Senescence drives immunotherapy resistance by inducing an immunosuppressive tumor microenvironment.

The potential of immune checkpoint inhibitors (ICI) may be limited in situations where immune cell fitness is impaired. Here, we show that the efficacy of cancer immunotherapies is compromised by the accumulation of senescent cells in mice and in the context of therapy-induced senescence (TIS). Resistance to immunotherapy is associated with a decrease in the accumulation and activation of CD8 T cells within tumors. Elimination of senescent cells restores immune homeostasis within the tumor micro-environment (TME) and increases mice survival in response to immunotherapy. Using single-cell transcriptomic analysis, we observe that the injection of ABT263 (Navitoclax) reverses the exacerbated immunosuppressive profile of myeloid cells in the TME. Elimination of these myeloid cells also restores CD8 T cell proliferation in vitro and abrogates immunotherapy resistance in vivo. Overall, our study suggests that the use of senolytic drugs before ICI may constitute a pharmacological approach to improve the effectiveness of cancer immunotherapies.

Comparison of Omicron breakthrough infection versus monovalent SARS-CoV-2 intramuscular booster reveals differences in mucosal and systemic humoral immunity.

Our understanding of the quality of cellular and humoral immunity conferred by COVID-19 vaccination alone versus vaccination plus SARS-CoV-2 breakthrough (BT) infection remains incomplete. While the current (2023) SARS-CoV-2 immune landscape of Canadians is complex, in late 2021 most Canadians had either just received a third dose of COVID-19 vaccine, or had received their two-dose primary series and then experienced an Omicron BT. Herein we took advantage of this coincident timing to contrast cellular and humoral immunity conferred by three doses of vaccine versus two doses plus BT. Our results show thatBT infection induces cell-mediated immune responses to variants comparable to an intramuscular vaccine booster dose. In contrast, BT subjects had higher salivary immunoglobulin (Ig)G and IgA levels against the Omicron spike and enhanced reactivity to the ancestral spike for the IgA isotype, which also reacted with SARS-CoV-1. Serumneutralizing antibody levels against the ancestral strain and the variants were also higher after BT infection. Our results support the need for the development of intranasal vaccines that could emulate the enhanced mucosal and humoral immunity induced by Omicron BT without exposing individuals to the risks associated with SARS-CoV-2 infection.

CNS Inflammation as the First Sign of Complement Factor I Deficiency: A Severe Myelitis Treated With Intense Immunotherapy and Eculizumab

OBJECTIVES: Complement factor I (CFI) deficiency is a rare autosomal recessive inborn error of immunity. In this report, we highlight that complete CFI deficiency may present with isolated and severe CNS inflammation without associated systemic features nor prior non-CNS episodes. This inflammation may respond to complement blockade therapy. METHODS: This is a case description of a young girl with severe longitudinal transverse myelitis treated with aggressive immunotherapy that included eculizumab. Published cases of CFI-associated CNS inflammation were reviewed and discussed. RESULTS: A primary immunodeficiency panel revealed 2 germline pathogenic variants in the CFI gene. Further complement testing of the index case and her family confirmed complete CFI deficiency. DISCUSSION: We describe a unique case of severe spinal inflammation secondary to complete CFI deficiency. Although rare, isolated CNS inflammation may be the primary manifestation of complete CFI deficiency. To halt the uncontrolled complement-mediated inflammation associated with CFI deficiency, prompt targeted blockade of the complement pathway using eculizumab may be life changing in the acute phase. Long-lasting blockade of the complement pathway is also essential to prevent relapse in this subgroup of patients.

Stat5 opposes the transcription factor Tox and rewires exhausted CD8+ T cells toward durable effector-like states during chronic antigen exposure.

