Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Spontaneous resolution of severe idiopathic T cell lymphopenia.

Potential etiologies of T-B+NK+ SCID include both hematopoietic defects and thymic aplasia. The management of patients with this phenotype, identified by newborn screen, may be unclear in the absence of a genetic diagnosis. We report an infant with lymphocyte flow cytometry consistent with T-B+NK+ SCID and reduced proliferative response to phytohemagglutinin. The patient had no genetic diagnosis after targeted panel and exome sequencing. The decision to trend laboratory values rather than move immediately to hematopoietic cell transplant was made given the absence of a genetic defect and the finding of a normal thymus on ultrasound. During the course of evaluation for transplant, the patient unexpectedly had normalization of T cell number and function. This case demonstrates a role for mediastinal ultrasound and the utility of trending laboratory values in patients with severe T cell lymphopenia but no genetic diagnosis, given the small but important possibility of spontaneous resolution.

B-cell differentiation and IL-21 response in IL2RG/JAK3 SCID patients after hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplant (HSCT) typically results in donor T-cell engraftment and function in patients with severe combined immunodeficiency (SCID), but humoral immunity, particularly when using donors other than matched siblings, is variable. B-cell function after HSCT for SCID depends on the genetic cause, the use of pre-HSCT conditioning, and whether donor B-cell chimerism is achieved. Patients with defects in IL2RG or JAK3 undergoing HSCT without conditioning often have poor B-cell function post-HSCT, perhaps as a result of impairment of IL-21 signaling in host-derived B cells. To investigate the effect of pre-HSCT conditioning on B-cell function, and the relationship of in vitro B-cell function to clinical humoral immune status, we analyzed 48 patients with IL2RG/JAK3 SCID who were older than 2 years after HSCT with donors other than matched siblings. T follicular helper cells (TFH) developed in these patients with kinetics similar to healthy young children; thus, poor B-cell function could not be attributed to a failure of TFH development. In vitro differentiation of B cells into plasmablasts and immunoglobulin secretion in response to IL-21 strongly correlated with the use of conditioning, donor B-cell engraftment, freedom from immunoglobulin replacement, and response to tetanus vaccine. Patients receiving immunoglobulin replacement who had normal serum immunoglobulin M showed poor response to IL-21 in vitro, similar to those with low serum IgM. In vitro response of B cells to IL-21 may predict clinically relevant humoral immune function in patients with IL2RG/JAK3 SCID after HSCT.

Unbalanced recovery of regulatory and effector T cells after allogeneic stem cell transplantation contributes to chronic GVHD.

The development and maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT) requires the balanced reconstitution of donor-derived CD4 regulatory T cells (CD4Tregs) as well as effector CD4 (conventional CD4 T cells [CD4Tcons]) and CD8 T cells. To characterize the complex mechanisms that lead to unbalanced recovery of these distinct T-cell populations, we studied 107 adult patients who received T-replete stem cell grafts after reduced-intensity conditioning. Immune reconstitution of CD4Treg, CD4Tcon, and CD8 T cells was monitored for a 2-year period. CD3 T-cell counts gradually recovered to normal levels during this period but CD8 T cells recovered more rapidly than either CD4Tregs or CD4Tcons. Reconstituting CD4Tregs and CD4Tcons were predominantly central memory (CM) and effector memory (EM) cells and CD8 T cells were predominantly terminal EM cells. Thymic generation of naive CD4Tcon and CD8 T cells was maintained but thymic production of CD4Tregs was markedly decreased with little recovery during the 2-year study. T-cell proliferation was skewed in favor of CM and EM CD4Tcon and CD8 T cells, especially 6 to 12 months after HSCT. Intracellular expression of BCL2 was increased in CD4Tcon and CD8 T cells in the first 3 to 6 months after HSCT. Early recovery of naive and CM fractions within each T-cell population 3 months after transplant was also strongly correlated with the subsequent development of chronic graft-versus-host disease (GVHD). These dynamic imbalances favor the production, expansion, and persistence of effector T cells over CD4Tregs and were associated with the development of chronic GVHD.

