Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), 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 ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.

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

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

Facile repurposing of peptide-MHC-restricted antibodies for cancer immunotherapy.

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 αß-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 αß-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.

Prevention of acute GVHD using an orthogonal IL-2/IL-2Rß system to selectively expand regulatory T cells in vivo.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for patients with hematological disorders and bone marrow (BM) failure syndromes. Graft-versus-host disease (GVHD) remains a leading cause of morbidity posttransplant. 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 interleukin 2 receptor ß (oIL-2Rß) that would selectively interact with oIL-2 cytokine and not wild-type (WT) IL-2. To test whether the orthogonal system would preferentially drive donor Treg expansion, we used a murine major histocompatibility complex-disparate GVHD model of lethally irradiated BALB/c mice given T cell-depleted BM from C57BL/6 (B6) mice alone or together with B6Foxp3+GFP+ Treg or oIL-2Rß-transduced Treg at low cell numbers that typically do not control GVHD with WT Treg. On day 2, B6 activated T cells (Tcons) were injected to induce GVHD. Recipients were treated with phosphate-buffered saline (PBS) or oIL-2 daily for 14 days, then 3 times weekly for an additional 14 days. Mice treated with oIL-2Rß Treg and oIL-2 compared with those treated with PBS had enhanced GVHD survival, in vivo selective expansion of Tregs, and greater suppression of Tcon expansion in secondary lymphoid organs and intestines. Importantly, oIL-2Rß Treg maintained graft-versus-tumor (GVT) responses in 2 distinct tumor models (A20 and MLL-AF9). These data demonstrate a novel approach to enhance the efficacy of Treg therapy in allo-HSCT using an oIL-2/oIL-2Rß system that allows for selective in vivo expansion of Treg leading to GVHD protection and GVT maintenance.

Induced CD45 Proximity Potentiates Natural Killer Cell Receptor Antagonism.

Natural killer (NK) cells are a major subset of innate immune cells that are essential for host defense against pathogens and cancer. Two main classes of inhibitory NK receptors (NKR), KIR and CD94/NKG2A, play a key role in suppressing NK activity upon engagement with tumor cells or virus-infected cells, limiting their antitumor and antiviral activities. Here, we find that single-chain NKR antagonists linked to a VHH that binds the cell surface phosphatase CD45 potentiate NK and T activities to a greater extent than NKR blocking antibodies alone in vitro. We also uncovered crosstalk between NKG2A and Ly49 that collectively inhibit NK cell activation, such that CD45-NKG2A and CD45-Ly49 bispecific molecules show synergistic effects in their ability to enhance NK cell activation. The basis of the activity enhancement by CD45 ligation may reflect greater antagonism of inhibitory signaling from engagement of MHC I on target cells, combined with other mechanisms, including avidity effects, tonic signaling, antagonism of weak inhibition from engagement of MHC I on non-target cells, and possible CD45 segregation within the NK cell-target cell synapse. These results uncover a strategy for enhancing the activity of NK and T cells that may improve cancer immunotherapies.

IL-2 receptor engineering enhances regulatory T cell function suppressed by calcineurin inhibitor.

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-2Rß 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-2Rß(+) 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.

Potentiating adoptive cell therapy using synthetic IL-9 receptors.

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 chemotherapy1,2. 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 (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rß-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.

Accumulation of γδ T cells in visceral fat with aging promotes chronic inflammation.

Adipose tissue dysfunction is strongly linked to the development of chronic inflammation and cardiometabolic disorders in aging. While much attention has been given to the role of resident adipose tissue immune cells in the disruption of homeostasis in obesity, age-specific effects remain understudied. Here, we identified and characterized a population of γδ T cells, which show unique age-dependent accumulation in the visceral adipose tissue (VAT) of both mice and humans. Diet-induced obesity likewise increased γδ T cell numbers; however, the effect was greater in the aged where the increase was independent of fat mass. γδ T cells in VAT express a tissue-resident memory T cell phenotype (CD44hiCD62LlowCD69+) and are predominantly IL-17A-producing cells. Transcriptome analyses of immunomagnetically purified γδ T cells identified significant age-associated differences in expression of genes related to inflammation, immune cell composition, and adipocyte differentiation, suggesting age-dependent qualitative changes in addition to the quantitative increase. Genetic deficiency of γδ T cells in old age improved the metabolic phenotype, characterized by increased respiratory exchange ratio, and lowered levels of IL-6 both systemically and locally in VAT. Decreased IL-6 was predominantly due to reduced production by non-immune stromal cells, primarily preadipocytes, and adipose-derived stem cells. Collectively, these findings suggest that an age-dependent increase of tissue-resident γδ T cells in VAT contributes to local and systemic chronic inflammation and metabolic dysfunction in aging.

