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Using anti-Tac, a monoclonal anti-interleukin 2 (IL-2) receptor antibody, we have explored the possibility that certain activated B cells display receptors for IL-2. Resting normal B cells and unselected B cell lines established from normal individuals were Tac antigen negative. In contrast, the cell surface Tac antigen expression was demonstrable on 6 of 10 B cell lines from patients with Burkitt's lymphoma, 5 of 6 B cell lines derived from patients with HTLV-I-associated adult T cell leukemia (including all four that had integrated HTLV-I into their genome), and on certain normal B cells activated with pokeweed mitogen. Furthermore, cloned Epstein-Barr virus-transformed B cell lines derived from Tac-positive normal B cells continued to express the Tac antigen in long-term cultures and manifested high affinity IL-2 receptors identified in binding studies with purified radiolabeled IL-2. The line 5B4 developed in the present study could be induced with purified JURKAT-derived or recombinant IL-2 to express a larger number of IL-2 receptors. Furthermore, the addition of IL-2 to the 5B4 B cell line augmented IgM synthesis, which could be blocked by the addition of anti-Tac. The size of the IL-2 receptors expressed on the cloned normal B cell lines was similar (53,000-57,000 daltons) to that of receptors on phytohemagglutinin-stimulated T cell lymphoblasts. Thus, certain malignant and activated normal B cells display the Tac antigen and manifest high affinity receptors for IL-2. These data suggest that IL-2 may play a role in the differentiation of activated B cells into immunoglobulin-synthesizing and -secreting cells.

Monoclonal antibodies J5, VIL-A1, and BA-3, known to react with the common acute lymphoblastic leukemia antigen (CALLA) were found to specifically stain normal human polymorphonuclear neutrophils (PMN). The antigen detected on PMN had a molecular weight (95,000-110,000 mol wt) close to that of CALLA (95,000-100,000 mol wt) and thus these surface membrane antigens are likely related, if not identical. The fluorescent staining intensity of PMN is comparable to that of CALLA-positive leukemic cells and the presence of PMN in patient samples could potentially produce false-positive results in diagnosis.

Progress in cytokine engineering is driving therapeutic translation by overcoming these proteins' limitations as drugs. The IL-2 cytokine is a promising immune stimulant for cancer treatment but is limited by its concurrent activation of both pro-inflammatory immune effector cells and antiinflammatory regulatory T cells, toxicity at high doses, and short serum half-life. One approach to improve the selectivity, safety, and longevity of IL-2 is complexing with anti-IL-2 antibodies that bias the cytokine toward immune effector cell activation. Although this strategy shows potential in preclinical models, clinical translation of a cytokine/antibody complex is complicated by challenges in formulating a multiprotein drug and concerns regarding complex stability. Here, we introduced a versatile approach to designing intramolecularly assembled single-agent fusion proteins (immunocytokines, ICs) comprising IL-2 and a biasing anti-IL-2 antibody that directs the cytokine toward immune effector cells. We optimized IC construction and engineered the cytokine/antibody affinity to improve immune bias. We demonstrated that our IC preferentially activates and expands immune effector cells, leading to superior antitumor activity compared with natural IL-2, both alone and combined with immune checkpoint inhibitors. Moreover, therapeutic efficacy was observed without inducing toxicity. This work presents a roadmap for the design and translation of cytokine/antibody fusion proteins.

Cytokine-mediated STAT5 protein activation is vital for lymphocyte development and function. In vitro tyrosine phosphorylation of a C-terminal tyrosine is critical for activation of STAT5A and STAT5B; however, the importance of STAT5 tyrosine phosphorylation in vivo has not been assessed. Here we generate Stat5a and Stat5b tyrosine-to-phenylalanine mutant knockin mice and find they have greatly reduced CD8 + T-cell numbers and profoundly diminished IL-2-induced proliferation of these cells, and this correlates with reduced induction of Myc, pRB, a range of cyclins and CDKs, and a partial G1→S phase-transition block. These mutant CD8 + T cells also exhibit decreased IL-2-mediated activation of pERK and pAKT, which we attribute in part to diminished expression of IL-2Rβ and IL-2Rγ. Our findings thus demonstrate that tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling. Moreover, our transcriptomic and proteomic analyses elucidate the molecular basis of the IL-2-induced proliferation of CD8 + T cells., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