Efficacy, durability, and response predictors of low-dose interleukin-2 therapy for chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is associated with inadequate reconstitution of tolerogenic CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs). Previous phase 1 studies identified a low daily dose of interleukin-2 (IL-2) that was well tolerated, did not exacerbate alloimmunity, augmented Treg in vivo, and was associated with improvement of active cGVHD. In the current phase 2 study, 35 adults with steroid-refractory cGVHD received daily IL-2 (1 × 10(6) IU/m(2)) for 12 weeks. Median time from transplantation and cGVHD onset was 616 days (range, 270-2145 days) and 317 days (range, 28-1880 days), respectively. Two patients withdrew and 5 required IL-2 dose reductions due to side effects. Twenty of 33 evaluable patients (61%) had clinical responses at multiple cGVHD sites (liver, skin, gastrointestinal tract, lung, joint/muscle/fascia). Three patients (9%) had progressive cGVHD. Compared with pretreatment levels, Treg and natural killer cell counts rose >fivefold (P < .001) and >fourfold (P < .001), respectively, without significant change in conventional CD4 T cells (Tcons) or CD8 T cells. The Treg:Tcon ratio rose >fivefold (P < .001). Clinical responders initiated IL-2 earlier (508 vs 917 days after transplantation, P = .005; 249 vs 461 days after cGVHD onset; P = .03). Treg:Tcon ratios ≥0.07 at baseline and ≥0.2 at week 1 also predicted clinical response (P = .003; P = .0003, respectively). After a 4-week treatment hiatus, clinical responders were eligible to continue IL-2 therapy indefinitely. During 2 years of extended IL-2 therapy, clinical and Treg immune responses persisted, while Tcon count and Treg:Tcon ratio gradually normalized. Low-dose IL-2 provides durable clinical improvement in active cGVHD and extended therapy is well-tolerated.

Combined immunodeficiency with EBV positive B cell lymphoma and epidermodysplasia verruciformis due to a novel homozygous mutation in RASGRP1.

RASGRP1 is a guanine-nucleotide-exchange factor essential for MAP-kinase mediated signaling in lymphocytes. We report the second case of RASGRP1 deficiency in a patient with a homozygous nonsense mutation in the catalytic domain of the protein. The patient had epidermodysplasia verruciformis, suggesting a clinically important intrinsic T cell function defect. Like the previously described patient, our proband also presented with CD4+ T cell lymphopenia, impaired T cell proliferation to mitogens and antigens, reduced NK cell function, and EBV-associated lymphoma. The severity of the disease and the development of EBV lymphoma in both patients suggest that hematopoietic stem cell transplantation should be performed rapidly in patients with RASGRP1 deficiency.

Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. In this study, we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps in a DNA-dependent manner. Thus, high-affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation.

Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma.

Our understanding of Ewing's sarcoma development mediated by the EWS/FLI fusion protein has been limited by a lack of knowledge regarding the tumor cell of origin. To circumvent this, we analyzed the function of EWS/FLI in Ewing's sarcoma itself. By combining retroviral-mediated RNA interference with reexpression studies, we show that ongoing EWS/FLI expression is required for the tumorigenic phenotype of Ewing's sarcoma. We used this system to define the full complement of EWS/FLI-regulated genes in Ewing's sarcoma. Functional analysis revealed that NKX2.2 is an EWS/FLI-regulated gene that is necessary for oncogenic transformation in this tumor. Thus, we developed a highly validated transcriptional profile for the EWS/FLI fusion protein and identified a critical target gene in Ewing's sarcoma development.

Tabelecleucel for EBV+ PTLD after allogeneic HCT or SOT in a multicenter expanded access protocol.

