Circulating epigenetic biomarkers for detection of recurrent colorectal cancer.

BACKGROUND: The sensitive detection of recurrent colorectal cancer (CRC) by the measurement of circulating tumor DNA (ctDNA) might improve the chance of a cure. This study compared a quantitative methylated ctDNA test with carcinoembryonic antigen (CEA) in the setting of surveillance for recurrence. METHODS: Blood samples collected either during surveillance or within 12 months of the confirmation of recurrence were assayed for ctDNA (methylated branched-chain amino acid transaminase 1 [BCAT1]/Ikaros family zinc-finger 1 protein [IKZF1]) and CEA. The optimal ctDNA threshold was determined by receiver operating characteristic analysis, and the test performance for the detection of recurrence was compared with CEA (5 ng/mL threshold). RESULTS: The study cohort comprised 144 eligible patients and included 50 recurrence events. The sensitivity of the methylated ctDNA test for recurrence was 66.0% (95% confidence interval [CI], 57.1%-69.3%), which was significantly higher than the sensitivity of CEA (31.9%; 95% CI, 22.8%-36.6%; P < .001). The sensitivity for resectable recurrence (n = 20) was also higher (ctDNA, 60.0%; CEA, 20.0%; P = .01). The specificity did not differ between the tests (ctDNA, 97.9%; 95% CI, 93.2%-99.6%; CEA, 96.4%; 95% CI, 91.4%-99.0%). When adjustments were made for other predictors of the presence of recurrence, a positive ctDNA test was an independent predictor (odds ratio, 155.7; 95% CI, 17.9-1360.6; P < .001), whereas CEA was not (odds ratio, 2.5; 95% CI, 0.3-20.6; P = .407). CONCLUSIONS: The quantitative ctDNA test showed superior sensitivity in comparison with CEA without a difference in the specificity for detecting recurrent CRC. Longitudinal studies are warranted to further assess the utility (specifically the survival benefit) of methylated BCAT1/IKZF1 ctDNA in the surveillance of patients with CRC.

Aiolos Overexpression in Systemic Lupus Erythematosus B Cell Subtypes and BAFF-Induced Memory B Cell Differentiation Are Reduced by CC-220 Modulation of Cereblon Activity.

BAFF is a B cell survival and maturation factor implicated in the pathogenesis of systemic lupus erythematosus (SLE). In this in vitro study, we describe that soluble BAFF in combination with IL-2 and IL-21 is a T cell contact-independent inducer of human B cell proliferation, plasmablast differentiation, and IgG secretion from circulating CD27+ memory and memory-like CD27-IgD- double-negative (DN) B cells, but not CD27-IgD+ naive B cells. In contrast, soluble CD40L in combination with IL-2 and IL-21 induces these activities in both memory and naive B cells. Blood from healthy donors and SLE patients have similar circulating levels of IL-2, whereas SLE patients exhibit elevated BAFF and DN B cells and reduced IL-21. B cell differentiation transcription factors in memory, DN, and naive B cells in SLE show elevated levels of Aiolos, whereas Ikaros levels are unchanged. Treatment with CC-220, a modulator of the cullin ring ligase 4-cereblon E3 ubiquitin ligase complex, reduces Aiolos and Ikaros protein levels and BAFF- and CD40L-induced proliferation, plasmablast differentiation, and IgG secretion. The observation that the soluble factors BAFF, IL-2, and IL-21 induce memory and DN B cell activation and differentiation has implications for extrafollicular plasmablast development within inflamed tissue. Inhibition of B cell plasmablast differentiation by reduction of Aiolos and Ikaros may have utility in the treatment of SLE, where elevated levels of BAFF and Aiolos may prime CD27+ memory and DN memory-like B cells to become Ab-producing plasmablasts in the presence of BAFF and proinflammatory cytokines.

Low Sensitivity of Fecal Immunochemical Tests and Blood-Based Markers of DNA Hypermethylation for Detection of Sessile Serrated Adenomas/Polyps.