Peripheral Blood Smear Detection of Asymptomatic Central Line Infection in a Patient With Sickle Cell Disease.

Sickle cell disease is a lifelong disorder which may be managed by chronic red cell transfusion including exchange transfusion. Chronic indwelling vascular catheters including ports offer convenient and reliable access for red cell exchange but confer risk of complications including infection and thrombosis. Detection of these complications is essential for preserving vascular access and relies on both clinical and laboratory observation. Here we describe a case of asymptomatic port infection detected by manual screening of a peripheral blood smear.

Lack of defined apheresis collection criteria in publicly available CAR-T cell clinical trial descriptions: Comprehensive review of over 600 studies.

BACKGROUND: Chimeric antigen receptor T (CAR-T) cell successes have encouraged continued clinical study. Apheresis collection of starting material for CAR-T cell therapy product manufacturing is critical but described approaches suggest variability and clinical guidelines are currently lacking. The goal of this study was to gather and assess variability in apheresis collection descriptions in publicly available CAR T-cell therapy clinical trials. STUDY DESIGN: We searched clinicaltrials.gov (a publicly available clinical trial database) for "chimeric antigen receptor T cells" on July 01, 2020 and studies accessed July 30, 2020-August 15, 2020. Data collected included date posted, study characteristics, apheresis mentions (number, location, and context), laboratory parameters and transfusion allowances. Apheresis context was analyzed using a qualitative inductive approach of grounded theory method with open coding. Text was classified into 37 context codes, grouped into 12 categories, and then consolidated into patient, procedure, product, and miscellaneous themes. RESULTS: Apheresis was mentioned 1044 times in 322 (51.9%) of 621 total studies. Laboratory parameters mentioned included white blood cells (100 studies), absolute neutrophil count (220 studies), absolute lymphocyte count (102 studies), CD3+ cell (38 studies), hemoglobin (233 studies, 54 studies specified transfusion allowance), and platelet (269 studies, 48 studies specified transfusion allowance). CONCLUSIONS: Apheresis collection of CAR-T cell products is not well-defined in clinical study descriptions and the context is inconsistent. Laboratory parameters useful for apheresis collection are variably present and do not consistently align with current practices. Further exploration, and clinical guideline development will encourage alignment of apheresis collections for CAR-T cell products.

A human orthogonal IL-2 and IL-2Rß system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia.

Interleukin-2 (IL-2) is a central T cell cytokine that promotes T cell proliferation and effector function; however, toxicity due to its pluripotency limits its application to enhance CAR T cell immunotherapy. Previously, mouse IL-2 and its cognate receptor were engineered to create an orthogonal (ortho) cytokine-cytokine receptor pair capable of delivering an IL-2 signal without toxicity. Here, we engineered a human orthogonal IL-2 (ortho-hIL-2) and human orthogonal IL-2Rß (ortho-hIL-2Rß) pair, containing human-specific mutations. Ortho-hIL-2 is selective toward ortho-hIL-2Rß­expressing cells with no appreciable signaling on wild-type T cells. Ortho-hIL-2 induces IL-2 receptor signaling and supports proliferation of both an IL-2­dependent cell line and primary T cells transduced to express the ortho-hIL-2Rß. Using CD19-specific chimeric antigen receptor (CAR) T cells, we show that ortho-hIL-2 induces a dose-dependent increase in ortho-hIL-2Rß+ CAR T cell expansion in vivo by as much as 1000-fold at 2 weeks after adoptive transfer into immunodeficient mice bearing CD19+ Nalm6 leukemia xenografts. Ortho-hIL-2 can rescue the antileukemic effect of an otherwise suboptimal CAR T cell dose. In addition, ortho-hIL-2 administration initiated at the time of leukemic relapse after CAR T cell therapy can rescue an otherwise failed antileukemic response. These data highlight the potential of combining an orthogonal cytokine approach with T cell­based immunotherapies to augment the antitumor efficacy of engineered T cells.

Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance.

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.

The tissue protective functions of interleukin-22 can be decoupled from pro-inflammatory actions through structure-based design.

Interleukin-22 (IL-22) acts on epithelial cells to promote tissue protection and regeneration, but can also elicit pro-inflammatory effects, contributing to disease pathology. Here, we engineered a high-affinity IL-22 super-agonist that enabled the structure determination of the IL-22-IL-22Rα-IL-10Rß ternary complex to a resolution of 2.6 Å. Using structure-based design, we systematically destabilized the IL-22-IL-10Rß binding interface to create partial agonist analogs that decoupled downstream STAT1 and STAT3 signaling. The extent of STAT bias elicited by a single ligand varied across tissues, ranging from full STAT3-biased agonism to STAT1/3 antagonism, correlating with IL-10Rß expression levels. In vivo, this tissue-selective signaling drove tissue protection in the pancreas and gastrointestinal tract without inducing local or systemic inflammation, thereby uncoupling these opposing effects of IL-22 signaling. Our findings provide insight into the mechanisms underlying the cytokine pleiotropy and illustrate how differential receptor expression levels and STAT response thresholds can be synthetically exploited to endow pleiotropic cytokines with enhanced functional specificity.

Structural basis for IL-12 and IL-23 receptor sharing reveals a gateway for shaping actions on T versus NK cells.

Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that are produced by antigen-presenting cells to regulate the activation and differentiation of lymphocytes, and they share IL-12Rß1 as a receptor signaling subunit. We present a crystal structure of the quaternary IL-23 (IL-23p19/p40)/IL-23R/IL-12Rß1 complex, together with cryoelectron microscopy (cryo-EM) maps of the complete IL-12 (IL-12p35/p40)/IL-12Rß2/IL-12Rß1 and IL-23 receptor (IL-23R) complexes, which reveal "non-canonical" topologies where IL-12Rß1 directly engages the common p40 subunit. We targeted the shared IL-12Rß1/p40 interface to design a panel of IL-12 partial agonists that preserved interferon gamma (IFNγ) induction by CD8+ T cells but impaired cytokine production from natural killer (NK) cells in vitro. These cell-biased properties were recapitulated in vivo, where IL-12 partial agonists elicited anti-tumor immunity to MC-38 murine adenocarcinoma absent the NK-cell-mediated toxicity seen with wild-type IL-12. Thus, the structural mechanism of receptor sharing used by IL-12 family cytokines provides a protein interface blueprint for tuning this cytokine axis for therapeutics.

Vedolizumab Dose Escalation Improves Therapeutic Response in a Subset of Patients with Ulcerative Colitis.

BACKGROUND: The Gemini trial failed to detect a significant difference in response rate for patients with ulcerative colitis (UC) randomized to standard (every 8 week) vedolizumab dosing vs escalated (every 4 week) dosing. Subsequent real-world data imply the Gemini trial design may have obscured a benefit of escalated dosing. AIMS: We investigated outcomes after vedolizumab dose escalation for patients with UC. We also explored potential clinical predictors of dose escalation requirement. METHODS: In this retrospective study, we included patients with UC who received vedolizumab between 1/2017-1/2019. We compared rates of clinical response (decrease in partial Mayo score by ≥ 2) and remission (partial Mayo < 2) for standard vs escalated dosing. RESULTS: Among the 90 patients reviewed, 52 achieved and maintained remission on standard dosing. The average time to remission with standard dosing was 33.3 ± 6.6 weeks. After an average of 56.3 ± 7.4 weeks standard dosing, 24 patients (22 "partial responders" and 2 "non-responders") were dose-escalated. Of the 22 "partial responders" dose-escalated, 10 (45%) achieved remission, 10 (45%) achieved further improvement. Neither "non-responder" demonstrated further clinical benefit. Prior anti-tumor necrosis factor (anti-TNF) biologic exposure predicted dose escalation requirement (p = 0.008). Patients requiring dose escalation had more severe disease at baseline as measured by both full Mayo (p = 0.009) and partial Mayo scores (p = 0.01). CONCLUSIONS: We show dose escalation benefited patients with UC who exhibit a "partial response" to standard dosing. Early vedolizumab dose escalation should be considered in both patients with severe disease and those with prior anti-TNF experience.

