Interleukin-4 (IL-4) plays a central role in type 2 immune responses. Despite its potential use for allergic and autoimmune diseases, its pleiotropic receptor binding complicates selective targeting of IL-4 signaling pathways. We developed a chemical synthesis of (i) IL-4 variants with atomically tailored side-chain modifications that deter specific receptor interactions and (ii) conditionally activatable IL-4 variants uncaged with 365-nanometer light. In primary cell studies, different variants elicited selective STAT5 or STAT6 phosphorylation in lymphocytes or neutrophils. In murine studies, photocaged IL-4 suppressed inflammation only upon UV irradiation, demonstrating precise on demand control. We accomplished the synthesis and folding of IL-4, a hydrophobic cytokine with three disulfide bonds, using the alpha-ketoacid-hydroxylamine (KAHA) ligation to assemble three segments. We introduced further conjugations, including PEGylation for half-life extension, through orthogonal ligations enabled by functionalized amino acid building blocks. This work highlights the flexibility of chemical protein synthesis to produce therapeutically valuable cytokines, including receptor-biased and spatiotemporally activatable IL-4 variants.

Acute viral infections are usually cleared by an efficient anti-pathogen immune response, following which immune homeostasis is restored. Occasionally, such pathogen-induced immune response fails to abate despite clinical recovery, but how this occurs in humans has not been thoroughly investigated. Here, we perform a detailed analysis of T cell homeostasis following severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, which reveals persistent activation and dyshomeostasis of CD4 + and CD8 + T cells for 6–12 months after acute infection. Compared to steady-state and unlike T cell responses following vaccination, interleukin (IL)−2 receptor and IL-7 receptor expression remains altered on both SARS-CoV-2-specific and bystander T cells for 6–12 months after acute infection. These alterations correlate with increased IL-7 and IL-15 serum levels and are reproduced by in vitro stimulation by IL-7 and IL-15, but surprisingly not by IL-2. Collectively, our study demonstrates prolonged T cell dyshomeostasis driven by dysregulated homeostatic cytokine signals following acute viral infection.

Signal transducer and activator of transcription (STAT) is a family of key transcriptional regulators in immune, epithelial and mesenchymal cells. Aberrant STAT activity is associated with malignancy, autoimmunity and immunodeficiency. The STAT signaling pathways are very attractive drug targets; however, validated tools to monitor real-time activation of STATs are lacking. Here, we developed a class of highly sensitive genetically encoded STAT biosensors, termed STATeLights, which allowed direct and continuous detection of STAT activity in live cells with high spatiotemporal resolution. Using human STAT5A, we demonstrate the versatility of STATeLight5A to quantify the activation of wild-type STAT5 versus disease-associated STAT5 mutants and to precisely select compounds targeting the STAT5 signaling pathway. Moreover, STATeLight5A also facilitated real-time tracking of STAT5 activation in human primary CD4+ T cells. Collectively, our biosensors open up unprecedented possibilities of studying STAT biology and druggability in various cellular contexts.

Radiotherapy (RT) can stimulate anti-cancer T cell responses, and cytokines, notably interleukin-2 (IL-2), are necessary for optimal T cell function and memory. However, timing and IL-2 receptor (IL-2R) bias of IL-2 signals are ill-defined. Using image-guided RT in a mouse colon cancer model, we observed single high-dose (20 Gy) RT transiently upregulated IL-2Rα (CD25) on effector CD8+ T cells, facilitating the use of CD25-biased IL-2 immunotherapy. Timed administration of CD25-biased IL-2 treatment after RT favored intratumoral expansion of CD8+ T cells over regulatory T cells, which resulted in comparable anti-tumor effects as with RT plus IL-2Rβ (CD122)-biased IL-2 immunotherapy. Moreover, intratumoral CD8+ T cells of animals receiving combined IL-2R-biased IL-2 and RT showed reduced markers of exhaustion. These combination treatments affected both primary irradiated and distant non-irradiated tumors and achieved durable responses. We demonstrate that timed IL-2R subunit-biased IL-2 immunotherapy synergizes with single high-dose RT to achieve potent anti-cancer immunity.

