microbial luminal environment. Inflammation results in an influx of immune cells including inflammatory M1 macrophages into the gut mucosa next to the crypt epithelium, but the direct effect of macrophages on crypt regeneration and renewal are poorly understood. METHODS: Using an in vitro macrophage-crypt coculture model, we show that homeostatic M2 macrophages and inflammatory M1 macrophages confer different effects on the crypt epithelium. RESULTS: Both M1 and M2 increase crypt cell proliferation, with M2 macrophages requiring physical contact with the crypt epithelium, whereas M1 macrophages exert their effect through a secreted factor. Only M1 macrophages reduce goblet and Tuft cell numbers and increase Lgr5 + crypt stem cell numbers, all dependent on physical contact with the crypt epithelium. Further studies showed that M1 macrophages increase the Wnt signaling pathways cyclin D1 and LEF1 through physical contact rather than a secreted factor. CONCLUSIONS: These findings highlight the importance of understanding distinct cellular interactions and direct dialogue between cells and increase our understanding of the contribution of different immune cell subtypes on crypt cell biology during inflammation.

Inflammatory macrophages but not homeostatic macrophages modulate crypt epithelial cell differentiation. Direct physical contact between an inflammatory macrophage and the crypt epithelium is required for regulation of differentiation, but crypt proliferation is via a secreted factor.

Intravenous Bacille Calmette-Guérin vaccination protects simian immunodeficiency virus-infected macaques from tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the most common cause of death in people living with human immunodeficiency virus (HIV). Intra-dermal Bacille Calmette-Guérin (BCG) delivery is the only licensed vaccine against tuberculosis; however, it offers little protection from pulmonary tuberculosis in adults and is contraindicated in people living with HIV. Intravenous BCG confers protection against Mtb infection in rhesus macaques; we hypothesized that it might prevent tuberculosis in simian immunodeficiency virus (SIV)-infected macaques, a model for HIV infection. Here intravenous BCG-elicited robust airway T cell influx and elevated plasma and airway antibody titres in both SIV-infected and naive animals. Following Mtb challenge, all 7 vaccinated SIV-naive and 9 out of 12 vaccinated SIV-infected animals were protected, without any culturable bacteria detected from tissues. Peripheral blood mononuclear cell responses post-challenge indicated early clearance of Mtb in vaccinated animals, regardless of SIV infection. These data support that intravenous BCG is immunogenic and efficacious in SIV-infected animals.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

BACKGROUND: Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical, host-derived regulators of the microbiota. However, mechanisms that support microbiota homeostasis in response to inflammatory stimuli, such as supraphysiologic oxygen, remain unclear. RESULTS: We show that supraphysiologic oxygen exposure to neonatal mice, or direct exposure of intestinal organoids to supraphysiologic oxygen, suppresses the intestinal expression of AMPs and alters intestinal microbiota composition. Oral supplementation of the prototypical AMP lysozyme to hyperoxia-exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. CONCLUSIONS: Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury in newborns. Together, these data support that intestinal AMPs modulate lung injury and repair. Video Abstract.

CD8+ cells and small viral reservoirs facilitate post-ART control of SIV replication in M3+ Mauritian cynomolgus macaques initiated on ART two weeks post-infection.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

Mathematical modeling indicates that regulatory inhibition of CD8+ T cell cytotoxicity can limit efficacy of IL-15 immunotherapy in cases of high pre-treatment SIV viral load.