Rewiring exhausted CD8+ T (Tex) cells toward functional states remains a therapeutic challenge. Tex cells are epigenetically programmed by the transcription factor Tox. However, epigenetic remodeling occurs as Tex cells transition from progenitor (Texprog) to intermediate (Texint) and terminal (Texterm) subsets, suggesting development flexibility. We examined epigenetic transitions between Tex cell subsets and revealed a reciprocally antagonistic circuit between Stat5a and Tox. Stat5 directed Texint cell formation and re-instigated partial effector biology during this Texprog-to-Texint cell transition. Constitutive Stat5a activity antagonized Tox and rewired CD8+ T cells from exhaustion to a durable effector and/or natural killer (NK)-like state with superior anti-tumor potential. Temporal induction of Stat5 activity in Tex cells using an orthogonal IL-2:IL2Rß-pair fostered Texint cell accumulation, particularly upon PD-L1 blockade. Re-engaging Stat5 also partially reprogrammed the epigenetic landscape of exhaustion and restored polyfunctionality. These data highlight therapeutic opportunities of manipulating the IL-2-Stat5 axis to rewire Tex cells toward more durably protective states.

Case report: Success of allogeneic hematopoietic stem cell transplantation for refractory systemic-onset juvenile idiopathic arthritis.

Objectives: This study reports cases of systemic-onset juvenile idiopathic arthritis (sJIA) who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) at our center and reviews published outcomes of allo-HSCT in sJIA. Methods: We present a case report of two patients with sJIA who underwent allo-HSCT at a tertiary pediatric hospital. Each patient's disease course and allo-HSCT protocol/outcome are described. Outcomes of published cases of allo-HSCT in sJIA were compared to our experience. Results: Two patients with sJIA had allo-HSCT. Both failed multiple lines of disease-modifying anti-rheumatic drugs and experienced severe disease/treatment-related complications. Despite post-HSCT complications, both recovered without sequelae. Five years post-HSCT, patient 1 is in complete remission (CR) and is off medications. Patient 2 was in CR until 11 months post-HSCT after which he developed three disease flares. At 4 years post-HSCT he is currently in CR on Adalimumab monotherapy. Engraftment was excellent with a donor chimerism of 100% for patient 1 and 93% for patient 2. In the literature, the outcome of allo-HSCT is reported in 13 sJIA patients. When merging those with our 2 patients, 1/15 patients died and 13/14 achieved CR, of which 12 are off medications (median [range] follow-up: 2.2 [0.2-7.0] years). Extended follow-up data on 11 of the 13 reported sJIA patients showed that an additional 3 patients flared at 3, 4, and 10 years post-HSCT. Conclusion: We report two patients with severe/refractory sJIA who underwent successful allo-HSCT and achieved CR. Allo-HSCT is a potential curative option for severe/refractory sJIA. It should be considered only after failure of conventional sJIA treatments and when an HLA-matched donor is available in order to lower transplant-related mortality. The outcomes of reported sJIA patients who received allo-HSCT are encouraging but long-term follow-up data are needed to better characterized the risk-benefit ratio of this procedure.

Combined IFN-γ and JAK inhibition to treat hemophagocytic lymphohistiocytosis in mice.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis is a life-threatening hyperinflammatory disease caused by genetic defects in the granule-mediated cytotoxic pathway. Success of hematopoietic cell transplantation, the only cure, is correlated with the extent of disease control before transplantation. Unfortunately, disease refractoriness and toxicities to standard chemotherapy-based regimens are fatal in a fraction of patients. Novel targeted immunotherapies, such as IFN-γ blocking antibodies or ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, are promising but only partially effective at controlling disease. OBJECTIVE: We asked whether combinations of cytokine-targeted therapies, using antibodies or JAK inhibitor, work synergistically to counteract HLH. METHODS: Genetically predisposed mice were infected and treated with distinct combinations of immunotherapies. Disease outcome was monitored and compared to monotherapies. RESULTS: We showed that inhibiting IL-6 or IL-18 signaling in combination with IFN-γ blockade or ruxolitinib did not increase disease control compared to anti-IFN-γ antibodies or ruxolitinib monotherapies. In contrast, clinically relevant doses of ruxolitinib combined with low doses of anti-IFN-γ blocking antibodies corrected cytopenias, prevented overt neutrophilia, limited cytokinemia, and resolved HLH immunopathology and symptomatology. CONCLUSIONS: Our findings demonstrate that IFN-γ blockade and ruxolitinib act synergistically to suppress HLH progression. This supports the use of combined cytokine-targeted therapies as a bridge to hematopoietic cell transplantation in severe familial hemophagocytic lymphohistiocytosis.