We develop and present a one-pot sandwich immunoassay (termed oneSTEP) to detect target biomolecules in complex biological fluids based on programmable zwitterionic polymer coacervates. We design these coacervates to selectively recruit target analytes with ultralow nonspecific adsorption. We show that dynamic compartmentalization combined with local target enrichment delivers a rapid and wash-free sandwich immunoassay with high specificity and a high signal-to-noise ratio. The fluorescence-based readout is performed using standard microscopy methods and flow cytometry. We demonstrate the capabilities of the oneSTEP assay by detecting complement component 5 in human serum and the spike protein of severe acute respiratory syndrome coronavirus 2 in artificial saliva with a limit of detection of 300 pM. The results highlight the potential of the oneSTEP sandwich immunoassay as complementary to bead-based approaches in high-throughput screening studies as well as clinical diagnostics.

Due to its stimulatory potential for immunomodulatory CD4+ regulatory T (Treg) cells, low-dose interleukin-2 (IL-2) immunotherapy has gained considerable attention for the treatment of autoimmune diseases. In this investigator-initiated single-arm non-placebo-controlled phase-2 clinical trial of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE) patients, we generated a comprehensive atlas of in vivo human immune responses to low-dose IL-2. We performed an in-depth study of circulating and cutaneous immune cells by imaging mass cytometry, high-parameter flow cytometry, transcriptomics, and targeted serum proteomics. Low-dose IL-2 stimulated various circulating immune cells, including Treg cells with a skin-homing phenotype that appeared in the skin of SLE patients in close interaction with endothelial cells. Analysis of surface proteins and transcriptomes revealed different IL-2-driven Treg cell activation programs, including gut-homing CD38+, skin-homing HLA-DR+, and highly proliferative inflammation-homing CD38+ HLA-DR+ Treg cells. Collectively, these data define the distinct human Treg cell subsets that are responsive to IL-2 immunotherapy.

Interleukin-4 (IL-4) is a cytokine that plays a central role in type 2 immune responses and is involved in regulating pleiotropic actions in our body by engaging multiple different IL-4 receptor (IL-4R) complexes. Targeting the IL-4R system has a high potential for therapeutic intervention for allergic and autoimmune diseases. A challenge in developing this pleiotropic cytokine for clinical application is the construction of variants tailored for engagement with specific receptor IL-4R subunits, which are necessary for selective activation of specific signaling pathways to treat disease with minimum side effects. To establish a platform for preparation of tailored IL-4 variants, we developed a modular and flexible chemical synthesis of IL-4 and applied this approach to the preparation of (i) IL-4 variants that act as receptor antagonist due to presence of unnatural residues that block specific interactions, and (ii) photocaged and in vivo half-life extended IL-4 variants that can be conditionally activated using UV light, achieved by the incorporation of a photocaged Gln116 residue. We were able to show that these different cytokine variants elicit differential STAT5 or STAT6 phosphorylation in lymphocytes or neutrophils in vitro with just one amino acid substitution. Furthermore, we demonstrated that the photocaged IL-4 can be activated by UV light and effectively suppresses neutrophils in an inflammation model in vivo. Collectively, this work demonstrated the flexibility and applicability of chemical protein synthesis by allowing us to broaden the scope of protein variants that can be accessed for the preparation and evaluation of therapeutically valuable proteins.

The key role of T cells in cancer immunotherapy is well established and is highlighted by the remarkable capacity of Ab-mediated checkpoint blockade to overcome T-cell exhaustion and amplify anti-tumor responses. However, total or partial tumor remission following checkpoint blockade is still limited to only a few types of tumors. Hence, concerted attempts are being made to devise new methods for improving tumor immunity. Currently, much attention is being focused on therapy with IL-2. This cytokine is a powerful growth factor for T cells and optimises their effector functions. When used at therapeutic doses for cancer treatment, however, IL-2 is highly toxic. Nevertheless, recent work has shown that modifying the structure or presentation of IL-2 can reduce toxicity and lead to effective anti-tumor responses in synergy with checkpoint blockade. Here, we review the complex interaction of IL-2 with T cells: first during normal homeostasis, then during responses to pathogens, and finally in anti-tumor responses.