ABSTRACT: Patients with Epstein-Barr virus (EBV)-positive posttransplant lymphoproliferative disease (EBV+ PTLD) in whom initial treatment fails have few options and historically low median overall survival (OS) of 0.7 months after allogeneic hematopoietic cell transplant (HCT) and 4.1 months after solid organ transplant (SOT). Tabelecleucel is an off-the-shelf, allogeneic EBV-specific cytotoxic T-lymphocyte immunotherapy for EBV+ PTLD. Previous single-center experience showed responses in patients with EBV+ PTLD after HCT or SOT. We now report outcomes from a multicenter expanded access protocol in HCT (n = 14) and SOT (n = 12) recipients treated with tabelecleucel for EBV+ PTLD that was relapsed/refractory (R/R) to rituximab with/without chemotherapy. The investigator-assessed objective response rate was 65.4% overall (including 38.5% with a complete and 26.9% with a partial response), 50.0% in HCT, and 83.3% in SOT. The estimated 1- and 2-year OS rates were both 70.0% (95% confidence interval [CI], 46.5-84.7) overall, both 61.5% (95% CI, 30.8-81.8) in HCT, and both 81.5% (95% CI, 43.5-95.1) in SOT (median follow-up: 8.2, 2.8, and 22.5 months, respectively). Patients responding to tabelecleucel had higher 1- and 2-year OS rates (94.1%) than nonresponders (0%). Treatment was well tolerated, with no reports of tumor flare, cytokine release syndrome, or rejection of marrow and SOT. Results demonstrate clinically meaningful outcomes across a broad population treated with tabelecleucel, indicating a potentially transformative and accessible treatment advance for R/R EBV+ PTLD after HCT or SOT. This trial was registered at www.ClinicalTrials.gov as #NCT02822495.

Antiviral cellular therapy for enhancing T-cell reconstitution before or after hematopoietic stem cell transplantation (ACES): a two-arm, open label phase II interventional trial of pediatric patients with risk factor assessment.

Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.

Treatment of recurrent pediatric myelodysplastic syndrome post hematopoietic stem cell transplantation.

Treatment of recurrent myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) remains challenging. We present a 4-year-old girl experiencing early MDS relapse post-HCT treated with a multimodal strategy encompassing a second HCT and innovative targeted therapies. We underscore the potential of a comprehensive treatment approach in managing recurrent pediatric MDS.

Real-world experience with low-dose IL-2 for children and young adults with refractory chronic graft-versus-host disease.

The majority of patients with chronic graft-versus-host disease (cGVHD) are steroid refractory (SR), creating a need for safe and effective therapies. Subcutaneous low-dose interleukin-2 (LD IL-2), which preferentially expands CD4+ regulatory T cells (Tregs), has been evaluated in 5 clinical trials at our center with partial responses (PR) in ∼50% of adults and 82% of children by week 8. We now report additional real-world experience with LD IL-2 in 15 children and young adults. We conducted a retrospective chart review of patients with SR-cGVHD at our center who received LD IL-2 from August 2016 to July 2022 not on a research trial. The median age at start of LD IL-2 was 10.4 years (range, 1.2-23.2 years) at a median of 234 days from cGVHD diagnosis (range, 11-542 days). Patients had a median of 2.5 (range, 1-3) active organs at LD IL-2 start and received a median of 3 (range, 1-5) prior therapies. The median duration of LD IL-2 therapy was 462 days (range, 8-1489 days). Most patients received 1 × 106 IU/m2 per day. There were no serious adverse effects. The overall response rate in 13 patients who received >4 weeks of therapy was 85% (complete response, n = 5; PR, n = 6) with responses in diverse organs. Most patients significantly weaned corticosteroids. Tregs preferentially expanded with a median peak fold increase of 2.8 in the ratio of Tregs to CD4+ conventional T cells (range, 2.0-19.8) by 8 weeks on therapy. LD IL-2 is a well-tolerated, steroid-sparing agent with a high response rate in children and young adults with SR-cGVHD.

Inefficiency of two-stage designs in phase II oncology clinical trials with high proportion of inevaluable patients.

BACKGROUND: Two-stage designs are commonly used for oncology Phase II clinical trials with a binary response endpoint. An issue that has not gained sufficient attention is the potential inefficiency in the usage of two-stage designs due to multiple enrollment suspensions when the proportion of patients inevaluable for response is high. METHODS: Simulation studies were used to assess the performance of Simon's two-stage designs, two-stage designs with a proposed modification, and a single-stage design in the context of Phase II clinical trials with a high proportion of patients inevaluable for response. RESULTS: Two-stage designs can require multiple enrollment disruptions when the inevaluable proportion is high, which can result in unacceptable inefficiency. The proposed modification provides a practical solution to this issue by enrolling an extra number of patients towards the end of the 1st stage, anticipating that a proportion of the patients pending response evaluation could be inevaluable. Single-stage designs with interim monitoring of futility that require no interim accrual suspension can be more efficient than two-stage designs, especially when the accrual and inevaluable rates are high. CONCLUSIONS: Planning of Phase II trials should consider the issue of inefficiency of the two-stage designs, especially for trials with a high inevaluable proportion. Designs with monitoring rules that do not require accrual suspensions may be given more considerations, especially in trials of agents that have already had some evidence for safety and efficacy in other populations.

