Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

A rare multisystemic disorder with chronic kidney disease: Karyomegalic interstitial nephritis due to homozygous FAN1 c.2260C>T variant.

Karyomegalic interstitial nephritis (KIN) is a rare entity associated with biallelic FAN1 (FANCD2/FANCI-Associated Nuclease 1) gene variants. In FAN1-related KIN, abnormal liver function tests and respiratory involvement are common, in addition to chronic kidney disease. Karyomegalic changes have also been reported in many other organs in patients with FAN1-related KIN in various studies. We report the case of a 35-year-old male with chronic kidney disease of unknown aetiology, concurrent recurrent upper and lower respiratory tract infections, and elevated liver function test results with unidentified aetiology. The patient's family history was remarkable for consanguineous parent marriage and history of kidney transplantation in his aunt. A kidney biopsy was performed, which was consistent with KIN. Clinical exome sequencing revealed a homozygous nonsense variant NM_014967.5 (FAN1): c. 2260C > T (p.Arg754Ter). According to the American College of Medical Genetics (ACMG) criteria, this variant is pathogenic and, to the best of our knowledge, has not been previously reported, homozygously. Therefore, the histopathological and clinical diagnoses of KIN were confirmed by genetic studies in our patient. This case report expands the genetic spectrum of FAN1-related KIN, and briefly reviews the current literature data.

Decline of humoral immune responses after natural SARS-CoV-2 infection can be efficiently reversed by vaccination.

Our aim was to analyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody level kinetics after coronavirus disease 2019 (COVID-19) infection and determine the efficiency of vaccination on SARS-CoV-2-specific antibody levels. The study included 50 SARS-CoV-2 infected and 70 uninfected cases. Levels of SARS-CoV-2-specific IgG nucleocapsid protein (IgG-NP), IgG spike protein (IgG-SP), IgM nucleocapsid protein (IgM-NP), and IgA spike protein (IgA-SP) antibodies were evaluated by an enzyme-linked immunosorbent assay in sera obtained at baseline, 1st, 3rd, and 6th month follow-up visits for infected cases and at postvaccination visits for all cases. In symptomatic cases (n = 50), IgG-SP levels were decreased in 6 months compared with baseline, while IgA-SP levels were significantly increased. IgG-NP levels were significantly decreased in symptomatic cases at the 6-month visit. After vaccination, IgG-SP levels were increased in symptomatic cases compared with prevaccination levels. Among subjects vaccinated with CoronaVac (the Sinovac COVID-19 vaccine), infected cases had approximately double the IgG-SP level of uninfected cases. SARS-CoV-2-specific antibody levels were higher at the baseline in symptomatic cases. Nevertheless, all infected cases showed significantly reduced IgG-SP levels at the 6th month. Vaccination effectively increased IgG-SP levels.

Contribution of Capillary Zone Electrophoresis Hyphenated with Drift Tube Ion Mobility Mass Spectrometry as a Complementary Tool to Microfluidic Reversed Phase Liquid Chromatography for Antigen Discovery.

The discovery of new antigens specific to multiple myeloma that could be targeted by novel immunotherapeutic approaches is currently of great interest. To this end, it is important to increase the number of proteins identified in the sample by combining different separation strategies. A capillary zone electrophoresis (CZE) method, coupled with drift tube ion mobility (DTIMS) and quadrupole time-of-flight mass spectrometry (QTOF), was developed for antigen discovery using the human myeloma cell line LP-1. This method was first optimized to obtain a maximum number of identifications. Then, its performance in terms of uniqueness of identifications was compared to data acquired by a microfluidic reverse phase liquid chromatography (RPLC) method. The orthogonality of these two approaches and the physicochemical properties of the entities identified by CZE and RPLC were evaluated. In addition, the contribution of DTIMS to CZE was investigated in terms of orthogonality as well as the ability to provide unique information. In conclusion, we believe that the combination of CZE-DTIMS-QTOF and microfluidic RPLC provides unique information in the context of antigen discovery.

Cut-off points for height, weight and body mass index adjusted bioimpedance analysis measurements of muscle mass with use of different threshold definitions.