High frequencies of stem-like memory T cells in infusion products correlate with superior patient outcomes across multiple T cell therapy trials. Herein, we analyzed a published CRISPR activation screening to identify transcriptional regulators that could be harnessed to augment stem-like behavior in CD8 + T cells. Using IFN-γ production as a proxy for CD8 + T cell terminal differentiation, LMO4 emerged among the top hits inhibiting the development of effectors cells. Consistently, we found that Lmo4 was downregulated upon CD8 + T cell activation but maintained under culture conditions facilitating the formation of stem-like T cells. By employing a synthetic biology approach to ectopically express LMO4 in antitumor CD8 + T cells, we enabled selective expansion and enhanced persistence of transduced cells, while limiting their terminal differentiation and senescence. LMO4 overexpression promoted transcriptional programs regulating stemness, increasing the numbers of stem-like CD8 + memory T cells and enhancing their polyfunctionality and recall capacity. When tested in syngeneic and xenograft tumor models, LMO4 overexpression boosted CD8 + T cell antitumor immunity, resulting in enhanced tumor regression. Rather than directly modulating gene transcription, LMO4 bound to JAK1 and potentiated STAT3 signaling in response to IL-21, inducing the expression of target genes (Tcf7, Socs3, Junb, and Zfp36) crucial for memory responses. CRISPR/Cas9-deletion of Stat3 nullified the enhanced memory signature conferred by LMO4, thereby abrogating the therapeutic benefit of LMO4 overexpression. These results establish LMO4 overexpression as an effective strategy to boost CD8 + T cell stemness, providing a new synthetic biology tool to bolster the efficacy of T cell-based immunotherapies., (© 2024. The Author(s).)

Axons undergo striking changes in their content and distribution of cell adhesion molecules (CAMs) and ion channels during myelination that underlies the switch from continuous to saltatory conduction. These changes include the removal of a large cohort of uniformly distributed CAMs that mediate initial axon-Schwann cell interactions and their replacement by a subset of CAMs that mediate domain-specific interactions of myelinated fibers. Here, using rodent models, we examine the mechanisms and significance of this removal of axonal CAMs. We show that Schwann cells just prior to myelination locally activate clathrin-mediated endocytosis (CME) in axons, thereby driving clearance of a broad array of axonal CAMs. CAMs engineered to resist endocytosis are persistently expressed along the axon and delay both PNS and CNS myelination. Thus, glia non-autonomously activate CME in axons to downregulate axonal CAMs and presumptively axo-glial adhesion. This promotes the transition from ensheathment to myelination while simultaneously sculpting the formation of axonal domains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies., (© 2024. The Author(s).)

Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance., Significance: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4., (©2023 The Authors; Published by the American Association for Cancer Research.)

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (T regs ) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and T regs , with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