BACKGROUND: Early detection and removal of precursor lesions reduce colorectal cancer morbidity and mortality. Sessile serrated adenomas/polyps (SSP) are a recognized precursor of cancer, but there are limited studies on whether current screening techniques detect this pathology. AIMS: To investigate the sensitivity of fecal immunochemical tests (FIT) and epigenetic biomarkers in blood for detection of SSP. METHODS: A prospective study offered FIT and a blood test (Colvera for methylated BCAT1 and IKZF1) to adults referred for colonoscopy. Sensitivity of FIT and the blood test were determined for four types of pathology: low-risk conventional adenoma, high-risk adenoma, SSP, and absence of neoplasia. Comparisons were made for FIT positivity at 10 and 20 µg hemoglobin (Hb)/g feces. RESULTS: One thousand eight hundred and eighty-two subjects completed FIT and underwent colonoscopy. One thousand four hundred and three were also tested for methylated BCAT1/IKZF1. The sensitivity of FIT (20 µg Hb/g feces) for SSP was 16.3%. This was lower than the sensitivity for high-risk adenomas (28.7%, p < 0.05), but no different to that for low-risk adenomas (13.1%) or no neoplasia (8.4%). A positive FIT result for SSP was not associated with demographics, morphology, concurrent pathology or intake of medications that increase bleeding risk. FIT sensitivity for SSP did not significantly increase through lowering the positivity threshold to 10 µg Hb/g feces (20.4%, p > 0.05). Sensitivity of the blood test for SSP was 8.8%, and 26.5% when combined with FIT. CONCLUSIONS: Both FIT and blood-based markers of DNA hypermethylation have low sensitivity for detection of SSP. Further development of sensitive screening tests is warranted.

Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to systemic lupus erythematosus.

OBJECTIVES: IKZF1 and IKZF3 (encoding transcription factors Ikaros and Aiolos) are susceptibility loci for systemic lupus erythematosus (SLE). The pharmacology of iberdomide (CC-220), a cereblon (CRBN) modulator targeting Ikaros and Aiolos, was studied in SLE patient cells and in a phase 1 healthy volunteer study. METHODS: CRBN, IKZF1 and IKZF3 gene expression was measured in peripheral blood mononuclear cells (PBMC) from patients with SLE and healthy volunteers. Ikaros and Aiolos protein levels were measured by Western blot and flow cytometry. Anti-dsDNA and anti-phospholipid autoantibodies were measured in SLE PBMC cultures treated for 7 days with iberdomide. Fifty-six healthy volunteers were randomised to a single dose of iberdomide (0.03-6 mg, n=6 across seven cohorts) or placebo (n=2/cohort). CD19+ B cells, CD3+ T cells and intracellular Aiolos were measured by flow cytometry. Interleukin (IL)-2 and IL-1ß production was stimulated with anti-CD3 and lipopolysaccharide, respectively, in an ex vivo whole blood assay. RESULTS: SLE patient PBMCs expressed significantly higher CRBN (1.5-fold), IKZF1 (2.1-fold) and IKZF3 (4.1-fold) mRNA levels compared with healthy volunteers. Iberdomide significantly reduced Ikaros and Aiolos protein levels in B cells, T cells and monocytes. In SLE PBMC cultures, iberdomide inhibited anti-dsDNA and anti-phospholipid autoantibody production (IC50 ≈10 nM). Single doses of iberdomide (0.3-6 mg) in healthy volunteers decreased intracellular Aiolos (minimum mean per cent of baseline: ≈12%-28% (B cells); ≈0%-33% (T cells)), decreased absolute CD19+ B cells, increased IL-2 and decreased IL-1ß production ex vivo. CONCLUSIONS: These findings demonstrate pharmacodynamic activity of iberdomide and support its further clinical development for the treatment of SLE. TRIAL REGISTRATION NUMBER: NCT01733875; Results.

Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors.

Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with ß-hemoglobinopathies.