AIM: Low muscle mass (LMM) is a prerequisite to define sarcopenia. We aimed to report muscle-mass reference cut-off points adjusted for height and weight as muscle-mass threshold best discriminating muscle-weakness and adjusted for body mass index (BMI) significantly lower than that of healthy young population. MATERIAL AND METHOD: We included young adults between 18 and 39 years and community dwelling older adults 60-99 years of age. Bioimpedance analysis (BIA) was used to assess skeletal muscle mass. Skeletal muscle mass index (SMMI) adjusted for height, weight, BMI were calculated [SMMI (height), SMMI (weight), SMMI (BMI)]. Handgrip strength was evaluated with Jamar hydraulic dynamometer for muscle-strength. SMMI (height) and SMMI (weight) cut-offs that predict low muscle-strength were calculated with receiver operator characteristic (ROC) analysis. Low muscle-strength was evaluated by three different thresholds, i.e. 32 kg/22 kg, 30 kg/20 kg, 26 kg/16 kg in males/females. SMMI (BMI) cut-offs were calculated as "mean young SMMI (BMI)-two standard deviation." RESULTS: The young and older reference groups included 301 and 992 individuals, respectively. LMM cut-points for SMMI (height) were (i) 10.8 vs. 8.9 kg/m2 for 32/22 kg; 10.8 vs. 9.4 kg/m2 for 30/20 kg and 11.1 vs. 8.9 kg/m2 for the 26/16 kg thresholds, in males and females, respectively. LMM cut-points for the SMMI (weight) were 40.6% and 33.2% for the all three studied muscle-strength thresholds for males and females, respectively. For all the analyses sensitivity, specificity and likelihood ratios were not sufficiently high in both genders. The SMMI (BMI) cut-points were 1.049 vs. 0.823 kg/BMI for males and females, respectively. CONCLUSIONS: We presented the very first cut-off thresholds for muscle-mass adjusted by height and weight that best discriminate muscle-weakness in the older adults and by BMI that is significantly lower than that of healthy young population. This study suggests that correlation between total skeletal muscle mass measured by BIA (either adjusted for height or weight) and muscle strength is low.

The effect of liver test abnormalities on the prognosis of COVID-19.

INTRODUCTION: COVID-19 caused by the SARS-CoV-2 continues to spread rapidly across the world. In our study, we aim to investigate the relationship between the liver enzymes on admission (AST, ALT, ALP, GGT) and severity of COVID-19. We evaluated course of disease, hospital stay, liver damage and mortality. MATERIALS AND METHODS: Our study included 614 patients who were hospitalized with the diagnosis of COVID-19 between 03.16.20 and 05.12.20. Patients with liver disease, hematological and solid organ malignancy with liver metastases were excluded, resulting in 554 patients who met our inclusion criteria. We retrospectively evaluated liver transaminase levels, AST/ALT ratio, cholestatic enzyme levels and R ratio during hospital admission and these were compared in terms of morbidity, mortality and clinical course. RESULTS: Mean age of 554 subjects were 66.21±15.45 years, 328 (59.2%) were men. The mean values of liver enzymes on admission were AST (36.2±33.6U/L), ALT (34.01±49.34U/L), ALP (78.8±46.86U/L), GGT (46.25±60.05U/L). Mortality rate and need for intensive care unit were statistically significant in subjects that had high ALT-AST levels during their admission to the hospital (p=0.001). According to the ROC analysis AST/ALT ratio was a good marker of mortality risk (AUC=0.713: p=0.001) and expected probability of intensive care unit admission (AUC=0.636: p=0.001). R ratio, which was used to evaluate prognosis, showed a poor prognosis rate of 26.5% in the cholestatic injury group, 36.1% in the mixed pattern group and 30% in the hepato-cellular injury group (p 0.001). CONCLUSIONS: ALT-AST elevation and AST/ALT ratio >1 was associated with more severe course and increased mortality in COVID-19.

Cut-off points for weight and body mass index adjusted bioimpedance analysis measurements of muscle mass.

