Cytokines dimerize two receptor chains to activate Janus kinases and signal transducer and activator of transcription (STAT) transcription factors that regulate immune cells, but they have therapeutic liabilities. We engineered "Trikines" to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs. Trikines coactivated phosphorylation of STAT5 (pSTAT5) and pSTAT3 signatures distinct from natural cytokines by assembling trimeric combinations of interleukin-2 (IL-2), IL-10, and IL-21 receptors. In preclinical models, an IL-2-based Trikine restrained terminal differentiation of T cells, promoted stemness, and enhanced durability of tumor control without observable toxicity. An IL-10-based Trikine induced immune infiltration into poorly immunogenic tumors, showing efficacy in preclinical models of small cell lung cancer and pancreatic cancer. Trikines obviate the need for cell engineering to customize STAT signatures and may hold potential for immunotherapy.

Regulatory T cell (Treg) therapy is an emerging platform for controlling immune overactivation. Persistence of infused Tregs is limited by insufficient IL-2, which is essential for Treg survival and function. IL-2 activates many immune cells, imposing a challenge for the selective provision of IL-2 to infused Tregs. In this study, we found that infusions of orthogonal (ortho) IL-2 failed to enhance Tregs expressing a corresponding orthoIL-2 receptor (IL-2R) in a mouse model of autoimmune diabetes. Engineering Tregs with an orthoIL-2 tethered to its receptor achieved selective autocrine signaling; increased CD25, CTLA-4, and Foxp3 expression; supported Treg persistence without exogenous IL-2 in vivo; and improved the efficacy of Treg prevention of autoimmune diabetes. Inserting the tethered orthoIL-2 construct into the Foxp3 locus enabled Treg-specific self-reinforced expression through the activation of the Foxp3 locus by increased IL-2 signaling. Together, these results illustrate a safe and effective cell-engineering solution for overcoming Tregs' dependency on exogenous IL-2, thereby achieving superior therapeutic efficacy.

Cytokines are signaling molecules that coordinate complex immune processes and are frequently dysregulated in disease. While cytokine blockade has become a common therapeutic modality, cytokine agonism has had limited utility due to the widespread expression of cytokine receptors with pleiotropic effects. To overcome this limitation, we devise an approach to engineer molecular switches, termed cytokine adaptors, that transform one cytokine signal into an alternative signal with a different functional output. Endogenous cytokines act to nucleate the adaptors, converting the cytokine–adaptor complex into a surrogate agonist for a different cytokine pathway. In this way, cytokine adaptors, which have no intrinsic agonist activity, can function as conditional, context-dependent agonists. We develop cytokine adaptors that convert IL-10 or TGF-β into IL-2 receptor agonists to reverse T cell suppression. We also convert the pro-inflammatory cytokines IL-23 or IL-17 into immunosuppressive IL-10 receptor agonists. Thus, we show that cytokine adaptors can convert immunosuppressive cytokines into immunostimulatory cytokines, or vice versa. Unlike other methods of immune conversion that require cell engineering, cytokine adaptors are soluble molecules that leverage endogenous cues from the microenvironment to drive context-specific signaling.

Building on the enhancing effects of the orthogonal IL2 (oIL2) system for T cell immunotherapy, we explored a viral vector-free strategy for generating orthogonal CAR (orthoCAR) T cells. We show that prime editing can generate orthogonal IL2Rβ (oIL2Rβ) mutations in T cells with an average efficiency of 72% (range 54-89%). The mutations are functional, enrich ex vivo when cultured with oIL2 and enhance the engraftment, efficacy and toxicity of CAR T cells in vivo comparable to orthoCAR T cells generated by co-transduction of the oIL2Rβ and CAR. We further show that insertion of a CD19-specific CAR with a chicken beta-globin promoter into the IL2 locus (IL2-CAR19) can be achieved by a CRISPR/Cas9-triggered homology-directed repair (HDR) with an average efficiency of 46% (range 30 – 65%) in primary T cells. IL2-CAR19 T cells show a > 3-log reduction in IL-2 production consistent with a >96% IL2 gene disruption. Despite the loss of autocrine IL2 secretion, IL2-CAR19 T cells rapidly and potently kill NALM-6 leukemic cells in vitro and in vivo without the need for exogenous IL2 support. Combining prime editing of oIL2Rβ with IL2-CAR19 T cells yields orthoCAR T cells that exhibit in vitro and in vivo function comparable to lentivirally-generated orthoCAR T cells. Prime editing of oIL2Rβ expands the potential application of the orthogonal IL2 system to existing T cell therapies. Moreover, robust functional activity of IL2-CAR19 cells highlights the dispensable nature of the IL2 gene in CAR T cells and its availability for target insertion of genetic payloads.

