We encountered a 51-year-old male with a family history of acute myeloid leukemia who presented with persistent neutrophilia and splenomegaly progressing over eight years. Despite extensive workup, no myeloid driver mutation was identified and repeated bone marrow biopsies revealed no clonal myeloid disorder. However, an IgA kappa plasma cell clone was detected, consistent with monoclonal gammopathy of clinical significance (MGCS). Reactive neutrophilia secondary to the abnormal plasma cell clone was suspected, hypothesized to be mediated by paraneoplastic granulocyte colony-stimulating factor (G-CSF) secretion. G-CSF levels were elevated before treatment, supporting the hypothesis that it might induce neutrophilia. The patient received antimyeloma therapy and exhibited significant hematologic improvement: reduction in neutrophil count, normalization of G-CSF levels, and improvement in splenomegaly. This is the first report to describe improvement of neutrophilia following antimyeloma therapy, providing novel evidence that MGCS could simulate chronic neutrophilic leukemia (CNL).

NK cells effectively eliminate infected cells or tumour cells. The acquisition of cytotoxic functions occurs throughout NK cell functional maturation, a process that is commonly defined by the sequential expression of CD27 and CD11b. In addition, NK cell cytotoxicity is finely tuned by the process of education, which is dependent on MHC I. Expression of effector molecules is not impaired in NK cells from MHC I-deficient mice, but their cytotoxic activity towards target cells is reduced. While the role of MHC I on NK cell education has been extensively studied, its impact on NK cell functional maturation has not been defined. Using two distinct MHC I-deficient mouse models, we confirm that MHC I is not required for expression of effector molecules by NK cells. However, based on CD27 and CD11b expression, we find that fewer NK cells reach the final stage of NK cell functional maturation. Moreover, in β2m-deficient mice, early-stage NK cells express higher levels of granzyme B. Together, these findings demonstrate that NK cell functional maturation is dependent on MHC I and that the expression of effector proteins can be uncoupled from the NK cell functional maturation stages.

Cardiotrophin-like cytokine factor 1 (CLCF1) is an essential gene that shows exceptional sequence conservation among vertebrates. CLCF1 loss-of-function mutations cause Crisponi/cold-induced sweating syndrome-2, a disorder with severe and complex phenotypes, underscoring the non-redundant roles of CLCF1. Initially identified as a member of the interleukin-6 (IL-6) cytokine family with activity in neurons and immune cells, CLCF1 is now recognized for broader roles in thermoregulation, glomerular function, myelopoiesis, oncogenesis, and muscle fitness. Despite growing interest in the multifaceted biology of CLCF1, the mechanisms regulating its transcription, translation, secretion, receptor binding, and signaling remain incompletely understood. This review integrates data from multiple open databases and functional studies to outline the current state of knowledge regarding CLCF1 regulatory landscape, receptor interactions, and downstream signaling. It also highlights recent discoveries about the CLCF1 interactome and intracellular functions and underlines its emerging potential as a therapeutic target.

Activation-induced marker (AIM) assays are a promising tool to track antigen-specific T cells, but methodological heterogeneity between research groups hinders their clinical utility. To evaluate AIM assay reproducibility, we conducted a multi-site study of SARS-CoV-2 and cytomegalovirus AIMs. We found inherent variability in AIM assays and optimized approaches to enhance reproducibility, including a standardized workflow to minimize technical variability and a generalizable Box-Cox transformation-based statistical method to optimize calculation of AIM stimulation responses. We further standardized AIM data analysis through the development of automated flow cytometric gating software and demonstrated its superior reproducibility compared to manual analysis. We also characterized antigen-responsive regulatory T cells (Tregs) as CD134+CD137+ cells among CD4+FOXP3+HELIOS+ cells. The combined methodology results in a high degree of reproducibility within and between research groups, providing a comprehensive foundation from which standardized AIM assays can be implemented across diverse scientific and clinical settings.