Aberrant T-cell exhaustion in severe combined immunodeficiency survivors with poor T-cell reconstitution after transplantation.

BACKGROUND: Severe combined immunodeficiency (SCID) comprises rare inherited disorders of immunity that require definitive treatment through hematopoietic cell transplantation (HCT) or gene therapy for survival. Despite successes of allogeneic HCT, many SCID patients experience incomplete immune reconstitution, persistent T-cell lymphopenia, and poor long-term outcomes. OBJECTIVE: We hypothesized that CD4+ T-cell lymphopenia could be associated with a state of T-cell exhaustion in previously transplanted SCID patients. METHODS: We analyzed markers of exhaustion in blood samples from 61 SCID patients at a median of 10.4 years after HCT. RESULTS: Compared to post-HCT SCID patients with normal CD4+ T-cell counts, those with poor T-cell reconstitution showed lower frequency of naive CD45RA+/CCR7+ T cells, recent thymic emigrants, and TCR excision circles. They also had a restricted TCR repertoire, increased expression of inhibitory receptors (PD-1, 2B4, CD160, BTLA, CTLA-4), and increased activation markers (HLA-DR, perforin) on their total and naive CD8+ T cells, suggesting T-cell exhaustion and aberrant activation, respectively. The exhaustion score of CD8+ T cells was inversely correlated with CD4+ T-cell count, recent thymic emigrants, TCR excision circles, and TCR diversity. Exhaustion scores were higher among recipients of unconditioned HCT, especially when further in time from HCT. Patients with fewer CD4+ T cells showed a transcriptional signature of exhaustion. CONCLUSIONS: Recipients of unconditioned HCT for SCID may develop late post-HCT T-cell exhaustion as a result of diminished production of T-lineage cells. Elevated expression of inhibitory receptors on their T cells may be a biomarker of poor long-term T-cell reconstitution.

Lentiviral Gene Therapy for Artemis-Deficient SCID.

BACKGROUND: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor ß chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

Symptomatology during previous SARS-CoV-2 infection and serostatus before vaccination influence the immunogenicity of BNT162b2 COVID-19 mRNA vaccine.

Public health vaccination recommendations for COVID-19 primary series and boosters in previously infected individuals differ worldwide. As infection with SARS-CoV-2 is often asymptomatic, it remains to be determined if vaccine immunogenicity is comparable in all previously infected subjects. This study presents detailed immunological evidence to clarify the requirements for one- or two-dose primary vaccination series for naturally primed individuals. The main objective was to evaluate the immune response to COVID-19 mRNA vaccination to establish the most appropriate vaccination regimen to induce robust immune responses in individuals with prior SARS-CoV-2 infection. The main outcome measure was a functional immunity score (zero to three) before and after vaccination, based on anti-RBD IgG levels, serum capacity to neutralize live virus and IFN-γ secretion capacity in response to SARS-CoV-2 peptide pools. One point was attributed for each of these three functional assays with response above the positivity threshold. The immunity score was compared based on subjects' symptoms at diagnosis and/or serostatus prior to vaccination. None of the naïve participants (n=14) showed a maximal immunity score of three following one dose of vaccine compared to 84% of the previously infected participants (n=55). All recovered individuals who did not have an immunity score of three were seronegative prior to vaccination, and 67% had not reported symptoms resulting from their initial infection. Following one dose of vaccine, their immune responses were comparable to naïve individuals, with significantly weaker responses than individuals who were symptomatic during infection. These results indicate that the absence of symptoms during initial infection and negative serostatus prior to vaccination predict the strength of immune responses to COVID-19 mRNA vaccine. Altogether, these findings highlight the importance of administering the complete two-dose primary regimen and following boosters of mRNA vaccines to individuals who experienced asymptomatic SARS-CoV-2 infection.

Evaluation of physiological Waldeyer's ring, mediastinal blood pool, thymic, bone marrow, splenic and hepatic activity with 18F-FDG PET/CT: exploration of normal range among pediatric patients.