Overexpression of Aiolos in Peripheral Blood Mononuclear Cell Subsets from Patients with Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Genetic studies demonstrate that the Aiolos polymorphisms contribute to the susceptibility to autoimmune diseases. The purpose of the study was to investigate the Aiolos expression in lymphocytes and monocytes in the peripheral blood from patients with SLE and RA, and to explore the correlation between Aiolos expression in cell subsets and laboratory measurements. Peripheral blood mononuclear cells (PBMC) from 32 patients with SLE, 35 patients with RA, and 37 healthy controls were purified. Aiolos expression in PBMC subsets was examined by flow cytometry. In SLE patients, a much higher percentage of Aiolos + CD8+ T cells and Aiolos + CD14+ monocytes was found, when compared with healthy controls (p = 8.29 × 10(-5) and p = 1.01 × 10(-5), respectively). Furthermore, the percentage of CD4+ and CD8+ T cells, CD19+ B cells, and CD14+ monocytes expressing Aiolos in RA patients was also determined and each found higher than that in healthy controls (p = 0.009, p = 4.11 × 10(-5), p = 0.001, and p = 1.11 × 10(-5), respectively). The percentage of Aiolos + CD8+ T cells was weakly correlated with ESR in SLE patients and RF in RA patients (r s = 0.37, p = 0.038; r s = 0.34, p = 0.044, respectively). On the other hand, the percentage of Aiolos + CD14+ monocytes was significantly correlated with multiple laboratory measurements, including ESR, creatinine, CRP, LDH, proteinuria, albumin, and ACCPA in patients (r s = 0.62, p < 0.001; r s = 0.65, p < 0.001; r s = 0.44, p = 0.010; r s = 0.42, p = 0.022; r s = 0.52, p = 0.013; r s = 0.34, p = 0.048, respectively). To our knowledge, it is the first study to demonstrate overexpression of Aiolos in PBMC subsets in SLE and RA patients. The results indicate that overexpression of Aiolos may contribute to pathogenesis of SLE and RA.

Dynamic Changes in Circulating Methylated Markers in Response to Antitumor Therapy of Rectal Cancer.

BACKGROUND: Rectal cancer (RC) occupies a leading position in the structure of oncological morbidity and mortality. Aberrant methylation of tumor-suppressor genes and hypomethylation of retrotransposons were shown to be detectable in cell-free DNA, circulating in the blood (cfDNA) of cancer patients, indicating the possibility to use them as diagnostic and prognosis markers. PURPOSE: Evaluation of the changes in the methylation level of LINE-1 elements and SEPTIN9 and IKZF1 genes in the cell-surface-bound cfDNA (csb-cfDNA) from the blood of RC patients after antitumor therapy at a long-term follow-up. METHODS: Blood samples were obtained from RC patients (n = 25) before treatment, after preoperative chemotherapy (3 courses according to the XELOX scheme), 10-15 days after surgery, and every 3 months during 12 months of dynamic observation. The methylation level of LINE-1, SEPTIN9, and IKZF1 in the csb-cfDNA was evaluated by quantitative methyl-specific PCR. RESULTS: The LINE-1 methylation level in the csb-cfDNA increased 1.6 times in RC patients after chemotherapy and 3 times after tumor resection versus methylation level before therapy. The SEPTIN9 gene methylation level in the csb-cfDNA decreased by 1.7 times in RC patients after chemotherapy and by 2.3 times after tumor resection compared with the values before the treatment. The IKZF1 gene methylation level decreased by 2 times in RC patients after combined therapy. Notably, all patients with relapses (n = 5) showed an increase in methylation level for the SEPTIN9 and IKZF1 genes and a decrease of methylation level for the LINE-1 elements by 2 times or more in comparison with the level 10-15 days after surgery. There were no changes in the circulating SEPTIN9, IKZF1, and LINE-1 methylation levels during the 12-month follow-up period after the combined therapy of RC patients (n = 20) without relapses. CONCLUSION: The results indicate that SEPTIN9, IKZF1, and LINE-1 methylation levels in the csb-cfDNA are potential markers of the effectiveness of antitumor therapy and early detection of relapse in RC patients.

A blood test measuring DNA methylation of BCAT1 and IKZF1 for detection of lung adenocarcinoma.

BACKGROUND: Colorectal (CRC) and lung adenocarcinoma share many genetic and pathological similarities. A circulating tumor DNA (ctDNA) test for CRC may also be useful for detection of lung adenocarcinoma. This study determined if a methylated BCAT1/IKZF1 ctDNA test for CRC can be used for detection of lung adenocarcinoma. PATIENTS AND METHODS: Circulating cell free DNA (ccfDNA) was extracted from plasma collected prospectively from healthy controls, patients in remission from CRC, patients with lung adenocarcinoma, and patients with isolated metastatic CRC lung lesions. Plasma ccfDNA was bisulfite converted and assessed for methylated BCAT1/IKZF1 by quantitative real-time PCR. Comparisons between the different patient groups for a positive ctDNA test (BCAT1 and/or IKZF1) and ctDNA levels (% of total ccfDNA), as well as any associations with clinicopathological and demographic features, were assessed. RESULTS: Methylated BCAT1/IKZF1 ctDNA was detected in 18/39 (46.2 %) patients with lung adenocarcinoma, which was significantly (p < 0.001) higher compared to healthy controls (49/606; 8.1 %) and patients in remission from CRC (22/171, 12.9 %). Patients with stage III/IV lung adenocarcinoma had higher BCAT1/IKZF1 ctDNA positivity compared to stage I/II cases (68.2 % vs 17.7 %, p < 0.01), where a significantly higher proportion tested positive for methylated IKZF1 ctDNA alone (54.6 % vs 5.9 %, p < 0.001). There was no difference in BCAT1/IKZF1 ctDNA test positivity between patients with stage IV primary lung adenocarcinoma (n = 17) compared to lung-metastasising CRC cases (n = 17; 70.6 % v 64.3 %). CONCLUSION: A ctDNA test measuring methylated BCAT1/IKZF1 can sensitively detect lung adenocarcinoma and may be a promising aid for detection of advanced disease. CLINICAL TRIAL REGISTRATIONS: Australian and New Zealand Clinical Trials Registry, www.anzctr.org.au, ACTRN12616001138471, ACTRN12611000318987.

Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells.

The Ikaros family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but Ikaros family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2, encoding the transcriptional activator and repressor Helios, which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8+ T cell lineage. Protein-protein interaction analysis revealed that the variant abolished heterodimerization of Helios with Ikaros and Aiolos and also prevented Helios binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, and chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis, we show that reduced Helios expression was associated with chronic T cell activation and increased production of proinflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed a profound reduction in circulating MAIT cell numbers. In summary, we show that this previously undescribed loss-of-function variant in Helios leads to an immunodeficiency with signs of immune overactivation.

Variables Associated with Detection of Methylated BCAT1 or IKZF1 in Blood from Patients Without Colonoscopically Evident Colorectal Cancer.

BACKGROUND: DNA methylated in BCAT1 and IKZF1 are promising circulating tumor DNA (ctDNA) biomarkers for colorectal cancer detection. This study tested for variables that might be associated with their detection in patients without colonoscopically evident colorectal cancer so-called false positives. METHODS: A retrospective review of demographic and clinical variables was conducted on patients who were assayed for these biomarkers prior to a colonoscopy for any indication. Potential relationships between detection of these biomarkers and patient variables in patients without colorectal cancer were identified by logistic regression. An age- and sex-matched case-control study was undertaken to identify additional associations. RESULTS: A total of 196 of 1,593 patients undergoing colonoscopy were positive for BCAT1 and/or IKZF1 methylation; 70 (35.7%) had confirmed diagnosis of colorectal cancer. Of the 126 false positives, biomarker levels were significantly lower than in those with colorectal cancer (P < 0.05), with the total cell-free circulating DNA concentration associated with biomarker detection (OR, 1.16; 95% CI, 1.10-1.22), and 83 (65.9%) of the non-colorectal cancer cases positive for methylated BCAT1 only. Age ≥70 years was the only demographic variable associated with biomarker detection (OR, 4.31; 95% CI, 1.50-12.41). No significant associations were seen with medications or comorbidities (P > 0.05). Four cases without colonoscopically evident colorectal cancer but with biomarker levels above the median for patients with colorectal cancer were diagnosed with metastatic adenocarcinoma within 1 year. CONCLUSIONS: False-positive results were most commonly associated with detection of methylated BCAT1 only, as well as age ≥70 years. IMPACT: In the absence of colonoscopically evident colorectal cancer, a high level of circulating methylated DNA warrants investigations for cancers at other sites.

Differences in Maturation Status and Immune Phenotypes of Circulating Helios+ and Helios- Tregs and Their Disrupted Correlations With Monocyte Subsets in Autoantibody-Positive T1D Individuals.

CD4 Tregs are involved in the regulation of various autoimmune diseases but believed to be highly heterogeneous. Studies have indicated that Helios controls a distinct subset of functional Tregs. However, the immunological changes in circulating Helios+ and Helios- Tregs are not fully explored in type 1 diabetes (T1D). Here, we elucidated the differences in maturation status and immune regulatory phenotypes of Helios+ and Helios- Tregs and their correlations with monocyte subsets in T1D individuals. As CD25-/low FOXP3+ Tregs also represent a subset of functional Tregs, we defined Tregs as FOXP3+CD127-/low and examined circulating Helios+ and Helios- Treg subpopulations in 68 autoantibody-positive T1D individuals and 68 age-matched healthy controls. We found that expression of both FOXP3 and CTLA4 diminished in Helios- Tregs, while the proportion of CD25-/low Tregs increased in Helios+ Tregs of T1D individuals. Although the frequencies of neither Helios+ nor Helios- Tregs were affected by investigated T1D genetic risk loci, Helios+ Tregs correlated with age at T1D diagnosis negatively and disease duration positively. Moreover, the negative correlation between central and effector memory proportions of Helios+ Tregs in healthy controls was disrupted in T1D individuals. Finally, regulatory non-classical and intermediate monocytes also decreased in T1D individuals, and positive correlations between these regulatory monocytes and Helios+/Helios- Treg subsets in healthy controls disappeared in T1D individuals. In conclusion, we demonstrated the alternations in maturation status and immune phenotypes in Helios+ and Helios- Treg subsets and revealed the missing association between these Treg subsets and monocyte subsets in T1D individuals, which might point out another option for elucidating T1D mechanisms.