CD4(-)CD8(-) TCR alpha beta(+) (double-negative [DN]) T cells represent a rare T cell population that promotes immunological tolerance through various cytotoxic mechanisms. In mice, autologous transfer of DN T cells has shown protective effects against autoimmune diabetes and graft-versus-host disease. Here, we characterized human DN T cells from people living with type 1 diabetes (PWT1D) and healthy controls. We found that while DN T cells and CD8(+) T cells share many similarities, DN T cells are a unique T cell population, both at the transcriptomic and protein levels. We also show that by using various cytokine combinations, human DN T cells can be expanded in vitro up to 1,000-fold (mean >250-fold) and remain functional post-expansion. In addition, we report that DN T cells from PWT1D display a phenotype comparable to that of healthy controls, efficiently expand, and are highly functional. As DN T cells are immunoregulatory and can prevent T1D in various mouse models, these observations suggest that autologous DN T cells may be amenable to therapy for the prevention or treatment of T1D.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases of the gastrointestinal tract believed to arise from an imbalance between its epithelial, immune and microbial components. It has been shown that biological differences (e.g. genetic, epigenetic, microbial, environmental) exist between patients with IBD. It is also known that there is important heterogeneity in the response to therapies that target very specific biological pathways (e.g. TNF‐alpha signaling, IL‐23R signaling, immune cell trafficking). The aim of this study was to identify potential biological differences associated with differential treatment response to the anti α4β7 integrin therapy known as vedolizumab. We performed targeted analyses of > 150 proteins and metabolites, and nontargeted analyses of > 1100 lipid entities in serum samples from 92 IBD patients (42 CD, 50 UC) immediately prior to initiation of therapy with vedolizumab (baseline samples) and at their first clinical assessment (week 14 samples). We detected that the baseline levels of multiple serum cytokines, amino acids, acylcarnitines and triglycerides were different between responders and nonresponders to treatment with vedolizumab. We also noted changes in serum analytes between baseline and week 14 samples that were different between these two groups of patients. Many of these serum analytes are markers of biological pathways that are involved in the activation, proliferation and metabolism of pro‐inflammatory cells. This study provides support for the hypothesis that biological differences between individuals not only impact the risk to develop IBD and IBD‐related clinical phenotypes but also an IBD patient's likelihood of responding to a biological therapy.

Cardiotrophin-like cytokine factor 1 (CLCF1) is a cytokine of the IL6/IL12 superfamily with pro-neurotrophic and immune-modulating functions. Although the pro-neurotrophic activities of CLCF1 are mediated through the ciliary neurotrophic factor receptor (CNTFR), the α receptor chain of the CNTFR, CNTFRα, is not expressed by immune cells. This suggests the presence of an alternative receptor or protein complex that mediates the immune activities of CLCF1. Using the BioID2 proximity-dependent biotinylation assay, we identified p40, the β subunit of IL12/IL23, as a potential interaction partner for CLCF1. We confirmed the protein-protein interaction between CLCF1 and p40 using co-immunoprecipitation and a proximity ligation assay. We also observed that the CLCF1-p40 complex forms both intracellularly and in the extracellular space. Furthermore, secretion of the CLCF1-p40 heterodimer was induced by the cytokine receptor-like factor 1 (CRLF1), leading to the release of a tripartite CLCF1-CRLF1-p40 complex. Lastly, we showed that a CLCF1-p40 fusion protein binds to both CNTFRα and IL12Rβ1. Taken together, our results uncover a new putative composite cytokine of the IL6/IL12 superfamily, which might affect our understanding of both CLCF1 activities and p40-associated pathologies. Moreover, our results reinforce the connection between the IL6 and IL12 cytokine families, and suggest that other possible protein interactions between these two families should be further investigated.

IntroductionReceptor-interacting protein kinase 3 (RIPK3) is a protein involved in cell death and inflammatory processes. The most recognized function of RIPK3 is the induction of necroptosis, an inflammatory type of cell death that is dependent on RIPK3 kinase activity. Deficiency in RIPK3-dependent pathways has been associated with protection from various inflammatory and autoimmune conditions. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the generation of autoantibodies to multiple intracellular antigens leading to multi-organ pathology. Little is known about the involvement of RIPK3-dependent pathways in SLE. We have previously shown that autoantibody generation in an induced model of murine lupus is impaired in RIPK3-deficient mice. The current study aimed to identify the RIPK3-dependent mechanisms that contribute to autoantibody generation in this induced model of murine lupus.MethodsSLE was induced in C57BL/6 (wild type), RIPK3-/-, RIPK3K51A/K51A, and MLKL-/- mice by subcutaneous immunization with a mixture of β2-glycoprotein I and lipopolysaccharide in order to evaluate the contribution of RIPK3 and MLKL to autoantibody production in this model. Bone marrow chimeras were generated to investigate the impact of RIPK3 deficiency within the hematopoietic compartment. Antigen presentation assays assessed the impact of RIPK3 deficiency in antigen presenting cells on T cell activation in vitro. T cells were evaluated ex vivo by flow cytometry following the induction of SLE in wild type and RIPK3-dependent pathway-deficient mice.ResultsGeneration of autoantibodies to SLE antigens following immunization with β2-glycoprotein I and lipopolysaccharide was found to be dependent on RIPK3 activity, but independent of MLKL (i.e., RIPK3-dependent necroptosis). Bone marrow chimeric experiments revealed that RIPK3 mediates autoantibody generation through both immune and non-immune compartments. RIPK3 deficiency within antigen presenting cells did not impact T cell activation in vitro. Moreover, early and late T cell activation ex vivo was not impaired in RIPK3-deficient mice following induction of murine lupus.ConclusionThese results suggest that RIPK3 contributes to autoantibody generation in our induced model of murine lupus through an interplay of pathways that appear to be independent of necroptosis and antigen presentation.