Haemophagocytic lymphohistiocytosis (HLH), a life-threatening hyperinflammatory disorder often driven by dysfunctional cytotoxic CD8+ T cells, is marked by cytokine storms, which may follow viral infections. In a study of a perforin-deficient mouse model of HLH with a viral trigger, we aimed to determine if CD8+ T cell behaviour could be modulated by targeted interleukin (IL)-2 treatment. We observed a paradoxical benefit that contrasted with IL-2's typical role in boosting T cell activity: targeted IL-2 delivery to CD8+ T cells led to reduced hyperinflammation and disease severity. Our results demonstrated that IL-2 induced exhaustion in overactive CD8+ T cells, thus mitigating hyperinflammation. These findings highlight the context-dependency of cytokine treatment and suggest new therapeutic strategies for HLH and other inflammatory diseases by leveraging cell exhaustion. ### Competing Interest Statement The authors have declared no competing interest.

Summary Radiotherapy (RT) can stimulate anti-cancer T cell responses that target primary and distant tumors. In addition to antigen-mediated stimulation of effector T cells, signals from stimulatory cytokines, notably interleukin-2 (IL-2), are necessary for optimal T cell function and memory. However, timing and IL-2 receptor (IL-2R) bias of such signals are ill-defined. Using image-guided RT in a mouse colon cancer model, we observed that single high-dose (1 x 20 Gy) RT transiently upregulated IL-2Rα (CD25) on effector CD8 + T cells, facilitating the use of CD25-biased IL-2 immunotherapy. Timed administration of CD25-biased IL-2 treatment after RT favored the expansion of tumor-infiltrating CD8 + T cells over regulatory T cells and IL-2Rβ (CD122) high CD8 + T cells, which resulted in comparable anti-tumor effects as with RT plus CD122-biased IL-2 immunotherapy. Moreover, intratumoral CD8 + T cells from animals receiving combined IL-2R-biased IL-2 and RT showed reduced signatures of T cell exhaustion. Finally, these combination treatments affected both primary irradiated and distant non-irradiated tumors, achieving durable responses. We demonstrate that timed and IL-2R subunit-biased IL-2 immunotherapy synergized with single high-dose RT to achieve potent anti-cancer immunity.

Profiling of plasma proteins in individuals with COVID-19 shows that complement activation and myeloid inflammation are major pathways in the pathogenesis of long COVID and identifies distinct profiles of immune dysregulation in individuals with long COVID, highlighting the heterogeneous and diverse nature of this disease.

Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte–platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.

c-Myb upregulation in MC38 tumor cells modulates genes involved in immune responses. A, Gene Ontology enrichment analysis of genes significantly dysregulated between tumor cells from mice on Dox- and Ctrl-chow using Enrichr tool (https://maayanlab.cloud/Enrichr). Sorted CD45−GFP+ tumor cells from subcutaneous MC38T/O tumors in C57BL/6J mice, after 21 days were analyzed by RNA-seq (n = 4). B, Using the list of 87 dysregulated genes as input to pathDIP (https://ophid.utoronto.ca/pathDIP), we identified significantly enriched pathways (Supplementary Table S3). Here we highlight pathway-gene associations for the top 10% significantly enriched pathways (i.e., more than 35 of the input DEGs present; listed in Supplementary Table S4). Similar pathways are grouped and color-coded (both pathway name and edge color that connects to corresponding DEGs). Number of gene–pathway associations (i.e., node degree) is reflected by node color shade from green (low degree; only a few pathways or a few genes) through red (high degree; many pathways or many genes). The node degree for genes ranges from 0 (MS4A7, KDM1B) to 90 (CD74, PTPRC, ITGB2), and for pathways from 35 (hematopoietic cell lineage, VEGFA-VEGFR2 Signaling, T-cell activation, costimulation by the CD28 family, TLR signaling, macrophage markers, T-cell antigen receptor pathway during Staphylococcus aureus infection, C-type lectin receptor signaling, RANKL/RANK Signaling) to 71 (chemokine signaling).