Background: Gene expression has long been known to be influenced by the relative proximity of DNA regulatory elements. Topologically associating domains (TADs) are self-interacting genomic regions involved in regulating gene expression by controlling the proximity of these elements. Prior studies of TADs and their biological roles have revealed correlations between TAD changes and cellular differentiation. Here, we used Hi-C and RNA-seq data to correlate TCR-induced changes in TAD structure and gene expression in human CD4 + T cells., Results: We developed a pipeline, Differentially Expressed Gene Enrichment Finder (DEGEF), that identifies regions of differentially expressed gene enrichment. Using DEGEF, we found that TCR-regulated genes cluster non-uniformly across the genome and that these clusters preferentially localized in regions of TAD rearrangement. Interestingly, clusters of upregulated genes preferentially formed new Hi-C contacts compared to downregulated clusters, suggesting that TCR-activated CD4 + T cells may regulate genes by changing stimulatory contacts rather than inhibitory contacts., Conclusions: Our observations support a significant relationship between TAD rearrangements and changes in local gene expression. These findings indicate potentially important roles for TAD rearrangements in shaping their local regulatory environments and thus driving differential expression of nearby genes during CD4 + T cell activation. Moreover, they provide new insights into global mechanisms that regulate gene expression., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Cytokines are secreted or membrane-presented molecules that mediate broad cellular functions, including development, differentiation, growth and survival. Accordingly, the regulation of cytokine activity is extraordinarily important both physiologically and pathologically. Cytokine and/or cytokine receptor engineering is being widely investigated to safely and effectively modulate cytokine activity for therapeutic benefit. IL-2 in particular has been extensively engineered, to create IL-2 variants that differentially exhibit activities on regulatory T cells to potentially treat autoimmune disease versus effector T cells to augment antitumour effects. Additionally, engineering approaches are being applied to many other cytokines such as IL-10, interferons and IL-1 family cytokines, given their immunosuppressive and/or antiviral and anticancer effects. In modulating the actions of cytokines, the strategies used have been broad, including altering affinities of cytokines for their receptors, prolonging cytokine half-lives in vivo and fine-tuning cytokine actions. The field is rapidly expanding, with extensive efforts to create improved therapeutics for a range of diseases., (© 2023. Springer Nature Limited.)

The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

CD4 + T helper 17 (T H 17) cells protect barrier tissues but also trigger autoimmunity. The mechanisms behind these opposing processes remain unclear. Here, we found that the transcription factor EGR2 controlled the transcriptional program of pathogenic T H 17 cells in the central nervous system (CNS) but not that of protective T H 17 cells at barrier sites. EGR2 was significantly elevated in myelin-reactive CD4 + T cells from patients with multiple sclerosis and mice with autoimmune neuroinflammation. The EGR2 transcriptional program was intricately woven within the T H 17 cell transcriptional regulatory network and showed high interconnectivity with core T H 17 cell-specific transcription factors. Mechanistically, EGR2 enhanced T H 17 cell differentiation and myeloid cell recruitment to the CNS by upregulating pathogenesis-associated genes and myelomonocytic chemokines. T cell-specific deletion of Egr2 attenuated neuroinflammation without compromising the host's ability to control infections. Our study shows that EGR2 regulates tissue-specific and disease-specific functions in pathogenic T H 17 cells in the CNS., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: The growing power and ever decreasing cost of RNA sequencing (RNA-Seq) technologies have resulted in an explosion of RNA-Seq data production. Comparing gene expression values within RNA-Seq datasets is relatively easy for many interdisciplinary biomedical researchers; however, user-friendly software applications increase the ability of biologists to efficiently explore available datasets., Results: Here, we describe ROGUE (RNA-Seq Ontology Graphic User Environment, https://marisshiny., Research: chop.edu/ROGUE/ ), a user-friendly R Shiny application that allows a biologist to perform differentially expressed gene analysis, gene ontology and pathway enrichment analysis, potential biomarker identification, and advanced statistical analyses. We use ROGUE to identify potential biomarkers and show unique enriched pathways between various immune cells., Conclusions: User-friendly tools for the analysis of next generation sequencing data, such as ROGUE, will allow biologists to efficiently explore their datasets, discover expression patterns, and advance their research by allowing them to develop and test hypotheses., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

In response to external stimuli during immune responses, monocytes can have multifaceted roles such as pathogen clearance and tissue repair. However, aberrant control of monocyte activation can result in chronic inflammation and subsequent tissue damage. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces monocyte differentiation into a heterogenous population of monocyte-derived dendritic cells (moDCs) and macrophages. However, the downstream molecular signals that dictate the differentiation of monocytes under pathological conditions is incompletely understood. We report here that the GM-CSF-induced STAT5 tetramerization is a critical determinate of monocyte fate and function. Monocytes require STAT5 tetramers to differentiate into moDCs. Conversely, the absence of STAT5 tetramers results in a switch to a functionally distinct monocyte-derived macrophage population. In the dextran sulfate sodium (DSS) model of colitis, STAT5 tetramer-deficient monocytes exacerbate disease severity. Mechanistically, GM-CSF signaling in STAT5 tetramer-deficient monocytes results in the overexpression of arginase I and a reduction in nitric oxide synthesis following stimulation with lipopolysaccharide. Correspondingly, the inhibition of arginase I activity and sustained supplementation of nitric oxide ameliorates the worsened colitis in STAT5 tetramer-deficient mice. This study suggests that STAT5 tetramers protect against severe intestinal inflammation through the regulation of arginine metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Monaghan, Zheng, Akhter, Wang, Ammer, Li, Lin, Hu, Leonard and Wan.)