Sphingosine‐1‐phosphate receptor 1 (S1P 1 ) ligands effectively reduce immunopathological damage in viral pneumonia models. Specifically, S1P 1 ligands inhibit cytokine storm and help preserve lung endothelial barrier integrity. We recently showed that the S1P receptor ligand ozanimod can be safely administered to hospitalized patients with coronavirus disease 2019 (COVID‐19) exhibiting severe symptoms of viral pneumonia, with potential clinical benefits. Here, we extend on this study and investigate the impact of ozanimod on key features of the immune response in patients with severe COVID‐19. We quantified circulating cytokine levels, peripheral immune cell numbers, proportions and activation status; we also monitored the quality of the humoral response by assessing anti–severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) antibodies. Our findings reveal that patients receiving ozanimod during acute SARS‐CoV‐2 infection exhibit significantly reduced numbers of circulating monocytes compared with those receiving standard care. Correspondingly, in the ozanimod‐treated group, circulating levels of C–C motif ligand 2 (CCL2) were decreased. While treatment with ozanimod negatively impacted the humoral response to COVID‐19 in unvaccinated patients, it did not impair the development of a robust anti–SARS‐CoV‐2 antibody response in vaccinated patients. These findings suggest that ozanimod influences key immune mechanisms during the acute phase of SARS‐CoV‐2 infection.

Cardiotrophin-like cytokine factor 1 (CLCF1) is a cytokine of the IL6/IL12 family with immune-modulating functions, mainly on B cells and myeloid cells. CLCF1 also plays a crucial role in the embryonic development of motor neurons, such that Clcf1-knock out mice are not viable. In order to further study the immune activities of CLCF1, we used a mouse model with a knock-out of Clcf1 in hematopoietic cells under the Vav promoter. While characterizing this model, we observed that CD4+ T cells from Clcf1-/- mice produced more IFNγ than those from Clcf1+/+ mice when activated in the presence of IL12. We also observed that CLCF1 induces a downregulation of IL12Rβ2 expression levels. We further demonstrated that CLCF1 interacts with IL12Rβ2 and promotes its degradation through the proteasome in a manner independent of ubiquitination. Altogether, these results suggest that CLCF1 can act as a negative regulator of IL12 activity, a role which could be exploited therapeutically to dampen the inflammatory response driven by Th1 cells. Our observations may also hint at a new role for CLCF1 as a mediator of protein degradation.

Leigh Syndrome French Canadian (LSFC) is a rare autosomal recessive metabolic disorder characterized by severe lactic acidosis crises and early mortality. LSFC patients carry variants in the Leucine Rich Pentatricopeptide Repeat Containing (LRPPRC) nuclear gene, which lead to defects in the respiratory chain complexes and mitochondrial dysfunction. Mitochondrial respiration modulates cellular metabolic activity, which impacts many cell processes, including the differentiation and function of immune cells. The purpose of this study is to define the role of Lrpprc on immune cell function. As genetic deletion of Lrpprc is not viable, we generated two conditional mouse models: a model for systemic deletion of Lrpprc and a knock-in (KI) model carrying the most common LSFC pathogenic variant in Quebec, NM_133259.4(LRPPRC):c.1061C > T (p.Ala354Val). We demonstrate that Lrpprc is an essential gene even in adult mice, as systemic deletion of Lrpprc leads to prominent weight loss and mortality. We also find an increase in lactate levels, a symptom of metabolic crises in LSFC. Lrpprc deletion and pathogenic variant affect various immune cell subsets, with a strong impact on B cell development and proliferation. We generated a viable disease-relevant mouse model to study the role of Lrpprc in vivo and find that disruption of Lrpprc strongly impairs B cell development and proliferation.