Early c-Myb upregulation is crucial for control of tumor growth. A, Experimental setup. C57BL/6J mice were subcutaneously injected with MC38T/O cells and fed Ctrl or Dox chow. For two groups, chow is switched at day 14 for the rest of the experiment, Dox→Ctrl (D→C) or Ctrl→Dox (C→D). B–D, Tumor growth curves, final tumor weight, representative pictures (B); flow cytometry analysis (C); and Bio-plex analysis (D) of indicated groups are shown. Heat maps are shown of representative samples. E, Flow cytometry analysis of subcutaneous MC38T/O tumors at day 14. Flow cytometry analysis for CD8+ T cells (CD45+CD11b−CD3e+CD8+) are represented as number of cells per gram of tumor tissue (C, E). CD8+PD1+/perforin+/granzymeB+ populations are represented as percentage of total CD8+ T cells (C, E). F, Long-term tumor growth was assessed in C57BL/6J mice subcutaneously injected with MC38T/O cells and fed Ctrl or Dox chow for the duration of the experiment; or Dox chow for the first 14 days and subsequently switched to Ctrl chow for the rest of the experiment (D → C). n = 5–6 (B), n = 4 (C), n = 3–4 (D), n = 8–10 (E), n = 7–8 (F) mice per group. Data are presented as mean values ± SEM. Two-way ANOVA was used for comparing tumor growth curves (B, F). One-way ANOVA with Kruskal–Wallis posttest was used (C–E) for statistical analysis. *, P < 0.05; **, P < 0.01.

The B cell response to different pathogens uses tailored effector mechanisms and results in functionally specialized memory B (B m ) cell subsets, including CD21 + resting, CD21 – CD27 + activated and CD21 – CD27 – B m cells. The interrelatedness between these B m cell subsets remains unknown. Here we showed that single severe acute respiratory syndrome coronavirus 2-specific B m cell clones showed plasticity upon antigen rechallenge in previously exposed individuals. CD21 – B m cells were the predominant subsets during acute infection and early after severe acute respiratory syndrome coronavirus 2-specific immunization. At months 6 and 12 post-infection, CD21 + resting B m cells were the major B m cell subset in the circulation and were also detected in peripheral lymphoid organs, where they carried tissue residency markers. Tracking of individual B cell clones by B cell receptor sequencing revealed that previously fated B m cell clones could redifferentiate upon antigen rechallenge into other B m cell subsets, including CD21 – CD27 – B m cells, demonstrating that single B m cell clones can adopt functionally different trajectories.

Transplantation of solid organs can be life-saving in patients with end-stage organ failure, however, graft rejection remains a major challenge. In this study, by pre-conditioning with interleukin-2 (IL-2)/anti-IL-2 antibody complex treatment biased toward IL-2 receptor α, we achieved acceptance of fully mismatched orthotopic lung allografts that remained morphologically and functionally intact for more than 90 days in immunocompetent mice. These allografts are tolerated by the actions of forkhead box p3 (Foxp3)+ regulatory T (Treg) cells that home to the lung allografts. Although counts of circulating Treg cells rapidly return to baseline following cessation of IL-2 treatment, Foxp3+ Treg cells persist in peribronchial and peribronchiolar areas of the grafted lungs, forming organized clusters reminiscent of inducible tertiary lymphoid structures (iTLS). These iTLS in lung allografts are made of Foxp3+ Treg cells, conventional T cells, and B cells, as evidenced by using microscopy-based distribution and neighborhood analyses. Foxp3-transgenic mice with inducible and selective deletion of Foxp3+ cells are unable to form iTLS in lung allografts, and these mice acutely reject lung allografts. Collectively, we report that short-term, high-intensity and biased IL-2 pre-conditioning facilitates acceptance of vascularized and ventilated lung allografts without the need of immunosuppression, by inducing Foxp3-controlled iTLS formation within allografts.