Type 2 T helper (Th2) cells and group 2 innate lymphoid cells (ILC2s) provide protection against helminth infection and are involved in allergic responses. However, their relative importance and crosstalk during type 2 immune responses are still controversial. By generating and utilizing mouse strains that are deficient in either ILC2s or Th2 cells, we report that interleukin (IL)-33-mediated ILC2 activation promotes the Th2 cell response to papain; however, the Th2 cell response to ovalbumin (OVA)/alum immunization is thymic stromal lymphopoietin (TSLP) dependent but independent of ILC2s. During helminth infection, ILC2s and Th2 cells collaborate at different phases of the immune responses. Th2 cells, mainly through IL-4 production, induce the expression of IL-25, IL-33, and TSLP, among which IL-25 and IL-33 redundantly promote ILC2 expansion. Thus, while Th2 cell differentiation can occur independently of ILC2s, activation of ILC2s may promote Th2 responses, and Th2 cells can expand ILC2s by inducing type 2 alarmins., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Connecting genes to their cis-regulatory elements has been enabled by genome-wide mapping of chromatin interactions using proximity ligation in ChIA-PET, Hi-C, and their derivatives. However, these methods require millions of input cells for high-quality data and thus are unsuitable for many studies when only limited cells are available. Conversely, epigenomic profiling via transposase digestion in ATAC-seq requires only hundreds to thousands of cells to robustly map open chromatin associated with transcription activity, but it cannot directly connect active genes to their distal enhancers. Here, we combine proximity ligation in ChIA-PET and transposase accessibility in ATAC-seq into ChIATAC to efficiently map interactions between open chromatin loci in low numbers of input cells. We validate ChIATAC in Drosophila cells and optimize it for mapping 3D epigenomes in human cells robustly. Applying ChIATAC to primary human T cells, we reveal mechanisms that topologically regulate transcriptional programs during T cell activation., (© 2023. The Author(s).)

Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine that acts on multiple cell lineages, including dendritic cells, T cells, B cells, neutrophils, mast cells, eosinophils and innate lymphoid cells, affecting their maturation, survival and recruitment. It is best known for its role in promoting type 2 immune responses such as in allergic diseases and, in 2021, a monoclonal antibody targeting TSLP was approved for the treatment of severe asthma. However, it is now clear that TSLP has many other important roles in a variety of settings. Indeed, several genetic variants for TSLP are linked to disease severity, and chromosomal alterations in TSLP are common in certain cancers, indicating important roles of TSLP in disease. In this Review, we discuss recent advances in TSLP biology, highlighting how it regulates the tissue environment not only in allergic disease but also in infectious diseases, inflammatory diseases and cancer. Encouragingly, therapies targeting the TSLP pathway are being actively pursued for several diseases., (© 2022. Springer Nature Limited.)

Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection 1 . Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1 + TCF1 + stem-like CD8 +  T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8 +  T cells that resemble highly functional effector CD8 +  T cells seen after an acute viral infection. The generation of these qualitatively superior CD8 + T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1 + TCF1 + stem-like CD8 + T cells, also referred to as precursors of exhausted CD8 + T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8 + T cells emerging from the stem-like CD8 + T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25-CD122-CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer 2,3 , and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14 V763A ) , a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14 V763A , suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse ( Med14 V769A ) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14 V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAM Q498H ) ; however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants., Competing Interests: Authors US, RA, and RS (Srinivasan) are employed by TATA Consultancy Services. SEB was a principal investigator on a research service agreement between TCS and the University of California, Berkley. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sertori, Lin, Martinez, Rana, Sharo, Kazemian, Sunderam, Andrake, Shinton, Truong, Dunbrack, Liu, Srinivasan, Brenner, Seroogy, Puck, Leonard and Wiest.)