Granulocyte concentrates (GCs) are leukocyte preparations enriched in neutrophils that can potentially save neutropenic patients from life-threatening, antimicrobial-resistant infections. The main challenge of GC transfusions is preserving the viability and antimicrobial activity of neutrophils beyond 24 h to reduce the logistical burden on collection centers and increase the availability of this cell therapy. Thus, the aim of this study was to explore extending the ex vivo viability and antimicrobial activity of GC neutrophils up to 72 h with a unique combination of the clinically approved additives Plasma-Lyte (PL), SAGM, AS-3, and Alburex. Neutrophils isolated from healthy donors were resuspended in autologous plasma at the same concentration as in GCs, diluted with various combinations of PL, SAGM, AS-3, and/or Alburex with or without the addition of buffers, and stored at room temperature for up to 72 h. During storage, neutrophil viability, phagocytosis, and intracellular reactive oxygen species production were measured by flow cytometry. Extracellular reactive oxygen species production was measured by spectrophotometry and chemotaxis by the number of calcein-stained neutrophils that migrated toward the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLF). The same assays were performed on pooled, residual leukocyte units generated by the Reveos system, after storage in the additive combination that most effectively preserved the viability and function of isolated neutrophils. The additive combination that best performed in the majority of the assays contained PL, buffers, and AS-3. Neutrophil viability was preserved for a maximum of 48 h and phagocytosis of opsonized bacteria and reactive oxygen species production up to 72 h of storage at room temperature. In contrast, fMLF-induced chemotaxis decreased by 20% after 24-h storage while extracellular reactive oxygen species production increased significantly within the same time period. Supplementing GCs prepared from pooled, residual leukocyte units with this storage solution after the standard 16- to 24-h processing period as per the blood center guidelines, did not significantly improve the preservation of neutrophil viability and function. Our findings provide proof of concept that mixtures of clinically approved additives can be tailored to significantly prolong the viability and function of freshly isolated neutrophils during room-temperature storage. The unique additive composition of this storage solution that we developed for freshly isolated neutrophils requires further optimization for use with pooled, residual leukocyte units as well as the timepoint at which the solution is added during processing to prolong the viability and functions of neutrophils in this blood product.

In this article for the Highlights of 2024 Series, we discuss strategies to enhance NK cell-based cancer therapies. These include (1) cytokine expression on bacterial membranes to boost NK cell activation in tumors, (2) optimizing CAR-NK cell manufacturing for improved efficacy, (3) using CRISPR-Cas9 to identify and target inhibitory genes, and (4) using tetraspecific engagers to enhance cytotoxicity and cytokine memory-like NK cells strengthening anti-tumor responses. This year's progress holds much promise for cancer treatments exploiting NK cells.

Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes and have enabled the identification of several loci associated with diabetes susceptibility, termed insulin-dependent diabetes (Idd). The generation of congenic mice has allowed the characterization of the impact of several loci on disease susceptibility. For instance, NOD.B6-Idd1 and B6.NOD-Idd1 congenic mice were instrumental in demonstrating that susceptibility alleles at the MHC locus (known as Idd1) are necessary but not sufficient for autoimmune diabetes progression. We previously showed that diabetes resistance alleles at the Idd2 locus provide significant protection from autoimmune diabetes onset, second to Idd1. In search of the minimal genetic factors required for T1D onset, we generated B6.Idd1.Idd2 double-congenic mice. Although the combination of Idd1 and Idd2 is not sufficient to induce diabetes onset, we observed immune infiltration in the exocrine pancreas of B6.Idd2 mice, as well as an increase in neutrophils and pancreatic tissue fibrosis. In addition, we observed phenotypic differences in T-cell subsets from B6.Idd1.Idd2 mice relative to single-congenic mice, suggesting epistatic interaction between Idd1 and Idd2 in modulating T-cell function. Altogether, these data show that Idd1 and Idd2 susceptibility alleles are not sufficient for autoimmune diabetes but contribute to inflammation and immune infiltration in the pancreas.