Immunotherapeutic cytokines can activate immune cells against cancers and chronic infections. N-803 is an IL-15 superagonist that expands CD8+ T cells and increases their cytotoxicity. N-803 also temporarily reduced viral load in a limited subset of non-human primates infected with simian immunodeficiency virus (SIV), a model of HIV. However, viral suppression has not been observed in all SIV cohorts and may depend on pre-treatment viral load and the corresponding effects on CD8+ T cells. Starting from an existing mechanistic mathematical model of N-803 immunotherapy of SIV, we develop a model that includes activation of SIV-specific and non-SIV-specific CD8+ T cells by antigen, inflammation, and N-803. Also included is a regulatory counter-response that inhibits CD8+ T cell proliferation and function, representing the effects of immune checkpoint molecules and immunosuppressive cells. We simultaneously calibrate the model to two separate SIV cohorts. The first cohort had low viral loads prior to treatment (≈3-4 log viral RNA copy equivalents (CEQ)/mL), and N-803 treatment transiently suppressed viral load. The second had higher pre-treatment viral loads (≈5-7 log CEQ/mL) and saw no consistent virus suppression with N-803. The mathematical model can replicate the viral and CD8+ T cell dynamics of both cohorts based on different pre-treatment viral loads and different levels of regulatory inhibition of CD8+ T cells due to those viral loads (i.e. initial conditions of model). Our predictions are validated by additional data from these and other SIV cohorts. While both cohorts had high numbers of activated SIV-specific CD8+ T cells in simulations, viral suppression was precluded in the high viral load cohort due to elevated inhibition of cytotoxicity. Thus, we mathematically demonstrate how the pre-treatment viral load can influence immunotherapeutic efficacy, highlighting the in vivo conditions and combination therapies that could maximize efficacy and improve treatment outcomes.

Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Development, Implementation and Evaluation of an Acute Care Physical Therapy 'Float' Placement during the COVID-19 Pandemic: A Case Report.

Clinical education is a mandatory component of physical therapy curricula globally. COVID-19 disrupted clinical education, jeopardizing students' abilities to meet graduation requirements. The objective of this case report is to outline the development, implementation and evaluation of a multiple clinical instructor (CI), multiple unit, acute care float clinical placement for a final year, entry-level physical therapy student and offer implementation recommendations. This placement included an eight-week, multiple CI (one primary, four supporting), multiple (five) unit clinical placement which was developed between St. Joseph's Healthcare and the McMaster University Masters of Science (Physiotherapy) Program between 10 August and 2 October 2020. Student evaluations and reflections by the student and CIs were collected and analyzed using interpretive description. Analysis from the reflections revealed six themes: (1) CI and student attributes; (2) increased feasibility; (3) varied exposure; (4) central communication and resources; (5) organization; and (6) managing expectations. An acute care clinical experience is required for students in Canadian entry-to-practice physical therapy programs. Due to COVID-19, placement opportunities were limited. The float placement allowed clinicians to offer supervision despite staff re-deployment and increased organizational and work-life pressures during the pandemic. This model provides an approach to extenuating circumstances and may also increase acute care placements during non-pandemic times for physical therapy and other similarly structured healthcare professions.

WNT2B Deficiency Causes Increased Susceptibility to Colitis in Mice and Impairs Intestinal Epithelial Development in Humans.

Background and aims: WNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. Methods: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3χ antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses. Results: Mice with WNT2B deficiency had significantly decreased Lgr5 expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3χ antibody was similar in Wnt2b KO and wild type (WT) mice. In contrast, the colonic response to DSS in Wnt2b KO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature. Conclusion: WNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine.WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization. Data Transparency Statement: All RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

Vaccination with intravenous BCG protects macaques with pre-existing SIV infection from tuberculosis.

Tuberculosis (TB) is the most common cause of death in people living with HIV. BCG delivered intradermally (ID) is the only licensed vaccine to prevent TB. However, it offers little protection from pulmonary TB in adults. Intravenous (IV) BCG, but not ID BCG, confers striking protection against Mycobacterium tuberculosis (Mtb) infection and disease in rhesus macaques. We investigated whether IV BCG could protect against TB in macaques with a pre-existing SIV infection. There was a robust influx of airway T cells following IV BCG in both SIV-infected and SIV-naïve animals, with elevated antibody titers in plasma and airways. Following Mtb challenge, all 7 SIV-naïve and 9 out of 12 SIV-infected vaccinated animals were completely protected, without any culturable bacilli in their tissues. PBMC responses post-challenge indicated early clearance of Mtb in vaccinated animals regardless of SIV infection. These data support that IV BCG is immunogenic and efficacious in SIV-infected animals.