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy 1,2 . Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γ c ) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γ c cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rβ-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Interferon γ (IFNγ) is an essential and pleiotropic activator of human monocytes, but little is known about the changes in cellular metabolism required for IFNγ-induced activation. We sought to elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. We found that IFNγ increased oxygen consumption rates (OCR) in monocytes, indicative of reactive oxygen species generation by both mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Transcriptional profiling revealed that this oxidative phenotype was driven by IFNγ-induced reprogramming of NAD+ metabolism, which is dependent on nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. Consistent with this pathway, monocytes from patients with gain-of-function mutations in STAT1 demonstrated higher-than-normal OCR, whereas chemical or genetic disruption of mitochondrial complex I (rotenone treatment or Leigh syndrome patient monocytes) or NADPH oxidase (diphenyleneiodonium treatment or chronic granulomatous disease [CGD] patient monocytes) reduced OCR. Interestingly, inhibition of NAMPT in healthy monocytes completely abrogated the IFNγ-induced oxygen consumption, comparable to levels observed in CGD monocytes. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for IFNγ activation of human monocytes., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Natural killer (NK) cells play key roles in immune surveillance against tumors and viral infection. NK cells distinguish abnormal cells from healthy cells by cell-cell interaction with cell surface proteins and then attack target cells via multiple mechanisms. In addition, extracellular vesicles (EVs) derived from NK cells (NK-EVs), including exosomes, possess cytotoxic capacity against tumor cells, but their characteristics and regulation by cytokines remain unknown. Here, we report that EVs derived from human NK-92 cells stimulated with IL-15 + IL-21 show enhanced cytotoxic capacity against tumor cells. Major cytolytic granules, granzyme B and granzyme H, are enriched by IL-15 + IL-21 stimulation in NK-EVs; however, knockout experiments reveal those cytolytic granules are independent of enhanced cytotoxic capacity. To find out the key molecules, mass spectrometry analyses were performed with different cytokine conditions, no cytokine, IL-15, IL-21, or IL-15 + IL-21. We then found that CD226 (DNAM-1) on NK-EVs is enriched by IL-15 + IL-21 stimulation and that blocking antibodies against CD226 reduced the cytolytic activity of NK-EVs. We also show NK-EVs are taken up by target cells via macropinocytosis. Collectively, our findings elucidate the novel properties of NK-EVs and the mechanism of their incorporation into target cells., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2021.)

Background: Pediatric endogenous Cushing syndrome (eCs) is mainly caused by pituitary corticotropin-producing adenomas, and most glucocorticoid-dependent effects progressively regress upon tumor removal. eCs reproduces long-term, high-dose glucocorticoid therapy, representing a clean, natural, and unbiased model in which to study glucocorticoid bona fide effects on immunity., Objective: We performed extensive immunologic studies in otherwise healthy pediatric patients with eCs before and 6 to 13 months after tumor resection, as well as in in vitro glucocorticoid-treated control cells., Methods: Flow cytometry, immunoblotting, enzyme-linked immunosorbent assay, real-time quantitative PCR, and RNA-Seq techniques were used to characterize patients' and in vitro glucocorticoid treated cells., Results: Reduced thymic output, decreased naive T cells, diminished proliferation, and increased T-cell apoptosis were detected before surgery; all these defects eventually normalized after tumor removal in patients. In vitro studies also showed increased T-cell apoptosis, with correspondingly diminished NF-κB signaling and IL-21 levels. In this setting, IL-21 addition upregulated antiapoptotic BCL2 expression and rescued T-cell apoptosis in a PI3K pathway-dependent manner. Similar and reproducible findings were confirmed in eCs patient cells as well., Conclusions: We identified decreased thymic output and lymphocyte proliferation, together with increased apoptosis, as the underlying causes to T-cell lymphopenia in eCs patients. IL-21 was decreased in both natural and in vitro long-term, high-dose glucocorticoid environments, and in vitro addition of IL-21 counteracted the proapoptotic effects of glucocorticoid therapy. Thus, our results suggest that administration of IL-21 in patients receiving long-term, high-dose glucocorticoid therapy may contribute to ameliorate lymphopenia and the complications associated to it., (Published by Elsevier Inc.)