Durability of clinical and immunologic responses to extended low-dose interleukin-2 therapy in patients with refractory chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) remains a frequent cause of non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation. In our single center trials of low-dose interleukin-2 (LD IL-2), the immunomodulatory properties of regulatory T cells (Tregs) have been harnessed to treat steroid-refractory cGVHD (SR-cGVHD) safely and effectively in adults and children. In these trials, 50-60% of patients showed clinical improvement of their cGVHD manifestations with partial responses at the primary response endpoint of 8-12 weeks. Many patients continued extended duration LD IL-2 therapy and achieved deeper clinical responses, including some complete responses. However, the durability of the clinical and immunologic improvement following IL-2 discontinuation has not been reported previously. We examined 20 adult and 2 pediatric patients who received extended duration LD IL-2 for a median of 103 weeks (range, 21-258) and had stable improvement or resolution of their cGVHD symptoms before discontinuing LD IL-2 therapy. The median follow-up after stopping IL-2 was 203 weeks (range 92-599). During this time, 16 patients (73%) were able to wean off all systemic immunosuppression without disease flare or progression. Among 13 patients with available immune cell data, the median fold change in absolute Treg count was 0.58 between 1 to 10 weeks after stopping IL-2 whereas CD4+ conventional T-cell (Tcon) and CD8+ T-cell numbers remained stable. Despite a decline in Treg numbers after IL-2 discontinuation, Treg numbers remained above the pre-treatment baseline. In addition, many patients had sustained clinical improvement after stopping IL-2, suggesting that extended IL-2 therapy can lead to immune tolerance.

Comparison of Pulmonary Toxicity after Total Body Irradiation- and Busulfan-Based Myeloablative Conditioning for Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients.

Pulmonary toxicity after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for childhood leukemia and myelodysplastic syndrome (MDS), along with the impact of different myeloablative conditioning regimens, remain incompletely described. Here we compared the acute and long-term incidence of pulmonary toxicity (PT) after total body irradiation (TBI)- and busulfan-based myeloablative conditioning. We conducted this retrospective cohort study of 311 consecutive pediatric patients with leukemia or MDS who underwent allo-HSCT at Dana-Farber Cancer Institute/Boston Children's Hospital between 2008 and 2018. PT was graded using Common Terminology Criteria for Adverse Events version 5.0. The primary objective was to compare the cumulative incidence of grade ≥3 and grade 5 PT after TBI-based and busulfan-based myeloablative conditioning using Gray's test. Secondary objectives were to determine factors associated with PT and overall survival (OS) using competing risk analysis and Cox regression analyses, respectively. There was no significant difference between the TBI-conditioned group (n = 227) and the busulfan-conditioned group (n = 84) in the incidence of grade ≥3 PT (29.2% versus 34.7% at 2 years; P = .26) or grade 5 pulmonary toxicity (6.2% versus 6.1% at 2 years; P = .47). Age (hazard ratio [HR], 1.70, 95% confidence interval [CI], 1.11 to 2.59; P = .01), grade ≥2 PT prior to allo-HSCT or preexisting pulmonary conditions (HR, 1.84, 95% CI, 1.24 to 2.72; P < .01), acute graft-versus-host disease (GVHD) (HR, 2.50; 95% CI, 1.51 to 4.14; P < .01), and chronic GVHD (HR, 2.61; 95% CI, 1.26 to 5.42; P = .01) were associated with grade ≥3 PT on multivariable analysis. Grade ≥3 PT was associated with worse OS (81.1% versus 61.5% at 2 years; P < .01). In pediatric allo-HSCT recipients, rates of PT were similar in recipients of TBI-based and recipients of busulfan-based myeloablative conditioning regimens. Age, the presence of PT or preexisting pulmonary conditions prior to transplantation, and the development of either acute or chronic GVHD were associated with grade ≥3 PT post-transplantation. Furthermore, the occurrence of grade 3-4 PT post-transplantation was associated with inferior OS.

Organ-specific response after low-dose interleukin-2 therapy for steroid-refractory chronic graft-versus-host disease.

