Phillip Scheinberg

Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections

Acute HIV infection is characterized by massive loss of CD4 T cells from the gastrointestinal (GI) tract. Th17 cells are critical in the defense against microbes, particularly at mucosal surfaces. Here we analyzed Th17 cells in the blood, GI tract, and broncheoalveolar lavage of HIV-infected and uninfected humans, and SIV-infected and uninfected sooty mangabeys. We found that (1) human Th17 cells are specific for extracellular bacterial and fungal antigens, but not common viral antigens; (2) Th17 cells are infected by HIV in vivo, but not preferentially so; (3) CD4 T cells in blood of HIV-infected patients are skewed away from a Th17 phenotype toward a Th1 phenotype with cellular maturation; (4) there is significant loss of Th17 cells in the GI tract of HIV-infected patients; (5) Th17 cells are not preferentially lost from the broncheoalveolar lavage of HIV-infected patients; and (6) SIV-infected sooty mangabeys maintain healthy frequencies of Th17 cells in the blood and GI tract. These observations further elucidate the immunodeficiency of HIV disease and may provide a mechanistic basis for the mucosal barrier breakdown that characterizes HIV infection. Finally, these data may help account for the nonprogressive nature of nonpathogenic SIV infection in sooty mangabeys.

Horse versus Rabbit Antithymocyte Globulin in Acquired Aplastic Anemia

BACKGROUND In severe acquired aplastic anemia, hematopoietic failure is the result of immune-mediated destruction of bone marrow stem and progenitor cells. Immunosuppressive therapy with antithymocyte globulin (ATG) plus cyclosporine is an effective alternative to stem-cell transplantation and improves blood counts and survival. Although horse ATG is the standard therapy, rabbit ATG is more potent in depleting peripheral-blood lymphocytes and is preferred in other clinical circumstances. METHODS From December 2005 through July 2010, we performed a randomized trial comparing these two ATG formulations in conventional regimens. Patients were treated at a single facility. The primary outcome was hematologic response at 6 months, as determined by blood counts. The study was designed to enroll 60 patients each for the rabbit-ATG and horse-ATG groups and was powered to detect a difference of 25 percentage points in the response rate. RESULTS A large, unexpected difference was observed in the rate of hematologic response at 6 months in favor of horse ATG (68%; 95% confidence interval [CI], 56 to 80) as compared with rabbit ATG (37%; 95% CI, 24 to 49; P<0.001). Overall survival at 3 years also differed, with a survival rate of 96% (95% CI, 90 to 100) in the horse-ATG group as compared with 76% (95% CI, 61 to 95) in the rabbit-ATG group (P=0.04) when data were censored at the time of stem-cell transplantation, and 94% (95% CI, 88 to 100) as compared with 70% (95% CI, 56 to 86; P=0.008) in the respective groups when stem-cell-transplantation events were not censored. CONCLUSIONS In a randomized study, rabbit ATG was inferior to horse ATG as a first treatment for severe aplastic anemia, as indicated by hematologic response and survival. (Funded by the Intramural Research Program of the National Institutes of Health; ClinicalTrials.gov number, NCT00260689.).

Massive ex Vivo Expansion of Human Natural Regulatory T Cells (T regs ) with Minimal Loss of in Vivo Functional Activity

A good manufacturing grade–compatible approach generates massive numbers of natural regulatory T cells that retain suppressive function in vivo. Cross-Checking Graft-Versus-Host Disease Fighting in hockey is a long-standing tradition: Stitches and gap-toothed smiles are badges of honor among these aggressive athletes. Yet, a balance must be maintained between the occasional high stick and an all-out melee. Black-and-white striped referees serve to uphold this balance, breaking up fights and preventing the bench-clearing brawl. Regulatory T cells (Tregs) are the referees of the adaptive immune system. They prevent the enforcers, cytotoxic T cells, from an overly exuberant response and, in the case of a bone marrow transplant, from attacking the patient’s own tissues. This process, called graft-versus-host disease (GVHD), is one of the risks of transplantation and differs from organ rejection. However, using Tregs to prevent GVHD has been limited by low Treg numbers and altered function after expansion in vitro. Hippen et al. now report a new way to expand Tregs to numbers much larger than those previously achieved while maintaining their ability to selectively suppress self-attacking cytotoxic T cells in vivo. Umbilical cord blood can be used to expand functional natural Tregs (nTregs); however, the initial number of nTregs in cord blood is limited. Therefore, the authors used peripheral blood as a source of nTregs for expansion. Using good manufacturing practice conditions and artificial antigen-presenting cells designed to stimulate T cell expansion, Hippen et al. expanded nTregs 80-fold after only one stimulation; they then showed that these multiplied cells maintained suppressor function. Stimulation of the nTreg population up to four times expanded the numbers of functional cells ~50 million–fold. When injected into mice at the same time as human T cells, these expanded Tregs significantly reduced mortality resulting from GVHD. Such large numbers of functional nTregs could be used to establish donor banks that would keep human GVHD and autoimmunity in check. Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4+CD25+ regulatory T cells (nTregs) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nTregs has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nTregs could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nTregs in UCB units is limited, and average yield after expansion was only 1 × 109 nTregs. Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nTregs. PB nTregs were purified under GMP conditions and expanded 80-fold to yield 19 × 109 cells using anti-CD3 antibody–loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. A single restimulation increased expansion to ~3000-fold and yield to >600 × 109 cells while maintaining Foxp3 expression and suppressor function. nTreg expansion was ~50 million–fold when flow sort–purified nTregs were restimulated four times with aAPCs. Indeed, cryopreserved donor nTregs restimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune-deficient mice. The capability to efficiently produce donor cell banks of functional nTregs could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.