Interleukin-15 is a pleiotropic cytokine type I four alpha-helical bundle cytokine that along with IL-2, IL-4, IL-7, IL-9, and IL-21 shares the common cytokine receptor γ chain, γ c . IL-15 is vital for the development, survival, and expansion of natural killer cells and for the development of CD8 + memory T cells. Whereas other family γ c cytokines signal by directly binding to their target cells, IL-15 is distinctive in that it binds to IL-15Rα, a sushi domain containing binding protein that is expressed on a number of cell types, including monocytes and dendritic cells as well as T cells, and then is trans-presented to responding cells that express IL-2Rβ and γ c . This distinctive mechanism for IL-15 relates to its role in signaling in the context of cell-cell interactions and signaling synapses. The actions of IL-15 and ways of manipulating its actions to potential therapeutic benefit are discussed., (2022 Published by Elsevier Inc.)

Signal tranducer and activator of transcription 5 (STAT5) plays a critical role in mediating cellular responses following cytokine stimulation. STAT proteins critically signal via the formation of dimers, but additionally, STAT tetramers serve key biological roles, and we previously reported their importance in T and natural killer (NK) cell biology. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a - Stat5b N-domain double knockin (DKI) mouse strain, we report here that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE). The mild EAE phenotype observed in DKI mice correlates with the impaired extravasation of pathogenic T-helper 17 (Th17) cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated STAT5 tetramerization regulates the production of CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune neuroinflammation., Competing Interests: The authors declare no competing interest.

The interleukin-21 receptor (IL-21R) can be upregulated in endothelial cells (EC) from ischemic muscles in mice following hind-limb ischemia (HLI), an experimental peripheral arterial disease (PAD) model, blocking this ligand-receptor pathway-impaired STAT3 activation, angiogenesis, and perfusion recovery. We sought to identify mRNA and microRNA transcripts that were differentially regulated following HLI, based on the ischemic muscle having intact, or reduced, IL-21/IL21R signaling. In this comparison, 200 mRNAs were differentially expressed but only six microRNA (miR)/miR clusters (and among these only miR-30b) were upregulated in EC isolated from ischemic muscle. Next, myoglobin-overexpressing transgenic (MgTG) C57BL/6 mice examined following HLI and IL-21 overexpression displayed greater angiogenesis, better perfusion recovery, and less tissue necrosis, with increased miR-30b expression. In EC cultured under hypoxia serum starvation, knock-down of miR-30b reduced, while overexpression of miR-30b increased IL-21-mediated EC survival and angiogenesis. In Il21r -/- mice following HLI, miR-30b overexpression vs. control improved perfusion recovery, with a reduction of suppressor of cytokine signaling 3, a miR-30b target and negative regulator of STAT3. Together, miR-30b appears both necessary and sufficient for IL21/IL-21R-mediated angiogenesis and may present a new therapeutic option to treat PAD if the IL21R is not available for activation.

Interleukin-4 (IL-4) is a four-α-helical bundle type I cytokine with broad pleiotropic actions on multiple lineages. Major actions of IL-4 were initially discovered for B and T cells, but this cytokine acts on more than a dozen different target cells spanning the innate and adaptive immune systems and is produced by multiple different cellular sources. While IL-4 was discovered just under 40 years ago in 1982, the interest in and discoveries related to this cytokine continue to markedly expand. There are important new advances related to its biological actions and to its mechanisms of signaling, including critical genes and downstream targets in a range of cell types. IL-4 is critical not only for careful control of immunoglobulin production but also related to inflammation, fibrosis, allergic reactions, and antitumor activity, with actions of IL-4 occurring through two different types of receptors, one of which is also used by IL-13, a closely related cytokine with partially overlapping actions. In this review, we cover critical older information but also highlight newer advances. An area of evolving interest relates to the therapeutic blockade of IL-4 signaling pathway to treat atopic dermatitis and asthma. Thus, this cytokine is historically important, and research in this area has both elucidated major biological pathways and led to therapeutic advances for diseases that affect millions of individuals., Competing Interests: The authors declare that they have no competing interests.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2021 Leonard WJ et al.)