Corneal HSV-1 infections are a leading cause of infectious blindness globally by triggering tissue damage due to the intense inflammation. HSV-1 infections are treated mainly with antiviral drugs that clear the infections but are inefficient as prophylactics. The body produces innate cationic host defence peptides (cHDP), such as the cathelicidin LL37. Various epithelia, including the corneal epithelium, express LL37. cHDPs can cause disintegration of pathogen membranes, stimulate chemokine production, and attract immune cells. Here, we selected GF17, a peptide containing the LL37 fragment with bioactivity but with minimal cytotoxicity, and added two cell-penetrating amino acids to enhance its activity. The resulting GF19 was relatively cell-friendly, inducing only partial activation of antigen presenting immune cells in vitro. We showed that HSV-1 spreads by tunneling nanotubes in cultured human corneal epithelial cells. GF19 given before infection was able to block infection, most likely by blocking viral entry. When cells were sequentially exposed to viruses and GF19, the infection was attenuated but not arrested, supporting the contention that the GF19 mode of action was to block viral entry. Encapsulation into silica nanoparticles allowed a more sustained release of GF19, enhancing its activity. GF19 is most likely suitable as a prevention rather than a virucidal treatment.

In this article for the Highlights of 2023 Series, we discuss how various factors affect the ability of natural killer (NK) cells to fight tumors. For instance, tumor cells can hinder NK cell function by reducing surface protrusions or increasing HLA-E expression via platelets. Lower UTX protein levels in male NK cells also decrease their cytotoxicity compared with females. Fortunately, recent advancements in therapeutic approaches have emerged, including the development of a comprehensive atlas of NK cell heterogeneity within the tumor microenvironment, as well as a trispecific engager molecule that has shown promise in enhancing the anti-tumor functions of NK cells.

Background: The gastrointestinal (GI) barrier can be damaged by chemotherapy or radiation therapy, causing fatigue, malnutrition, sepsis, dose-limiting toxicity, and, occasionally, death. Glucagon-like peptide-2 (GLP-2) promotes mucosal epithelium growth and repair in the GI tract. Here, we examined the GI-protective effects of apraglutide, a long-acting peptide GLP-2 analog, in murine models of chemotherapy, and total body irradiation followed by allogeneic transplantation. Material/Methods: The impact of apraglutide on cytarabine or melphalan chemotherapy-induced intestinal damage was assessed in BALB/c mice, and the effect on allogeneic transplantation in BALB/cJ and C57BL/6J mice. Outcomes included survival, and changes in body weight, intestinal function and morphology, including colon length and bacterial composition of the intestinal microbiota. Results: Adding apraglutide to chemotherapy significantly improved survival rates and reduced weight loss, with no impact on leukocyte counts (and, therefore, no effect on chemotherapy-induced immunosuppression), compared with chemotherapy alone in mice. These benefits were associated with preservation of the morphological integrity of the GI mucosa, attenuation of the negative impact of cytarabine on the intestinal microbiota, and significant improvement in plasma levels of citrulline. In addition, in a model of irradiation followed by allogeneic transplantation, mice in groups receiving apraglutide had improved survival, reduced weight loss, and increased colon length compared with those that did not. Conclusions: Apraglutide protects intestinal function and improves survival in mice following allogeneic transplantation or chemotherapy with cytarabine or melphalan. The potential effect of apraglutide on chemotherapy efficacy and on engraftment following allogeneic transplantation has been investigated in a parallel manuscript.

In this article for the Highlights of 2023 Series, we discuss four recent articles that investigated thymic B cells, in both mice and humans. These studies provide important novel insights into the biology of this unique B-cell population, from their activation and differentiation to their role in promoting the negative selection of thymocytes and the generation of regulatory T cells. image

Cardiotrophin-like cytokine factor 1 (CLCF1) is an IL-6 family cytokine with neurotrophic and immuno-modulating functions. CLCF1 mRNA has been detected in primary and secondary lymphoid organs, and up-regulation of CLCF1 mRNA levels has been associated with the T helper (Th) 17 polarization. However, information regarding CLCF1 expression by immune cells at the protein level remains scarce. We have developed a methodology that uses a monoclonal antibody (mAb) directed against CLCF1 for the detection of human and mouse CLCF1 by flow cytometry. We have successfully detected CLCF1 protein expression in cells from the mouse pro-B cell line Ba/F3 that were transduced with CLCF1 cDNA. Interestingly, we found that the anti-CLCF1 mAb inhibits CLCF1 biological activity in vitro by binding to an epitope that encompasses site III of the cytokine. Moreover, we have detected CLCF1 expression in mouse splenic T cells, as well as in vitro differentiated Th1 cells. The specificity of the fluorescence signal was demonstrated using Clcf1-deficient lymphocytes generated using a conditional knock-out mouse model. The detection of CLCF1 protein by flow cytometry will be a valuable tool to study CLCF1 expression during normal and pathological immune responses.