AIM: Low skeletal muscle mass (LMM) is a criterion to define both sarcopenia and malnutrition. Muscle mass varies with gender, height, weight or fat mass, and many indices of adjusted-muscle mass have been proposed. We aimed to find reference cut-off points of the skeletal muscle mass index (SMMI) adjusted for weight and body mass index (BMI) in Turkish population. MATERIALS AND METHODS: Adults between 18 and 39 years of age and community-dwelling older adults of 60-99 years of age were included. Body composition was assessed with bioimpedance analysis (BIA). SMMI adjusted for weight and BMI were calculated [SMMI (weight) and SMMI (BMI)]. Muscle strength was assessed by hand-grip-strength with hand dynamometer. SMMI (weight) cut points were calculated from the healthy young adults' data as "mean SMMI-2 standard deviation (SD)". SMMI (BMI) cut points that predict low muscle strength were calculated with ROC analysis. To define low muscle strength, we used three currently suggested low muscle-strength thresholds, i.e., 32 kg/22 kg, 30 kg/20 kg, 26 kg/16 kg in males/females, respectively. RESULTS: 301 healthy young adults (187 male, 114 female) and 992 older people (308 male, 684 female) were included. LMM cut points for SMMI (weight) were 37.4% and 33.6% for males and females, respectively. SMMI (BMI) cut points that best predict the low grip-strength for 32 kg/22 kg; 30 kg/20 kg; 26 kg/16 kg thresholds were1.017 kg/BMI and 0.677 kg/BMI; 1.014 kg/BMI and 0.710 kg/BMI; 1.036 kg/BMI and 0.770 kg/BMI for males and females, respectively. CONCLUSIONS: Muscle-mass adjustment methods and techniques show diversity among the studies and have impact on the LMM cut-off points. This study presents population specific LMM thresholds for skeletal muscle mass adjusted for weight and BMI aiming to increase and improve the general applicability of the leading sarcopenia consensus definitions.

Endocan: A Novel Marker of Endothelial Dysfunction in C1-Inhibitor-Deficient Hereditary Angioedema.

BACKGROUND: Hereditary angioedema (HAE) related to C1-inhibitor deficiency is a rare autosomal dominant disorder. Vascular cell adhesion molecules (VCAM) are known as endothelial activation markers. Endocan (also called ESM-1) is proposed as an endothelial dysfunction indicator. We aimed to investigate endothelial activation in attack-free periods in HAE patients by measuring their levels of endocan and VCAM-1. METHODS: Twenty-six HAE patients (22 female, mean age 40 ± 13 years) and 38 healthy control patients (13 female, mean age 36.9 ± 12 years) were included in the study. Peripheral blood samples were collected from HAE patients during symptom-free periods and control subjects. Endocan and VCAM-1 levels were measured using the enzyme-linked immunosorbent assay method. RESULTS: The median serum levels of endocan (647 ± 101 ng/mL) and VCAM-1 (500 ± 79 ng/mL) in the HAE patients were significantly higher than in the control patients (391 ± 41 and 325 ± 4; p < 0.001 for both). CONCLUSION: The increased endocan and VCAM-1 levels may reflect an endothelial activation even in attack-free periods in HAE patients.

Acquired FVIII and FIX Inhibitors after Pregnancy: A Case Report.

Acquired hemophilia is a relatively rare clinical presentation, and most cases present with acquired FVIII inhibitor. The co-occurrence of inhibitors to multiple coagulation factors is uncommon. These autoantibodies may induce spontaneous life-threatening bleeding in patients who have had no previous bleeding disorder. Herein, we present a patient with postpartum acquired FVIII and FIX inhibitors who developed intramuscular hematoma and hemothorax during follow-up. She was then treated with activated prothrombin complex concentrate and methylprednisolone.

Case report of fatal Mycobacterium tilburgii infection.

There are few reports concerning Mycobacterium tilburgii infection in humans because this bacterium is non-cultivatable. Herein, using new molecular techniques, we report the case of an immunocompromised patient with fatal disseminated lymphadenitis that was caused by M. tilburgii.26 years old Caucasian HIV negative female patient presented with abdominal pain. Her clinical assessment revealed disseminated lymphadenitis, that was acid fast bacilli positive. Further molecular evaluation showed the causative agent as M. tilburgii. Despite anti mycobacterial therapy and careful management of intervening complications patient died because of an intraabdominal sepsis. This is the first fatal M. tilburgii infection in the literature. This case points the importance of careful management of patient's immune status and intervening infections besides implementation of effective drug treatment.