Human papillomavirus (HPV) infections underlie a wide spectrum of both benign and malignant epithelial diseases. In this report, we describe the case of a young man who had encephalitis caused by herpes simplex virus during adolescence and currently presented with multiple recurrent skin and mucosal lesions caused by HPV. The patient was found to have a pathogenic germline mutation in the X-linked interleukin-2 receptor subunit gamma gene ( IL2RG ), which was somatically reverted in T cells but not in natural killer (NK) cells. Allogeneic hematopoietic-cell transplantation led to restoration of NK cytotoxicity, with normalization of the skin microbiome and persistent remission of all HPV-related diseases. NK cytotoxicity appears to play a role in containing HPV colonization and the ensuing HPV-related hyperplastic or dysplastic lesions. (Funded by the National Institutes of Health and the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources.)., (Copyright © 2021 Massachusetts Medical Society.)

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients 1 . Both the number of transferred T cells and their differentiation state are critical determinants of effective responses 2,3 . T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells 4,5 and lower therapeutic efficacy 6 , whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial 7 . Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8 +  T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8 + T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2021.)

Interleukin-2 is a pleiotropic cytokine that mediates both pro- and anti-inflammatory functions. Immune cells naturally differ in their sensitivity to IL-2 due to cell type and activation state-dependent expression of receptors and signaling pathway components. To probe differences in IL-2 signaling across cell types, we used structure-based design to create and profile a series of IL-2 variants with the capacity to titrate maximum signal strength in fine increments. One of these partial agonists, IL-2-REH, specifically expanded Foxp3+ regulatory T cells with reduced activity on CD8+ T cells due to cell type-intrinsic differences in IL-2 signaling. IL-2-REH elicited cell type-dependent differences in gene expression and provided mixed therapeutic results: showing benefit in the in vivo mouse dextran sulfate sodium (DSS) model of colitis, but no therapeutic efficacy in a transfer colitis model. Our findings show that cytokine partial agonists can be used to calibrate intrinsic differences in response thresholds across responding cell types to narrow pleiotropic actions, which may be generalizable to other cytokine and growth factor systems., Competing Interests: CG, LS, SM Coauthor on a patent which includes discoveries described in this manuscript (WO/2019/104092). HW, FM, PL, MP, PH, IK, NN, TH, LP, MK, PB, LS, EM, JP, WL No competing interests declared, KG Coauthor on a patent which includes discoveries described in this manuscript (WO/2019/104092). Founder of Synthekine.

Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.

Thymic stromal lymphopoietin (TSLP) is a cytokine that acts directly on CD4 + T cells and dendritic cells to promote progression of asthma, atopic dermatitis, and allergic inflammation. However, a direct role for TSLP in CD8 + T-cell primary responses remains controversial and its role in memory CD8 + T cell responses to secondary viral infection is unknown. Here, we investigate the role of TSLP in both primary and recall responses in mice using two different viral systems. Interestingly, TSLP limited the primary CD8 + T-cell response to influenza but did not affect T cell function nor significantly alter the number of memory CD8 + T cells generated after influenza infection. However, TSLP inhibited memory CD8 + T-cell responses to secondary viral infection with influenza or acute systemic LCMV infection. These data reveal a previously unappreciated role for TSLP on recall CD8 + T-cell responses in response to viral infection, findings with potential translational implications., Competing Interests: RE, EW, RS, PL, JO, MK, DG, PS, ND, DM, WL No competing interests declared

Inter-individual differences in T helper (Th) cell responses affect susceptibility to infectious, allergic and autoimmune diseases. To identify factors contributing to these response differences, here we analyze in vitro differentiated Th1 cells from 16 inbred mouse strains. Haplotype-based computational genetic analysis indicates that the p53 family protein, p73, affects Th1 differentiation. In cells differentiated under Th1 conditions in vitro, p73 negatively regulates IFNγ production. p73 binds within, or upstream of, and modulates the expression of Th1 differentiation-related genes such as Ifng and Il12rb2. Furthermore, in mouse experimental autoimmune encephalitis, p73-deficient mice have increased IFNγ production and less disease severity, whereas in an adoptive transfer model of inflammatory bowel disease, transfer of p73-deficient naïve CD4 + T cells increases Th1 responses and augments disease severity. Our results thus identify p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation may contribute to susceptibility to autoimmune disease.