Ectopic Rod Photoreceptor Development in Mice with Genetic Deficiency of WNT2B.

Wnt/ß-catenin signaling is essential for embryonic eye development in both the anterior eye and retina. WNT2B, a ligand and activator of the Wnt/ß-catenin pathway, assists in the development of the lens and peripheral regions of the eye. In humans WNT2B mutations are associated with coloboma and WNT2B may also assist in retinal progenitor cell differentiation in chicken, yet the potential role of WNT2B in retinal neuronal development is understudied. This study explored the effects of WNT2B on retinal neuronal and vascular formation using systemic Wnt2b knockout (KO) mice generated by crossing Wnt2bflox/flox (fl/fl) mice with CMV-cre mice. Wnt2b KO eyes exhibited relatively normal anterior segments and retinal vasculature. Ectopic formation of rod photoreceptor cells in the subretinal space was observed in Wnt2b KO mice as early as one week postnatally and persisted through nine-month-old mice. Other retinal neuronal layers showed normal organization in both thickness and lamination, without detectable signs of retinal thinning. The presence of abnormal photoreceptor genesis was also observed in heterozygous Wnt2b mice, and occasionally in wild type mice with decreased Wnt2b expression levels. Expression of Wnt2b was found to be enriched in the retinal pigment epithelium compared with whole retina. Together these findings suggest that WNT2B is potentially involved in rod photoreceptor genesis during eye development; however, potential influence by a yet unknown genetic factor is also possible.

CD8+ cells and small viral reservoirs facilitate post-ART control of SIV in Mauritian cynomolgus macaques.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). For six months after ART withdrawal, we observed undetectable or transient viremia in seven of eight MCMs. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the PTC was mediated, at least in part, by CD8α+ cells. We found that MCMs had smaller acute viral reservoirs than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. The mechanisms by which unusually small viral reservoirs and CD8α+ cell-mediated virus suppression enable PTC can be investigated using this MHC-haplomatched MCM model. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical host-derived regulators of the microbiota. However, mechanisms that support homeostasis of the microbiota in response to inflammatory stimuli such as supraphysiologic oxygen remain unclear. Here, we show that neonatal mice breathing supraphysiologic oxygen or direct exposure of intestinal organoids to supraphysiologic oxygen suppress the intestinal expression of AMPs and alters the composition of the intestinal microbiota. Oral supplementation of the prototypical AMP lysozyme to hyperoxia exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury. Together, these data support that intestinal AMPs modulate lung injury and repair. In Brief: Using a combination of murine models and organoids, Abdelgawad and Nicola et al. find that suppression of antimicrobial peptide release by the neonatal intestine in response to supra-physiological oxygen influences the progression of lung injury likely via modulation of the ileal microbiota. Highlights: Supraphysiologic oxygen exposure alters intestinal antimicrobial peptides (AMPs).Intestinal AMP expression has an inverse relationship with the severity of lung injury.AMP-driven alterations in the intestinal microbiota form a gut-lung axis that modulates lung injury.AMPs may mediate a gut-lung axis that modulates lung injury.

A sepsis trigger tool reduces time to antibiotic administration in the NICU.

OBJECTIVE: Prolonged time to antibiotic administration is associated with increased morbidity and mortality. Interventions to decrease the time to antibiotic administration may improve mortality and morbidity. STUDY DESIGN: We identified possible change concepts for reducing time to antibiotic usage in the NICU. For the initial intervention, we developed a sepsis screening tool based on NICU-specific parameters. The main goal of the project was to reduce time to antibiotic administration by 10%. RESULTS: The project was conducted from April 2017 until April 2019. There were no missed cases of sepsis in the project period. Time to antibiotic administration for patients who were started on antibiotics decreased during the project, with the mean shifting from 126 to 102 min, a reduction of 19%. CONCLUSIONS: We successfully reduced time to antibiotic delivery in our NICU using a trigger tool to identifying potential cases of sepsis in the NICU environment. The trigger tool requires broader validation.