Immune effector cell (IEC) therapies provide increasingly important treatment options for patients with certain hematologic malignancies. Apheresis comprises a critical step, providing the starting material from which peripheral-blood derived IEC therapies are manufactured. Regulatory and accreditation bodies providing laws, regulations, guidance, and standards appropriately impose high quality goals for apheresis collection facilities, but they are often not all congruent with clinical trial requirements. Multiple efforts are underway to standardize apheresis procedures for IEC therapies. However, few publications give practical guidance on establishing quality indicators for apheresis collection facilities. Recognizing this, the American Society for Apheresis (ASFA) Clinical Applications Committee (Immune Effector Cell Therapy Subcommittee) sought to review and define the quality indicators that can be applied at each phase of the collection process. This paper is primarily focused on apheresis collection facilities in the United States (US), but general concepts may be applicable outside of the US as well. These recommendations are also endorsed by the Foundation for the Accreditation of Cellular Therapy (FACT), Association for the Advancement of Blood and Biotherapies (AABB), and International Society for Cell & Gene Therapy (ISCT).

Background: Recombinant activated factor VIIa (rFVIIa, NovoSeven®) is an FDA-approved medication for managing bleeding in patients with hemophilia A or B with inhibitors and for those with congenital factor VII deficiency. Its use has expanded off-label in the perioperative context to manage unexpected bleeding complications. However, there is still a gap in real-world data regarding institutional practices, dosing, and outcomes associated with rFVIIa. Methods: With IRB approval, we identified patients who received rFVIIa in the perioperative period between 2020 to 2024 at Mayo Clinic. A detailed chart review was conducted to gather demographic data, comorbid conditions, bleeding disorders, timing, and dosing of perioperative rFVIIa (pre-, intra-, and postoperative), along with postoperative outcomes. We focused on thrombotic and bleeding events, hospital readmissions, and return to the operating room (OR). Descriptive statistics were used for patient characteristics, rFVIIa dosing patterns, and postoperative complications. Type of surgical interventions were stratified based on UCLA perioperative risk stratifications, with a risk score of 5 equating to very high risk surgery and a score of 1 equating to very low risk surgery. Results: Of the 55 patients included, the median age at surgery was 42.3 years (SD 27.4), and 69% were male. The majority were non-Hispanic white (91%). The most common bleeding diatheses were Factor VII deficiency (54.6%), Factor VIII inhibitor (12.7%), and Glanzmann thrombasthenia (7.3%). Less frequent conditions included Hemophilia A (3.6%) and Hemophilia A with inhibitors (3.6%). Among patients with Factor VII deficiency, mild deficiency accounted for 65.9% of cases, followed by severe (19.5%) and moderate (14.6%) forms. Adjunctive medications to control bleeding included emicizumab, FEIBA, and rituximab. Mean estimated blood loss was 136 mL (SD 211 mL), and mean length of stay was 5.7 days (SD 6.4). Surgical risk strongly correlated with perioperative outcomes: blood loss increased from 19 mL in very low-risk surgeries to 352 mL in very high-risk surgeries, while mean length of stay increased from 4.5 days (risk 1) to 19 days (risk 4) and 11 days (risk 5). Actual rFVIIa dosing ranged from 7.2 to 7211.5 mcg/kg per dose, with a median of 67.4 mcg/kg and a mean of 852.8 mcg/kg, reflecting variability in clinical practice. When stratified by surgical risk, the highest median doses were observed in very low- and low-risk surgeries (84.4 and 86.8 mcg/kg, respectively), while very high-risk surgeries had the lowest median dose (30.0 mcg/kg). In the postoperative period, thrombotic events did not occur in the first 90 days but were documented in 1.8% of patients between 90 and 365 days and in 1.8% after 365 days. Bleeding complications were frequent and demonstrated predominance in the immediate post-op period. Within the first 30 days after surgery, 18.2% of patients experienced bleeding complications, with 3.6% classified as major and 14.5% classified as minor, based on ISTH criteria. Between 30 and 90 days, bleeding complications occurred in 9.1% of patients (3.6% major and 5.5% minor), between 90 and 365 days, bleeding was observed in 10.9% of patients, all of which were minor, and beyond one year, bleeding complications were documented in 23.6% of patients, with 3.6% major and 21.8% minor. Readmissions were also common and followed a similar time distribution. In the first 30 days, 7.3% of patients had readmissions related to the prior surgery, and 3.6% had unrelated readmissions. Between 30 and 90 days, 3.6% of readmissions were related and 3.6% were unrelated. Between 90 and 365 days, 3.6% were related and 14.5% were unrelated to the initial procedure. Returns to the OR occurred in 1.8% of patients between 30 and 90 days and in 1.8% between 90 and 365 days. Conclusions: In conclusion, perioperative rFVIIa was most frequently used for Factor VII deficiency but was also administered to patients with Factor VIII inhibitor and Glanzmann's thrombasthenia, as well as several rare congenital and acquired bleeding disorders. Postoperative thrombotic events were rare, while ISTH-defined bleeding complications and readmissions were common. Most bleeding complications were minor, but their frequency was highest in the early postoperative period and again beyond one year. High-risk surgeries were associated with greater blood loss, prolonged hospitalization, and a higher burden of postoperative complications.