A Case Study: Rare Lepiota brunneoincarnata Poisoning.

Amatoxin poisoning from the genus Lepiota may have a deadly outcome, although this is not seen as often as it is from the genus Amanita. In this report, we present a patient who was poisoned by a sublethal dose of Lepiota brunneoincarnata mushrooms. The patient was hospitalized 12 hours after eating the mushrooms. The patient's transaminase levels increased dramatically starting on day 4. Aspartate transaminase peaked at 78 hours. Starting at 1265 IU/L, alanine transaminase peaked at 90 hours at 5124 IU/L. The patient was discharged on day 8 to outpatient care, and his transaminase levels returned to normal ranges in the subsequent days. A toxin analysis was carried out on the mushrooms that the patient claimed to have eaten. Using reversed-phase high-performance liquid chromatography analysis, an uptake of approximately 19.9 mg of amatoxin from nearly 30 g of mushrooms was calculated. This consisted of 10.59 mg of α-amanitin, 9.18 mg of ß-amanitin, and 0.16 mg of γ-amanitin. In conclusion, we present a patient from Turkey who was poisoned by L. brunneoincarnata mushrooms.

Evaluation of malnutrition with blood ghrelin and fecal elastase levels in acute decompensated heart failure patients.

OBJECTIVES: Exocrine pancreatic dysfunction may contribute to malnutrition and lack of appetite in the advanced stages of heart failure. Nutritional assessment was carried out on patients diagnosed with mild or moderate/severe heart failure. Fecal elastase levels are an indicator of pancreatic exocrine function and ghrelin is an appetite hormone which is also investigated for its contribution to malnutrition. STUDY DESIGN: This is an observational study. 52 patients (32 males, 20 females) aged over eighteen years and hospitalized for acute decompensated heart failure (ADHF) were included in the study. They were compared with 31 people (16 male, 15 female) of the same age as Control Group (C). Patients in New York Heart Association (NYHA) stages 1 and 2 were grouped as mild (miADHF), while those in NYHA stages 3 and 4 were grouped as moderate/severe ADHF (seADHF). Fecal and blood samples were taken at admission. In ADHF patients, exocrine pancreatic functions and their relationship with malnutrition were evaluated. Statistical analyses were performed using Tukey's test, the independent-sample t-test, the Kruskal-Wallis test, the Mann-Whitney U-test, the chi-square test and Pearson's bivariate correlation analysis. RESULTS: Significantly decreased fecal elastase levels were found when moderate/severe ADHF patients and the control group were compared. (C 278.9±144.8, miADHF 336.6±181.7, seADHF 168.7±153.6, p=0.002). 10 seADHF patients (50%) had severe, 4 (20%) moderate, and 6 (30%) mild pancreatic insufficiency. Ghrelin levels were higher in seADHF patients compared to C and miADHF patients (C 69.7±34.6, miCHF 82.5±48.2, SeADHF 105.0±78.1 p=0.361). CONCLUSION: Fecal elastase and ghrelin hormone levels can contribute to the determination of malnutrition in ADHF patients.

Uncomplicated diabetes does not accelerate age-related sarcopenia.

BACKGROUND: Diabetes is reported to accelerate sarcopenia (age-related loss of muscle mass and function). We aimed to assess muscle mass and strength in elderly diabetics, elderly non-diabetics, younger diabetics and healthy subjects, and to define correlates of muscle mass and strength in these subjects. METHODS: Sixteen elderly diabetics, 16 younger diabetics, 16 elderly non-diabetics and 18 younger non-diabetics were included. Elderly and diabetic subjects were first evaluated with exercise testing. Isokinetic leg extension and flexion tests were performed using a Cybex 350 dynamometer. Muscle mass was calculated using bioelectric impedance analysis. RESULTS: Muscle mass was similar between all groups; however, muscle strength was significantly lower in diabetic and non-diabetic elderly subjects compared with younger diabetic subjects and non-diabetics. Muscle strength was positively correlated with albumin, metabolic equivalent and hemoglobin, and inversely correlated with age, HbA1c, functional capacity and CRP. Independent correlates of muscle strength were age and hemoglobin. There was no clinically significant correlate of muscle mass. Presence or duration of diabetes was not associated with muscle mass or strength. CONCLUSIONS: Uncomplicated diabetes does not seem to accelerate aging-related muscle mass or strength loss. Exercise test parameters may be useful markers in the screening of sarcopenia.