Transient T Cell Expansion, Activation, and Proliferation in Therapeutically Vaccinated Simian Immunodeficiency Virus-Positive Macaques Treated with N-803.

Vaccine strategies aimed at eliciting human immunodeficiency virus (HIV)-specific CD8+ T cells are one major target of interest in HIV functional cure strategies. We hypothesized that CD8+ T cells elicited by therapeutic vaccination during antiretroviral therapy (ART) would be recalled and boosted by treatment with the interleukin 15 (IL-15) superagonist N-803 after ART discontinuation. We intravenously immunized four simian immunodeficiency virus-positive (SIV+) Mauritian cynomolgus macaques receiving ART with vesicular stomatitis virus (VSV), modified vaccinia virus Ankara strain (MVA), and recombinant adenovirus serotype 5 (rAd-5) vectors all expressing SIVmac239 Gag. Immediately after ART cessation, these animals received three doses of N-803. Four control animals received no vaccines or N-803. The vaccine regimen generated a high-magnitude response involving Gag-specific CD8+ T cells that were proliferative and biased toward an effector memory phenotype. We then compared cells elicited by vaccination (Gag specific) to cells elicited by SIV infection and unaffected by vaccination (Nef specific). We found that N-803 treatment enhanced the frequencies of both bulk and proliferating antigen-specific CD8+ T cells elicited by vaccination and the antigen-specific CD8+ T cells elicited by SIV infection. In sum, we demonstrate that a therapeutic heterologous prime-boost-boost (HPBB) vaccine can elicit antigen-specific effector memory CD8+ T cells that are boosted by N-803. IMPORTANCE While antiretroviral therapy (ART) can suppress HIV replication, it is not a cure. It is therefore essential to develop therapeutic strategies to enhance the immune system to better become activated and recognize virus-infected cells. Here, we evaluated a novel therapeutic vaccination strategy delivered to SIV+ Mauritian cynomolgus macaques receiving ART. ART was then discontinued and we delivered an immunotherapeutic agent (N-803) after ART withdrawal with the goal of eliciting and boosting anti-SIV cellular immunity. Immunologic and virologic analysis of peripheral blood and lymph nodes collected from these animals revealed transient boosts in the frequency, activation, proliferation, and memory phenotype of CD4+ and CD8+ T cells following each intervention. Overall, these results are important in educating the field of the transient nature of the immunological responses to this particular therapeutic regimen and the similar effects of N-803 on boosting T cells elicited by vaccination or elicited naturally by infection.

Control of Simian Immunodeficiency Virus Infection in Prophylactically Vaccinated, Antiretroviral Treatment-Naive Macaques Is Required for the Most Efficacious CD8 T Cell Response during Treatment with the Interleukin-15 Superagonist N-803.

The IL-15 superagonist N-803 has been shown to enhance the function of CD8 T cells and NK cells. We previously found that in a subset of vaccinated, ART-naive, SIV+ rhesus macaques, N-803 treatment led to a rapid but transient decline in plasma viremia that positively correlated with an increase in the frequency of CD8 T cells. Here, we tested the hypothesis that prophylactic vaccination was required for the N-803 mediated suppression of SIV plasma viremia. We either vaccinated rhesus macaques with a DNA prime/Ad5 boost regimen using vectors expressing SIVmac239 gag with or without a plasmid expressing IL-12 or left them unvaccinated. The animals were then intravenously infected with SIVmac239M. 6 months after infection, the animals were treated with N-803. We found no differences in the control of plasma viremia during N-803 treatment between vaccinated and unvaccinated macaques. Interestingly, when we divided the SIV+ animals based on their plasma viral load set-points prior to the N-803 treatment, N-803 increased the frequency of SIV-specific T cells expressing ki-67+ and granzyme B+ in animals with low plasma viremia (<104 copies/mL; SIV controllers) compared to animals with high plasma viremia (>104 copies/mL; SIV noncontrollers). In addition, Gag-specific CD8 T cells from the SIV+ controllers had a greater increase in CD8+CD107a+ T cells in ex vivo functional assays than did the SIV+ noncontrollers. Overall, our results indicate that N-803 is most effective in SIV+ animals with a preexisting immunological ability to control SIV replication. IMPORTANCE N-803 is a drug that boosts the immune cells involved in combating HIV/SIV infection. Here, we found that in SIV+ rhesus macaques that were not on antiretroviral therapy, N-803 increased the proliferation and potential capacity for killing of the SIV-specific immune cells to a greater degree in animals that spontaneously controlled SIV than in animals that did not control SIV. Understanding the mechanism of how N-803 might function differently in individuals that control HIV/SIV (for example, individuals on antiretroviral therapy or spontaneous controllers) compared to settings where HIV/SIV are not controlled, could impact the efficacy of N-803 utilization in the field of HIV cure.