INTRODUCTION: While 18F-FDG PET/CT pediatrics applications have increased in number and indications, few studies have addressed normal maximum standardized uptake values (SUVmax) of referral organs in children. The purpose of this study is to assess these in a cohort of pediatric patients. MATERIAL AND METHODS: 285 18F-FDG PET/CT scans in 229 patients were reviewed. SUVmax were assessed for mediastinal blood pool (MBP), thymus (T), liver (L), spleen (S), bone marrow (BM) and Waldeyer's Ring (Wald). L/MBP and S/L ratios were calculated. Same day complete blood counts (CBC) were available for 132 studies and compared to BM and S. Means, standard deviations and correlation coefficients with age, weight and body surface area (BSA) were calculated. RESULTS: Weak correlation with age, weight or BSA was found for Wald. Strong correlations with weight/BSA more than with age were demonstrated for MBP, L and BM and moderate for S and T. After initial decrease between age 0 and 2, thymic activity peaked at age 11 years then involuted. No correlation was found between CBC ad BM or S. In 28 studies, L was less or equal to MBP. In 74 S was superior to L. CONCLUSIONS: Referral organs 18F-FDG uptake varies in children more in relation with weight and BSA than with age for key referral organs, such as L, S and MBP. In a significant number of studies, L activity may impede evaluation of treatment response in comparison with MBP or inflammation/infection evaluation in comparison with S.

The REinfection in COVID-19 Estimation of Risk (RECOVER) study: Reinfection and serology dynamics in a cohort of Canadian healthcare workers.

BACKGROUND: Understanding the immune response to natural infection by SARS-CoV-2 is key to pandemic management, especially in the current context of emerging variants. Uncertainty remains regarding the efficacy and duration of natural immunity against reinfection. METHODS: We conducted an observational prospective cohort study in Canadian healthcare workers (HCWs) with a history of PCR-confirmed SARS-CoV-2 infection to (i) measure the average incidence rate of reinfection and (ii) describe the serological immune response to the primary infection. RESULTS: Our cohort comprised 569 HCWs; median duration of individual follow-up was 371 days. We detected six cases of reinfection in absence of vaccination between August 21, 2020, and March 1, 2022, for a reinfection incidence rate of 4.0 per 100 person-years. Median duration of seropositivity was 415 days in symptomatics at primary infection compared with 213 days in asymptomatics (p < 0.0001). Other characteristics associated with prolonged seropositivity for IgG against the spike protein included age over 55 years, obesity, and non-Caucasian ethnicity. CONCLUSIONS: Among unvaccinated healthcare workers, reinfection with SARS-CoV-2 following a primary infection remained rare.

The mutational landscape of SARS-CoV-2 variants diversifies T cell targets in an HLA-supertype-dependent manner.

The rapid, global dispersion of SARS-CoV-2 has led to the emergence of a diverse range of variants. Here, we describe how the mutational landscape of SARS-CoV-2 has shaped HLA-restricted T cell immunity at the population level during the first year of the pandemic. We analyzed a total of 330,246 high-quality SARS-CoV-2 genome assemblies, sampled across 143 countries and all major continents from December 2019 to December 2020 before mass vaccination or the rise of the Delta variant. We observed that proline residues are preferentially removed from the proteome of prevalent mutants, leading to a predicted global loss of SARS-CoV-2 T cell epitopes in individuals expressing HLA-B alleles of the B7 supertype family; this is largely driven by a dominant C-to-U mutation type at the RNA level. These results indicate that B7-supertype-associated epitopes, including the most immunodominant ones, were more likely to escape CD8+ T cell immunosurveillance during the first year of the pandemic.

Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice.

Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease.

Association of outcomes in acute flaccid myelitis with identification of enterovirus at presentation: a Canadian, nationwide, longitudinal study.