How I treat acquired aplastic anemia

Survival in severe aplastic anemia (SAA) has markedly improved in the past 4 decades because of advances in hematopoietic stem cell transplantation, immunosuppressive biologics and drugs, and supportive care. However, management of SAA patients remains challenging, both acutely in addressing the immediate consequences of pancytopenia and in the long term because of the diseases natural history and the consequences of therapy. Recent insights into pathophysiology have practical implications. We review key aspects of differential diagnosis, considerations in the choice of first- and second-line therapies, and the management of patients after immunosuppression, based on both a critical review of the recent literature and our large personal and research protocol experience of bone marrow failure in the Hematology Branch of the National Heart, Lung, and Blood Institute.

Eltrombopag and Improved Hematopoiesis in Refractory Aplastic Anemia

BACKGROUND Severe aplastic anemia, which is characterized by immune-mediated bone marrow hypoplasia and pancytopenia, can be treated effectively with immunosuppressive therapy or allogeneic transplantation. One third of patients have disease that is refractory to immunosuppression, with persistent, severe cytopenia and a profound deficit in hematopoietic stem cells and progenitor cells. Thrombopoietin may increase the number of hematopoietic stem cells and progenitor cells. METHODS We conducted a phase 2 study involving patients with aplastic anemia that was refractory to immunosuppression to determine whether the oral thrombopoietin mimetic eltrombopag (Promacta) can improve blood counts. Twenty-five patients received eltrombopag at a dose of 50 mg, which could be increased, as needed, to a maximum dose of 150 mg daily, for a total of 12 weeks. Primary end points were clinically significant changes in blood counts or transfusion independence. Patients with a response continued to receive eltrombopag. RESULTS Eleven of 25 patients (44%) had a hematologic response in at least one lineage at 12 weeks, with minimal toxic effects. Nine patients no longer needed platelet transfusions (median increase in platelet count, 44,000 per cubic millimeter). Six patients had improved hemoglobin levels (median increase, 4.4 g per deciliter); 3 of them were previously dependent on red-cell transfusions and no longer needed transfusions. Nine patients had increased neutrophil counts (median increase, 1350 per cubic millimeter). Serial bone marrow biopsies showed normalization of trilineage hematopoiesis in patients who had a response, without increased fibrosis. Monitoring of immune function revealed no consistent changes. CONCLUSIONS Treatment with eltrombopag was associated with multilineage clinical responses in some patients with refractory severe aplastic anemia. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT00922883.).

Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia.

BACKGROUND In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia. METHODS We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients. RESULTS Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- and BCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients. CONCLUSIONS Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.).

Predicting response to immunosuppressive therapy and survival in severe aplastic anaemia

Horse anti‐thymocyte globulin (h‐ATG) and ciclosporin are the initial therapy for most patients with severe aplastic anaemia (SAA), but there is no practical and reliable method to predict response to this treatment. To determine whether pretreatment blood counts discriminate patients with SAA who have a higher likelihood of haematological response at 6 months to immunosuppressive therapy (IST), we conducted a single institution retrospective analysis on 316 SAA patients treated with h‐ATG‐based IST from 1989 to 2005. In multivariate analysis, younger age, higher baseline absolute reticulocyte count (ARC), and absolute lymphocyte count (ALC) were highly predictive of response at 6 months. Patients with baseline ARC ≥ 25 × 109/l and ALC ≥ 1 × 109/l had a much greater probability of response at 6 months following IST compared to those with lower ARC and ALC (83% vs. 41%, respectively; P < 0·001). This higher likelihood of response translated to greater rate of 5‐year survival in patients in the high ARC/ALC group (92%) compared to those with a low ARC/ALC (53%). In the era of IST, the baseline ARC and ALC together serve as a simple predictor of response following IST, which should guide in risk stratification among patients with SAA.