IL-15 Superagonist N-803 Enhances IFN-γ Production of MAIT Cells in SIV+ Macaques.

Mucosal associated invariant T (MAIT) cells are innate T cells that recognize bacterial metabolites and secrete cytokines and cytolytic enzymes to destroy infected target cells. This makes MAIT cells promising targets for immunotherapy to combat bacterial infections. Here, we analyzed the effects of an immunotherapeutic agent, the IL-15 superagonist N-803, on MAIT cell activation, trafficking, and cytolytic function in macaques. We found that N-803 could activate MAIT cells in vitro and increase their ability to produce IFN-γ in response to bacterial stimulation. To expand upon this, we examined the phenotypes and functions of MAIT cells present in samples collected from PBMC, airways (bronchoalveolar lavage [BAL] fluid), and lymph nodes (LN) from rhesus macaques that were treated in vivo with N-803. N-803 treatment led to a transient 6 to 7-fold decrease in the total number of MAIT cells in the peripheral blood, relative to pre N-803 time points. Concurrent with the decrease in cells in the peripheral blood, we observed a rapid decline in the frequency of CXCR3+CCR6+ MAITs. This corresponded with an increase in the frequency of CCR6+ MAITs in the BAL fluid, and higher frequencies of ki-67+ and granzyme B+ MAITs in the blood, LN, and BAL fluid. Finally, N-803 improved the ability of MAIT cells collected from PBMC and airways to produce IFN-γ in response to bacterial stimulation. Overall, N-803 shows the potential to transiently alter the phenotypes and functions of MAIT cells, which could be combined with other strategies to combat bacterial infections.

Premature Infants Have Normal Maturation of the T Cell Receptor Repertoire at Term.

Premature infants are known to have immature immune systems compared to term infants; however, the impacts of ex utero immune development are not well characterized. Our previous retrospective clinical review showed prolonged T cell lymphopenia in a subset of extremely premature infants, suggesting that they may have lasting abnormalities in their T cell compartments. We used T cell receptor (TCR) repertoire sequencing to analyze the composition of the T cell compartment in premature and term infants in our NICU. We collected twenty-eight samples from individual subjects and analyzed the number of clonotypes, repertoire diversity, CDR3 length, and V gene usage between groups based on gestational age at birth and postmenstrual age at the time of sample collection. Further, we examined the TCR repertoire in infants with severe bronchopulmonary dysplasia (BPD) and those with abnormal T cell receptor excision circle (TREC) assays. Former extremely premature infants who were corrected to term postmenstrual age had TCR repertoire diversity that was more similar to term born infants than extremely premature infants, supporting normal maturation of the repertoire. Infants with severe BPD did not appear to have increased abnormalities in repertoire diversity. Decreased TCR repertoire diversity was associated with repeatedly abnormal TREC screening, although the diversity was within the normal range for subjects without low TRECs. This study suggests that extremely premature infants demonstrate normal maturation of the T cell repertoire ex utero. Further work is needed to better characterize postnatal T cell development and function in this population.