BACKGROUND: Acute flaccid myelitis (AFM) is characterised by rapid onset of limb weakness with spinal cord grey-matter abnormalities on MRI scan. We aimed to assess whether detection of enterovirus in respiratory or other specimens can help predict prognosis in children with AFM. METHODS: In this nationwide, longitudinal study, we evaluated the significance of detection of enterovirus in any sample in predicting outcomes in a cohort of Canadian children younger than 18 years presenting with AFM to tertiary paediatric hospitals in Canada in 2014 and 2018. All patients fulfilled the 2015 US Centers for Disease Control and Prevention case definition for definite AFM or probable AFM. Clinical data, laboratory findings, treatment, and neuroimaging results were collected (follow up period up to 5 years). We assessed neurological function and motor outcomes using Kurtzke's Expanded Disability Status Scale (EDSS) and a Weakest Limb Score. FINDINGS: 58 children with AFM (median age 5·1 years, IQR 3·8-8·3) were identified across five of Canada's ten provinces and three territories. 25 (43%) children had enterovirus detected in at least one specimen: 16 (64%) with EV-D68, two (8%) with EV-A71, two (8%) with coxsackievirus, 10 (40%) with untyped enterovirus. Children who were enterovirus positive were more likely than those that were negative to have had quadriparesis (12 [48%] of 25 vs four [13%] of 30; p=0·028), bulbar weakness (11 [44%] of 25 vs two [7%] of 30; p=0·028), bowel or bladder dysfunction (14 [56%] of 25 vs seven [23%] of 30; p=0·040), cardiovascular instability (nine [36%] of 25 vs one [3%] of 30; p=0·028), and were more likely to require intensive care unit admission (13 [52%] of 25 vs 5 [17%] of 30; p=0·028). On MRI, most children who were enterovirus positive showed brainstem pontine lesions (14 [61%] of 23), while other MRI parameters did not correlate with enterovirus status. Median EDSS of enterovirus positive (EV+) and enterovirus negative (EV-) groups was significantly different at all timepoints: baseline (EDSS 8·5, IQR 4·1-9·5 vs EDSS 4·0, IQR 3·0-6·0; p=0·0067), 3 months (EDSS 4·0, IQR 3·0-7·4 vs EDSS 3·0, IQR 1·5-4·3; p=0·0067), 6 months (EDSS 3·5, IQR 3·0-7·0 vs EDSS 3·0, IQR 1·0-4·0; p=0·029), and 12 months (EDSS 3·0, IQR 3·0-6·9 vs EDSS 2·5 IQR 0·3-3·0; p=0·0067). Kaplan-Meier survival analysis of a subgroup of patients showed significantly poorer motor recovery among children who tested positive for enterovirus than for those who tested negative (p=0·037). INTERPRETATION: Detection of enterovirus in specimens from non-sterile sites at presentation correlated with more severe acute motor weakness, worse overall outcomes and poorer trajectory for motor recovery. These results have implications for rehabilitation planning as well as counselling of families of children with these disorders. The findings of this study support the need for early testing for enterovirus in non-CNS sites in all cases of AFM. FUNDING: None.

Microbiota stimulation generates LCMV-specific memory CD8+ T cells in SPF mice and determines their TCR repertoire during LCMV infection.

Both mouse and human harbour memory phenotype CD8+ T cells specific for antigens in hosts that have not been previously exposed to these antigens. The origin and the nature of the stimuli responsible for generation of CD44hi CD8+ T cells in specific pathogen-free (SPF) mice remain controversial. It is known that microbiota plays a crucial role in the prevention and resolution of systemic infections by influencing myelopoiesis, regulating dendritic cells, inflammasome activation and promoting the production of type I and II interferons. By contrast, here we suggest that microbiota has a direct effect on generation of memory phenotype CD44hiGP33+CD8+ T cells. In SPF mice, it generates a novel GP33+CD44hiCD8+ T cell sub-population associating the properties of innate and genuine memory cells. These cells are highly enriched in the bone marrow, proliferate rapidly and express immediate effector functions. They dominate the response to LCMV and express particular TCRß chains. The sequence of these selected TCRß chains overlaps with that of GP33+CD8+ T cells directly selected by microbiota in the gut epithelium of SPF mice, demonstrating a common selection mechanism in gut and peripheral CD8+ T cell pool. Therefore microbiota has a direct role in priming T cell immunity in SPF mice and in the selection of TCRß repertoires during systemic infection. We identify a mechanism that primes T cell immunity in SPF mice and may have a major role in colonization resistance and protection from infection.

Human models of NUP98-KDM5A megakaryocytic leukemia in mice contribute to uncovering new biomarkers and therapeutic vulnerabilities.

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.