Isolated renal intravascular lymphoma: a case report and review of the literature.

Intravascular large B-cell lymphoma (IVLBCL) is a very rare subtype of extranodal large B-cell lymphoma. It may involve various organ systems such as skin, liver, lung or kidney. Isolated kidney involvement of IVLBCL is also very rare. Herein we report a very rare case of isolated renal IVLBCL presented with fever of unknown origin, acute kidney injury and nephrotic syndrome. Diagnosis was suspected with isolated high renal (18)F fluorodeoxyglucose uptake in positron emission tomography and confirmed with renal biopsy. Complete remission was obtained with combined chemotherapy including rituximab. We reviewed the English literature in terms of IVLBCL with renal involvement and we could only find 16 such cases. Accordingly, fever, AKI and nephritic syndrome are the most common presenting symptoms in renal intravascular lymphoma.

LEP rs7799039 and LEPR rs1137101 gene variants are not associated with clinical features in patients with metabolic syndrome in the Turkish population.

OBJECTIVES: Genetic predisposition plays a role in the etiology of metabolic syndrome (MetS), an important health problem worldwide. Leptin (LEP), produced by adipose tissue, plays a crucial role in the development of MetS. In this study, we evaluated the effects of LEP and LEP receptor (LEPR) variants on clinical findings and risk of developing MetS in the Turkish population. METHODS: A total of 320 patients were included in the study, of whom 150 were patients with MetS and 170 were healthy controls. DNA was extracted from blood samples. LEP rs7799039 and LEPR rs1137101 variants were genotyped using the polymerase chain reaction-based restriction fragment length polymorphism method. The genotype distributions of these variants and clinical and laboratory findings were compared. RESULTS: The LEP rs7799039 GA and AA genotypes and A allele frequencies were higher in participants with MetS than in the control group. For LEP rs7799039, the genotype AA-GA was higher in males, and the GG genotype was higher in females. On analyzing the clinical outcomes associated with these variants, it was observed that individuals possessing LEP rs7799039 GA and AA genotypes displayed elevated levels of triglycerides. In addition, those with the AG-GG genotype of LEPR rs1137101 had lower mean hemoglobin levels. CONCLUSION: Our results showed that the LEP rs7799039 and LEPR rs1137101 variants may be associated with both the risk of MetS development and clinical findings. Among the various contributors to MetS, a genetic predisposition is commonly recognized as the primary cause.

Acute Intermittent Porphyria Attack Triggered by COVID-19 Infection.

A 45-year-old male patient who was diagnosed with acute intermittent porphyria (AIP) four years ago and had his last episode two years prior presented to our clinic with an AIP attack complicated with rhabdomyolysis triggered by coronavirus disease 2019 (COVID-19) infection. Although there are well-known factors that might trigger an AIP attack, some studies also showed an association of COVID-19 with porphyria. These studies suggest that the accumulation of by-products in the heme synthesis pathway during COVID-19 infection may cause attacks mimicking acute intermittent porphyria. In respect to that, in the early phases of the pandemic, hypotheses emerged arguing the treatment of severe COVID-19 infections with hemin as the treatment of an AIP attack. In our instance, after a two-year period during which there had not been an episode, there was no evident cause other than COVID-19 infection. We believe that patients with porphyria are particularly prone to exacerbations during a COVID-19 infection and should be monitored carefully.

The suppressor of cytokine signaling-1 (SOCS1) gene polymorphism and promoter methylation correlate with the course of COVID-19.