Interleukin (IL)-2 and IL-21 dichotomously shape CD8 + T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (T SCM ) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1 While deletion of Ldha prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of T SCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21., Competing Interests: Competing interest statement: W.J.L. is an inventor on patents or patent applications related to IL-2 and IL-21. D.H., S.G., L.M.N., L.G., and W.J.L. are inventors on patent application(s) related to the LDH inhibitor described herein. L.G. is an inventor on a patent related to methods for generating TSCM cells.

T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1 + Tcf-1 + CD8 + T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1 + CD8 + T cells initially differentiated into a transitory population of CD101 - Tim3 + cells that later converted into CD101 + Tim3 + cells. Recently generated CD101 - Tim3 + cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101 - Tim3 + CD8 + T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Uncontrolled interferon γ (IFNγ)-mediated T-cell responses to commensal microbiota are a driver of inflammatory bowel disease (IBD). Interleukin-10 (IL-10) is crucial for controlling these T-cell responses, but the precise mechanism of inhibition remains unclear. A better understanding of how IL-10 exerts its suppressive function may allow identification of individuals with suboptimal IL-10 function among the heterogeneous population of IBD patients. Using cells from patients with an IL10RA deficiency or STAT3 mutations, we demonstrate that IL-10 signaling in monocyte-derived dendritic cells (moDCs), but not T cells, is essential for controlling IFNγ-secreting CD4 + T cells. Deficiency in IL-10 signaling dramatically increased IL-1β release by moDCs. IL-1β boosted IFNγ secretion by CD4 + T cells either directly or indirectly by stimulating moDCs to secrete IL-12. As predicted a signature of IL-10 dysfunction was observed in a subgroup of pediatric IBD patients having higher IL-1β expression in activated immune cells and macroscopically affected intestinal tissue. In agreement, reduced IL10RA expression was detected in peripheral blood mononuclear cells and a subgroup of pediatric IBD patients exhibited diminished IL-10 responsiveness. Our data unveil an important mechanism by which IL-10 controls IFNγ-secreting CD4 + T cells in humans and identifies IL-1β as a potential classifier for a subgroup of IBD patients.

We have recently defined a novel population of PD-1 (programmed cell death 1)+ TCF1 (T cell factor 1)+ virus-specific CD8 T cells that function as resource cells during chronic LCMV infection and provide the proliferative burst seen after PD-1 blockade. Such CD8 T cells have been found in other chronic infections and also in cancer in mice and humans. These CD8 T cells exhibit stem-like properties undergoing self-renewal and also differentiating into the terminally exhausted CD8 T cells. Here we compared the epigenetic signature of stem-like CD8 T cells with exhausted CD8 T cells. ATAC-seq analysis showed that stem-like CD8 T cells had a unique signature implicating activity of HMG (TCF) and RHD (NF-κB) transcription factor family members in contrast to higher accessibility to ETS and RUNX motifs in exhausted CD8 T cells. In addition, regulatory regions of the transcription factors Tcf7 and Id3 were more accessible in stem-like cells whereas Prdm1 and Id2 were more accessible in exhausted CD8 T cells. We also compared the epigenetic signatures of the 2 CD8 T cell subsets from chronically infected mice with effector and memory CD8 T cells generated after an acute LCMV infection. Both CD8 T cell subsets generated during chronic infection were strikingly different from CD8 T cell subsets from acute infection. Interestingly, the stem-like CD8 T cell subset from chronic infection, despite sharing key functional properties with memory CD8 T cells, had a very distinct epigenetic program. These results show that the chronic stem-like CD8 T cell program represents a specific adaptation of the T cell response to persistent antigenic stimulation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)