Immune receptor inhibition through enforced phosphatase recruitment.

Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

Should a fully covered self-expandable biliary metal stent be anchored with a double-pigtail plastic stent? A retrospective study.

BACKGROUND: The migration rate of fully covered self-expandable metal stents (FCSEMSs) has been reported to be between 14% to 37%. Anchoring of FCSEMSs using a double-pigtail plastic stent (DPS) may decrease migration. AIM: To compare stent migration rates between patients who received FCSEMS alone and those who received both an FCSEMS and anchoring DPS. METHODS: We conducted a retrospective analysis of endoscopy reporting system and medical records of 1366 patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) with FCSEMS placement at the University of Kentucky health care. Between July 2015 and April 2017, 203 patients with FCSEMS insertion for the treatment of malignant biliary stricture, benign biliary stricture, post-sphincterotomy bleeding, bile leak, and cholangitis drainage were identified. The review and analysis were conducted through our endoscopy reporting system (ProVation® MD) and medical records. Categorical data were analyzed using Chi-Square and Fischer exact test and continuous data using non-parametric tests. A regression analysis was performed to identify factors independently associated with increased risk of stent migration. We determined an FCSEMS migration endoscopically if the stent was no longer visible in the major papilla. RESULTS: 1366 patients had undergone ERCP by three advanced endoscopists over 21-mo period; among these, 203 patients had FCSEMSs placed. 65 patients had FCSEMSs with DPS, and 138 had FCSEMSs alone. 65 patients had FCSEMSs with DPS, and 138 had FCSEMSs alone. 95 patients had a malignant stricture, 82 patients had a benign stricture, 12 patients had bile leak, 12 patients had cholangitis, and nine patients had post-sphincterotomy bleeding. The migration rate in patients with anchored FCSEMSs with DPS was 6%, and those without anchoring DPS was 10% (P = 0.35). Overall, migration was reported in 18 patients with FCSEMSs placement out of 203 patients with an overall migration rate of 9.7%. There was no significant association between anchoring the FCSEMSs with DPS and the risk of stent migration. Only patients with the previous sphincterotomy and begin biliary stricture were found to have a statistically significant difference in the migration rate between patients who had FCSEMS with DPS and FCSEMS alone (P = 0.01). CONCLUSION: The risk of migration of biliary FCSEMS was 9.7 %. Anchoring an FCSEMS with DPS does not decrease the risk of stent migration.

Structure of the IFNγ receptor complex guides design of biased agonists.

The cytokine interferon-γ (IFNγ) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFNγ pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFNγ receptor IFNγR1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFNγ-IFNγR1-IFNγR2 signalling complex at 3.25 Å resolution. The structure reveals the mechanism underlying deficits in IFNγ responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFNγR2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFNγ variants to tune IFNγ receptor signalling output. Unexpectedly, we found that several partial IFNγ agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFNγ for therapeutic applications.

Successful treatment of severe refractory autoimmune hemolytic anemia after hematopoietic stem cell transplant with abatacept.

BACKGROUND: After hematopoietic stem cell transplantation (HSCT) autoimmune hemolytic anemia (AIHA) is a known and fairly common complication. It is often refractory to conventional therapies including corticosteroids, intravenous immunoglobulin, splenectomy, and the more recently described use of monoclonal antibodies. The high morbidity associated with these severe persistent cases elucidates the gaps in alternative therapies available for treatment. STUDY DESIGN AND METHODS: We described the successful use of abatacept for severe refractory AIHA after HSCT in three patients. RESULTS: Three pediatric patients with refractory AIHA after allogeneic stem cell transplantation were observed to be unresponsive to multitude immunosuppressive therapies, resulting in persistent transfusion dependency. Treatment with abatacept, a fusion protein that inhibits T-cell activation by binding to CD80/CD86 on antigen-presenting cells (APCs), thus blocking the required CD28 interaction between APCs and T cells, resulted in the resolution of hemolysis. CONCLUSION: Abatacept may provide significant clinical benefit in the management of AIHA after HSCT.