The suppressor of the cytokine signaling-1 (SOCS1) gene is a short sequence located on chromosome 16 that functions to induce an appropriate immune response and is an essential physiological regulator of interferon (IFN) signaling. In addition to comparing the global DNA and SOCS1 gene promoter methylation status between our patients with coronavirus disease 2019 (COVID-19) and healthy controls, this study demonstrates the effect of the SOCS1 rs33989964 polymorphism on patients with COVID-19. The study group included 139 patients diagnosed with COVID-19 in our hospital's clinics between June and December 2020, and the control group included 78 healthy individuals. After comparing the initial gene polymorphisms of the patients with the healthy control group, three separate clinical subgroups were formed. The gene polymorphism distribution and the methylation status of SOCS1 were examined in these clinical subgroups. Hypomethylation of the SOCS1 gene was observed in the COVID-19 patient group compared to the healthy control group (p = 0.001). Between the patients divided into two separate clinical subgroups, those with severe and mild infections, the Del/Del genotype of the SOCS1 gene was more common in patients with severe infection than in patients with mild infection (p = 0.018). Patients with the CA/CA and CA/Del genotypes were 0.201 times more likely to have a severe infection (95% CI: 0.057-0.716, p = 0.007). Having a non-Del/Del genotype was a protective factor against severe infection. The effect of the SOCS1 rs33989964 polymorphism and methylation status of the SOCS1 gene throughout the COVID-19 pandemic could be significant contributions to the literature.

The effect of DNA repair gene variants on COVID-19 disease: susceptibility, severity, and clinical course.

Oxidative stress (OS), which leads to DNA damage, plays a role in the pathogenesis of Coronavirus disease 2019 (COVID-19). We aimed to evaluate the role of DNA repair gene variants [X-ray repair cross complementing 4 (XRCC4) rs28360071, rs6869366, and X-ray cross-complementary gene 1 (XRCC1) rs25487] in susceptibility to COVID-19 in a Turkish population. We also evaluated its effect on the clinical course of the disease. A total of 300 subjects, including 200 COVID-19 patients and 100 healthy controls, were included in this study. These variants were genotyped using polymerase chain reaction (PCR) and/or PCR-restriction fragment length polymorphism (RFLP) methods. The patients were divided into three groups: those with a mild or severe infection; those who died or lived at the 28-day follow-up; those who required inpatient treatment or intensive care. There were 87 women (43.5%) and 113 men (56.5%) in the patient group. Hypertension was the most common comorbidity (26%). In the patient group, XRCC4 rs6869366 G/G genotype and G allele frequency were increased compared to controls, while XRCC4 rs6869366 G/T and T/T genotype frequencies were found to be higher in controls compared to patients. For XRCC1 rs25487, the A/A and A/G genotypes were significantly associated with COVID-19 disease. All of the patients hospitalized in the intensive care unit had the XRCC4 rs6869366 G/G genotype. In this study, we evaluated for the first time the impact of DNA repair gene variants on COVID-19 susceptibility. Results suggested that XRCC4 rs6869366 and XRCC1 rs25487 were associated with COVID-19 suspectibility and clinical course.

Post-transplant cyclophosphamide prevents xenogeneic graft-versus-host disease while depleting proliferating regulatory T cells.

Graft-versus-host disease (GVHD) remains a serious limitation of allogeneic hematopoietic cell transplantation (allo-HCT). While post-transplant administration of cyclophosphamide (PTCy) is increasingly used as GVHD prophylaxis, its precise mechanisms of action and its impact on graft-versus-leukemia effects have remained debated. Here, we studied the mechanisms of xenogeneic GVHD (xGVHD) prevention by PTCy in different humanized mouse models. We observed that PTCy attenuated xGVHD. Using flow cytometry and single-cell RNA-sequencing, we demonstrated that PTCy depleted proliferative CD8+ and conventional CD4+ T cells but also proliferative regulatory T cells (Treg). Further, T-cell receptor ß variable region sequencing (TCRVB) analyses demonstrated that highly xenoreactive T-cell clones were depleted by PTCy. Although Treg frequencies were significantly higher in PTCy-treated than in control mice on day 21, xGVHD attenuation by PTCy was not abrogated by Treg depletion. Finally, we observed that PTCy did not abrogate graft-versus-leukemia effects.