Despite new therapeutic options, treatment of steroid-refractory chronic graft-versus-host disease (SR-cGVHD) remains challenging as organ involvement and clinical manifestations are highly variable. In previous trials of low-dose interleukin-2 (LD IL-2), we established the safety and efficacy of LD IL-2 for the treatment of SR-cGVHD. In the present report, we combined five phase 1 or 2 clinical trials conducted at our center to investigate organ-specific response rate, coinvolvement of organs, predictors of organ-specific response, and its possible association with immune response. For the 105 adult patients included in this report, the overall response rate after 8 or 12 weeks of LD IL-2 was 48.6% and 53.3%, including late responses in patients who continued treatment for extended periods. Skin was the most frequent organ involved (84%). The organ-specific response rate was highest in liver (66.7%) followed by the gastrointestinal tract (62.5%), skin (36.4%), joint/muscle/fascia (34.2%), and lung (19.2%). In multivariable analysis, shorter time from diagnosis of cGVHD to IL-2 initiation, shorter time from transplant to IL-2 initiation, and fewer prior therapies were associated with overall response as well as skin response. For immunologic correlates, the ratio of regulatory T cells:conventional T cells (ie, CD4Treg:CD4Tcon) ratio at 1 week was significantly higher in patients with overall and skin response; skin response was significantly associated with lower number of total CD3 T cells, CD4Tcon cells, and CD8 T cells and a higher number of B cells. For lung responders, terminal effector memory cell counts were lower within all T-cell populations compared with nonresponders. Organ-specific mechanisms of injury should be investigated, and organ-specific targeted therapies need to be developed.

Genetic diagnosis of immune dysregulation can lead to targeted therapy for interstitial lung disease: A case series and single center approach.

In recent years, a growing number of monogenic disorders have been described that are characterized by immune dysregulation. A subset of these "primary immune regulatory disorders" can cause severe interstitial lung disease, often recognized in late childhood or adolescence. Patients presenting to pulmonary clinic may have long and complex medical histories, but lack a unifying genetic diagnosis. It is crucial for pulmonologists to recognize features suggestive of multisystem immune dysregulation and to initiate genetic workup, since targeted therapies based on underlying genetics may halt or even reverse pulmonary disease progression. Through such an approach, our center has been able to diagnose and treat a cohort of patients with interstitial lung disease from gene defects that affect immune regulation. Here we present representative cases related to pathogenic variants in three distinct pathways and summarize disease manifestations and treatment approaches. We conclude with a discussion of our perspective on the outstanding challenges for diagnosing and managing these complex life-threatening and chronic disorders.

A phase 1 study of donor regulatory T-cell infusion plus low-dose interleukin-2 for steroid-refractory chronic graft-vs-host disease.

Chronic graft-versus-host disease (cGVHD) remains a frequent cause of nonrelapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Despite recent advances, options for steroid-refractory (SR) cGVHD are limited. In previous trials of low-dose interleukin-2 (LD IL-2), the immunomodulatory properties of regulatory T cells (Tregs) have been harnessed to treat SR-cGVHD safely and effectively. In the present study, we combined a single infusion of Treg-enriched lymphocytes (Treg DLI) from the original stem cell donor with in vivo Treg expansion using LD IL-2 (1 × 106 IU/m2 per day for 8 weeks) in 25 adult patients with SR-cGVHD. Treg were not expanded ex vivo. Treg DLI was initiated at 0.1 × 106 cells per kg patient and escalated to a maximum dose of 1 × 106 cells per kg. Treg DLI plus LD IL-2 was well tolerated and led to partial responses (PR) in 5 of 25 patients (20%) after 8 weeks of therapy. Ten additional patients (40%) had stable disease with minor responses not meeting PR criteria. Patients at all dose levels had similar Treg expansion without significant changes in CD4+ conventional T cells or CD8+ T cells. High-throughput sequencing of the T-cell receptor ß locus showed selective improvement of Treg diversity. A subset of DLI-derived Treg clones showed preferential expansion at week 8 and long-term persistence 1-year postinfusion. We demonstrate for the first time that infusion of polyclonal healthy donor Tregs followed by expansion with LD IL-2 is safe in patients with SR-cGVHD, thus establishing a foundation for future adoptive Treg therapies in the posttransplant setting. This trial was registered at www.clinicaltrials.gov as #NCT01937468.