Building on the enhancing effects of the orthogonal interleukin-2 (oIL-2) system for T-cell immunotherapy, we explored a viral vector-free strategy for generating orthogonal chimeric antigen receptor (orthoCAR) T cells. We show that prime editing can generate orthogonal IL-2Rβ (oIL-2Rβ) mutations in T cells with an average efficiency of 72% (range, 54%-89%). The mutations are functional, enrich ex vivo when cultured with oIL-2 and enhance the engraftment, efficacy, and toxicity of CAR T cells in vivo comparable to orthoCAR T cells generated by cotransduction of the oIL-2Rβ and CAR. We further show that the insertion of a CD19-specific CAR with a chicken β-globin promoter into the IL-2 locus (IL-2-CAR19) can be achieved by a CRISPR/CRISPR-associated protein 9 (Cas9)-triggered homology-directed repair with an average efficiency of 46% (range, 30%-65%) in primary T cells. IL-2-CAR19 T cells show a >3-log reduction in IL-2 production consistent with a >96% IL-2 gene disruption. Despite the loss of autocrine IL-2 secretion, IL-2-CAR19 T cells rapidly and potently kill NALM-6 leukemic cells in vitro and in vivo without the need for exogenous IL-2 support. Combining the prime editing of oIL-2Rβ with IL-2-CAR19 T cells yields orthoCAR T cells that exhibit in vitro and in vivo function comparable to lentivirally generated orthoCAR T cells. The prime editing of oIL-2Rβ expands the potential application of the orthogonal IL-2 system to existing T-cell therapies. Moreover, the robust functional activity of IL-2-CAR19 cells highlights the dispensable nature of the IL-2 gene in CAR T cells and its availability for the target insertion of genetic payloads.

Cytokines and their receptors enable precise tuning of T cell function. Leveraging this biology holds tremendous promise for optimizing antitumor immunity. Arming T cells with a synthetically orthogonal interleukin (IL)-9 receptor (o9R), for instance, permits facile engraftment and potent anti-tumor functions. Exploiting the paucity of wild-type IL-9R expression and the safety of high doses of IL-9, here, we showed that, compared with o9R, T cells engineered with wild-type IL-9R exhibited superior tissue infiltration, stemness, and anti-tumor activity. These qualities were consistent with a stronger Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal, which included canonically IL-12-driven STAT4 in addition to STAT1/3/5. IL-9R T cells were exquisitely sensitive to perturbations of proximal signaling, including structure-guided attenuation, amplification, and rebalancing of JAK/STAT signals. Biased IL-9R mutants showed that STAT1 acts as a rheostat between stem-like and effector states. In summary, we identify IL-9/IL-9R as a naturally orthogonal cytokine-receptor pair with an optimal JAK/STAT signaling profile for engineered T cell therapy.