Retreatment with rabbit anti‐thymocyte globulin and ciclosporin for patients with relapsed or refractory severe aplastic anaemia

The management of patients with severe aplastic anaemia (SAA) who do not have a matched sibling donor and fail a course of horse anti‐thymocyte globulin (h‐ATG)/ciclosporin (CsA) is uncertain. Repeated courses of ATG‐based immunosuppression are often employed; in children and increasingly in adults, alternative donor haematopoietic stem cell transplantation is an option. We analysed the success rate of retreatment with rabbit ATG (r‐ATG)/CsA in 43 patients treated at our institution in the last 5 years; 22 were refractory (20 adults; two children) to h‐ATG/CsA‐based regimens and 21 (17 adults; four children) had relapsed after h‐ATG/CsA‐based regimens. The overall response rate was 30% in patients who were refractory to h‐ATG and 65% in patients who had relapsed following h‐ATG. The 1000‐d survival in patients who responded to r‐ATG was 90% compared with 65% in non‐responders. Six patients developed a clonal haematological disorder; two were responders, two were non‐responders and in two the evolution occurred before the response could be assessed at 3 months following r‐ATG. Thirteen patients died; three were responders, six were non‐responders and four patients died prior to 3 months when response was assessed. In our study, the response rate in refractory patients was inferior to what has been previously reported.

Eltrombopag restores trilineage hematopoiesis in refractory severe aplastic anemia that can be sustained on discontinuation of drug

About a quarter of patients with severe aplastic anemia remain pancytopenic despite immunosuppressive therapy. We have previously demonstrated that eltrombopag has efficacy in this setting with 44% (11/25) of patients having clinically significant hematologic responses. We now report safety and efficacy data on a further 18 patients and long-term follow-up on the entire cohort of 43 patients. The overall response rate was 17 of 43 patients (40%) at 3 to 4 months, including tri- and bilineage responses. The majority of patients who remained on eltrombopag in an extension study (14/17) continued to show improvement, and 7 eventually had significant increases in neutrophil, red cell, and platelet lineages. Five patients with robust near-normalization of blood counts had drug discontinued at a median of 28.5 months after entry (range, 9-37 months), and all maintained stable counts a median of 13 months (range, 1-15 months) off eltrombopag. Eight patients, including 6 nonresponders and 2 responders, developed new cytogenetic abnormalities on eltrombopag, including 5 with chromosome 7 loss or partial deletion. None evolved to acute myeloid leukemia to date. Eltrombopag is efficacious in a subset of patients with aplastic anemia refractory to immunosuppressive therapy, with frequent multilineage responses and maintenance of normalized hematopoiesis off treatment. This study is registered at www.clinicaltrials.gov as #NCT00922883.

Association of Telomere Length of Peripheral Blood Leukocytes With Hematopoietic Relapse, Malignant Transformation, and Survival in Severe Aplastic Anemia

CONTEXT Critically short telomeres produce apoptosis, cell senescence, and chromosomal instability in tissue culture and animal models. Variations in telomere length have been reported in severe aplastic anemia but their clinical significance is unknown. OBJECTIVE To investigate the relationship between telomere length and clinical outcomes in severe aplastic anemia. DESIGN, SETTING, AND PATIENTS Single institution analysis of 183 patients with severe aplastic anemia who were treated in sequential prospective protocols at the National Institutes of Health from 2000 to 2008. The pretreatment leukocyte age-adjusted telomere length of patients with severe aplastic anemia consecutively enrolled in immunosuppression protocols with antithymocyte globulin plus cyclosporine for correlation with clinical outcomes were analyzed. MAIN OUTCOME MEASURES Hematologic response, relapse, clonal evolution, and survival. RESULTS There was no relationship between hematologic response and telomere length with response rates of 56.5% of 46 patients in the first, 54.3% of 46 in the second, 60% of 45 in the third, and 56.5% of 46 in the fourth quartiles. Multivariate analysis demonstrated that telomere length was associated with relapse, clonal evolution, and mortality. Evaluated as a continuous variable, telomere length inversely correlated with the probability of hematologic relapse (hazard ratio [HR], 0.16; 95% confidence interval [CI], 0.03-0.69; P = .01). The probability of clonal evolution was higher in patients in the first quartile (24.5%; 95% CI, 8.7%-37.5%) than in quartiles 2 through 4 (8.4%; 95% CI, 3.2%-13.3%; P = .009), and evolution to monosomy 7 or complex cytogenetics was more common in the first quartile (18.8%; 95% CI, 3.5%-31.6%) [corrected] than in quartiles 2 through 4 (4.5%; 95% CI, 0.5%-8.2%; P = .002) [corrected]. Survival between these 2 groups differed, with 66% (95% CI, 52.9%-82.5%) surviving 6 years in the first quartile compared with 83.8% (95% CI, 77.3%-90.9%) in quartiles 2 through 4 (P = .008). CONCLUSION In a cohort of patients with severe aplastic anemia receiving immunosuppressive therapy, telomere length was unrelated to response but was associated with risk of relapse, clonal evolution, and overall survival.