Distinct Alterations in the Composition of Mucosal Innate Lymphoid Cells in Newly Diagnosed and Established Crohn's Disease and Ulcerative Colitis.

BACKGROUND AND AIMS: Innate lymphoid cells [ILC] have been suggested to play a role in inflammatory bowel disease [IBD]. Here, we investigated the ILC compartment in intestinal biopsies and blood from distinct patient groups with Crohn's disease [CD] and ulcerative colitis [UC], either newly diagnosed or with disease established for at least 1 year. This approach allowed us to simultaneously investigate temporal, disease-specific, and tissue-specific changes in ILC composition in IBD. METHODS: ILC subset frequencies, phenotype, and transcription factor profile in blood and intestinal biopsies were investigated by multi-parameter flow cytometry analysis. Endoscopic disease severity was judged using the ulcerative colitis endoscopic index of severity and the simple endoscopic score for Crohn's disease. RESULTS: The frequency of NKp44+ILC3 was decreased in inflamed tissue, both in patients with CD and those with UC, already at the time of diagnosis, and correlated with disease severity. Simultaneously, the frequency of ILC1 was increased in patients with CD, whereas the frequency of ILC2 was increased in patients with UC. However, in patients with established UC or CD, both ILC1 and ILC2 were increased. In contrast to the ILC composition in inflamed tissue, ILC in non-inflamed tissue or blood were unchanged compared with non-IBD controls. Finally, in patients undergoing treatment with an anti-α4ß7 antibody the frequencies of ILC in peripheral blood remained unchanged. CONCLUSIONS: We report both shared and distinct changes in ILC composition depending on diagnosis and disease duration. The alterations in ILC composition in IBD occur selectively at inflamed sites in the gut.

Decreased Helios Expression in Regulatory T Cells in Acute Coronary Syndrome.

Regulatory T cells (Tregs) play an essential role in acute coronary syndrome (ACS). However, there is debate about which Treg subsets are truly critical to ACS. Helios, a transcription factor, was recently reported to be a bona fide marker for natural Tregs or activated Tregs with a suppression function, but little is known about its role in ACS. We therefore examined Helios+ Tregs in patients with ACS, patients with stable angina, and control subjects. 73 patients with ACS, 30 patients with stable angina, and 48 control subjects were enrolled. The frequencies and estimated absolute numbers of different Treg subsets in peripheral blood were measured by flow cytometry. Plasma cytokine level was measured by ELISA. The mRNA expression of Foxp3 and Helios in purified CD4+ T cells was determined by RT-PCR. Helios+ Tregs was decreased significantly in patients with ACS. The frequency and estimated absolute numbers of CD4+Foxp3+Helios+ Tregs were negatively correlated with IL-6 and positively correlated with circulating level of TGF-beta1 and HDL-C. The mRNA expression of Foxp3 and Helios was decreased in CD4+ T cells from patients with ACS. In summary, Helios+ Tregs was downregulated in patients with ACS and may play a role in ACS.

A cross-sectional study comparing a blood test for methylated BCAT1 and IKZF1 tumor-derived DNA with CEA for detection of recurrent colorectal cancer.