Purpose:Autologous serum tears (ASTs) are produced from serum obtained by blood draw and used to treat severe dry eye diseases. Multiple cases of failed AST production occurred due to serum becoming a solidified, gel-like substance after centrifugation. The aim of this study was to identify risk factors associated with inability to compound AST.Methods:From January 2019 to December 2022, 99 patients received AST produced at Mayo Clinic in Arizona. Patient demographics, medical diagnoses, medications, laboratory results, and events surrounding blood draws were obtained through an IRB-approved retrospective chart review. The laboratory and pharmacy protocol procedures were observed in person after the occurrence of the last production issue.Results:Ten total clotted serum episodes occurred in 4 patients, each with multiple occurrences. The indication for AST was chronic ocular graft-vs-host disease (n = 3) and Sj & ouml;gren syndrome (n = 1). Eighty percent of episodes were associated with either a systemic infection (n = 7) or pulmonary embolism (n = 1) within the prior month. At the time of blood draw, all 4 patients were treated with antiplatelet and/or anticoagulation medications, including rivaroxaban, apixaban, aspirin, and low-molecular-weight heparin.Conclusions:The AST protocol was increased from 30 to 60 minutes of resting time to allow clotting before centrifugation. Inadequate clotting during this stasis period likely resulted in fibrinogen remaining suspended in the serum, leading to solidified plasma instead of liquid serum. We hypothesize risk of inadequate clotting would be increased in patients taking anticoagulation or antiplatelet medications. Our recommendations include ensuring 60-minute collection tube resting time for all patients, including those on anticoagulation medication.

The use of normothermic machine perfusion (NMP) over static cold storage in liver transplantation has been shown to reduce posttransplant risks of early allograft dysfunction, primary nonfunction, and ischemic cholangiopathy, and its increasing use has played a role in the expanded utilization of marginal livers. While studies have demonstrated improved clinical outcomes using NMP over static cold storage preservation, real-time intraoperative data reflecting the quality and viability of NMP livers is limited. This retrospective, single-center study compared NMP versus static cold storage livers in first-time recipients of liver transplants through the evaluation of synthetic coagulation function as measured by thromboelastography and conventional coagulation testing. Secondarily, transfusion utilization between the 2 cohorts was reviewed. One hundred eighty-six recipients of liver transplants receiving allografts from donors after circulatory death were included in the study, of which 99 (53%) allografts were preserved in static cold storage, and 87 (47%) allografts were placed on the TransMedics Organ Care System. Study findings showed NMP livers supported with the TransMedics Organ Care System were associated with increased synthetic coagulation function and less excess fibrinolysis in the postreperfusion period compared to static cold storage livers, and that these findings were better reflected in real-time with thromboelastography monitoring versus conventional coagulation testing. Following reperfusion, there was a significant decrease in the transfusion of blood products in the NMP group compared with that in the static cold storage group. Overall, we determined that the use of intraoperative thromboelastography can provide real-time data to assess one aspect of reperfusion liver quality and viability.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (βc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of βc dimers. These findings provide insights into IL-5 and βc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged βc signaling.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for patients with hematological disorders and bone marrow failure syndromes. Graft-versus-Host Disease (GVHD) remains a leading cause of morbidity post-transplant. Regulatory T cell (Treg) therapies are efficacious in ameliorating GVHD but limited by variable suppressive capacities and the need for a high therapeutic dose. Here, we sought to expand Treg in vivo by expressing an orthogonal IL-2Rβ receptor (oIL2Rβ) that would selectively interact with orthogonal IL-2 (oIL2) cytokine and not wildtype IL2. To test whether the orthogonal system would preferentially drive donor Treg expansion, we used a murine MHC-disparate GVHD model of lethally irradiated BALB/c mice given T-cell depleted bone marrow from C57BL/6 (B6) mice alone, or together with B6Foxp3+GFP+ Treg or oIL2Rβ transduced Treg at low cell numbers that typically do not control GVHD with WT Treg. On day 2, B6 Tcons were injected to induce GVHD. Recipients were treated with PBS or oIL2 daily for 14 days, then 3 times weekly for an additional 14 days. Mice treated with oIL2Rβ Treg and oIL2 compared to PBS had enhanced GVHD survival, in vivo selective expansion of Tregs, and greater suppression of Tcon expansion in secondary lymphoid organs, and intestines. Importantly, oIL2Rβ Treg maintained graft-versus-tumor (GVT) responses in two distinct tumor models (A20, MLL-AF9). These data demonstrate a novel approach to enhance the efficacy of Treg cell therapy in allo-HSCT using an oIL2/oIL2Rβ system that allows for selective in vivo expansion of Treg leading to GVHD protection and GVT maintenance.

Rewiring exhausted CD8+ T (Tex) cells toward functional states remains a therapeutic challenge. Tex cells are epigenetically programmed by the transcription factor Tox. However, epigenetic remodeling occurs as Tex cells transition from progenitor (Texprog) to intermediate (Texint) and terminal (Texterm) subsets, suggesting development flexibility. We examined epigenetic transitions between Tex cell subsets and revealed a reciprocally antagonistic circuit between Stat5a and Tox. Stat5 directed Texint cell formation and re-instigated partial effector biology during this Texprog-to-Texint cell transition. Constitutive Stat5a activity antagonized Tox and rewired CD8+ T cells from exhaustion to a durable effector and/or natural killer (NK)-like state with superior anti-tumor potential. Temporal induction of Stat5 activity in Tex cells using an orthogonal IL-2:IL2Rβ-pair fostered Texint cell accumulation, particularly upon PD-L1 blockade. Re-engaging Stat5 also partially reprogrammed the epigenetic landscape of exhaustion and restored polyfunctionality. These data highlight therapeutic opportunities of manipulating the IL-2-Stat5 axis to rewire Tex cells toward more durably protective states.

Interleukin-21 (IL-21) plays a critical role in generating immunological memory by promoting the germinal center reaction, yet clinical use of IL-21 remains challenging because of its pleiotropy and association with autoimmune disease. To better understand the structural basis of IL-21 signaling, we determine the structure of the IL-21–IL-21R–γc ternary signaling complex by X-ray crystallography and a structure of a dimer of trimeric complexes using cryo-electron microscopy. Guided by the structure, we design analogs of IL-21 by introducing substitutions to the IL-21–γc interface. These IL-21 analogs act as partial agonists that modulate downstream activation of pS6, pSTAT3, and pSTAT1. These analogs exhibit differential activity on T and B cell subsets and modulate antibody production in human tonsil organoids. These results clarify the structural basis of IL-21 signaling and offer a potential strategy for tunable manipulation of humoral immunity.

Monoclonal antibodies (Abs) that recognize major histocompatability complex (MHC)-presented tumor antigens in a manner similar to T cell receptors (TCRs) have great potential as cancer immunotherapeutics. However, isolation of ‘TCR-mimic’ (TCRm) Abs is laborious because Abs have not evolved the structurally nuanced peptide–MHC restriction of αβ-TCRs. Here, we present a strategy for rapid isolation of highly peptide-specific and ‘MHC-restricted’ Abs by re-engineering preselected Abs that engage peptide–MHC in a manner structurally similar to that of conventional αβ-TCRs. We created structure-based libraries focused on the peptide-interacting residues of TCRm Ab complementarity-determining region (CDR) loops, and rapidly generated MHC-restricted Abs to both mouse and human tumor antigens that specifically killed target cells when formatted as IgG, bispecific T cell engager (BiTE) and chimeric antigen receptor-T (CAR-T). Crystallographic analysis of one selected pMHC-restricted Ab revealed highly peptide-specific recognition, validating the engineering strategy. This approach can yield tumor antigen-specific antibodies in several weeks, potentially enabling rapid clinical translation.

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.

Clinical trials utilizing regulatory T cell (Treg) therapy in organ transplantation have shown promising results, however, the choice of a standard immunosuppressive regimen is still controversial. Calcineurin inhibitors (CNIs) are one of the most common immunosuppressants for organ transplantation, although they may negatively affect Tregs by inhibiting IL-2 production by conventional T cells. As a strategy to replace IL-2 signaling selectively in Tregs, we have introduced an engineered orthogonal IL-2 (ortho IL-2) cytokine/cytokine receptor (R) pair that specifically binds with each other but does not bind with their wild-type counterparts. Murine Tregs were isolated from recipients and retrovirally transduced with ortho IL-2Rβ during ex vivo expansion. Transduced Tregs (ortho Tregs) were transferred into recipient mice in a mixed hematopoietic chimerism model with tacrolimus administration. Ortho IL-2 treatment significantly increased the ortho IL-2Rβ(+) Treg population in the presence of tacrolimus without stimulating other T cell subsets. All the mice treated with tacrolimus plus ortho IL-2 achieved heart allograft tolerance, even after tacrolimus cessation, whereas those receiving tacrolimus treatment alone did not. These data demonstrate that Treg therapy can be adopted into a CNI-based regimen by utilizing cytokine receptor engineering.