Recurrence will develop in 30-50% of colorectal cancer (CRC) cases despite apparent clearance following treatment. Carcinoembryonic antigen (CEA) is the only guideline-recommended blood test for monitoring cases for recurrence, but its sensitivity and specificity are suboptimal. This observational study compared a novel 2-gene (methylated BCAT1 and IKZF1 DNA) blood test with CEA for detection of recurrent CRC. We conducted a paired comparison of the BCAT1/IKZF1 test with CEA (cut-off 5 ng/mL) in blood from patients in remission after treatment for primary CRC and undergoing surveillance. Blood collected in the 12 months prior to or 3 months after complete investigational assessment of recurrence status were assayed and the results compared by McNemar's test. Of 397 patients enrolled, 220 underwent satisfactory assessment for recurrence and 122 had blood testing performed within the prescribed period. In 28 cases with recurrent CRC, CEA was positive in 9 (32%; 95% CI 16-52%) compared to 19 (68%; 95% CI 48-84%) positive for methylated BCAT1/IKZF1 (P = 0.002). All samples that were CEA positive were also BCAT1/IKZF1 positive. In 94 patients without clinically detectable recurrence, CEA was positive in 6 (6%, 95% CI 2-13%) and BCAT1/IKZF1 in 12 (13%, 95% CI 7-21%), P = 0.210. The odds ratio of a positive CEA test for recurrence was 6.9 (95% CI 2-22) compared to 14.4 (5-39) for BCAT1/IKZF1. The BCAT1/IKZF1 test was more sensitive for recurrence than CEA and the odds of recurrence given a positive test was twice that of CEA. The BCAT1/IKZF1 test should be further considered for monitoring cases for recurrence.

Low-dose oral cholecalciferol is associated with higher numbers of Helios(+) and total Tregs than oral calcitriol in renal allograft recipients: an observational study.

BACKGROUND: Regulatory T cells (Tregs) are a cornerstone of graft acceptance. High numbers of Tregs are associated with better long-term graft survival. Recently, Vitamin D was suggested as an immunomodulator, in addition to its classical role in calcium metabolism. Vitamin D modulates Tregs and might, thereby, promote graft acceptance and long-term graft survival. METHODS: One hundred twenty-three renal allograft recipients attending either Heidelberg nephrology or Giessen internal medicine clinic were enrolled in this cross- sectional study. Sixteen healthy controls were studied in addition. Sixty-nine patients were receiving no vitamin D, 38 calcitriol, and 16 cholecalciferol supplementations. We evaluated whether there was a difference in the absolute numbers of Helios(+), Helios(-), CTLA-4(+), IFNg(+), and total Tregs among the patient groups. RESULTS: Cholecalciferol supplementation was associated with higher absolute numbers of Helios(+), CTLA-4(+), and total Tregs than calcitriol (p < 0.001, p = 0.004, p = 0.001 respectively). Helios(+) Tregs were also higher in cholecalciferol than no vitamin D supplementation patients (p = 0.001), whereas CTLA-4(+) and total Tregs were similar in both groups (p = NS). Helios(+), Helios(-), CTLA-4(+), IFNg(+), and total Tregs were similar in the cholecalciferol and healthy control groups (p = NS). CONCLUSION: Our findings indicate that cholecalciferol, even when administered at low dosages, has a stabilizing effect on Tregs (particularly the Helios + subset), in contrast to calcitriol which showed neither a stabilizing nor a proliferation-inducing effect on the same cell population.

Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP.

Regulatory T cells (Tregs) are key players of immune regulation/dysregulation both in physiological and pathophysiological settings. Despite significant advances in understanding Treg function, there is still a pressing need to define reliable and specific markers that can distinguish different Treg subpopulations. Herein we show for the first time that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are expressed on CD4+FoxP3- T cells expressing Helios (FoxP3-Helios+) in the steady state. Following TCR activation, GARP/LAP are up-regulated on CD4+Helios+ T cells regardless of FoxP3 expression (FoxP3+/-Helios+). We show that CD4+GARP+/-LAP+ Tregs make IL-10 immunosuppressive cytokine but not IFN-γ effector cytokine. Further characterization of FoxP3/Helios subpopulations showed that FoxP3+Helios+ Tregs proliferate in vitro significantly less than FoxP3+Helios- Tregs upon TCR stimulation. Unlike FoxP3+Helios- Tregs, FoxP3+Helios+ Tregs secrete IL-10 but not IFN-γ or IL-2, confirming they are bona fide Tregs with immunosuppressive characteristics. Taken together, Helios, and not FoxP3, is the marker of activated Tregs expressing GARP/LAP, and FoxP3+Helios+ Tregs have more suppressive characteristics, compared with FoxP3+Helios- Tregs. Our work implies that therapeutic modalities for treating autoimmune and inflammatory diseases, allergies and graft rejection should be designed to induce and/or expand FoxP3+Helios+ Tregs, while therapies against cancers or infectious diseases should avoid such expansion/induction.