HLA-DR Research Articles

Causal role of immune cells in schizophrenia: Mendelian randomization (MR) study

BackgroundComplex immune-brain interactions that affect neural development, survival and function might have causal and therapeutic implications for psychiatric illnesses. However, previous studies examining the association between immune inflammation and schizophrenia (SCZ) have yielded inconsistent findings.MethodsComprehensive two-sample Mendelian randomization (MR) analysis was performed to determine the causal association between immune cell signatures and SCZ in this study. Based on publicly available genetic data, we explored causal associations between 731 immune cell signatures and SCZ risk. A total of four types of immune signatures (median fluorescence intensities (MFI), relative cell (RC), absolute cell (AC), and morphological parameters (MP)) were included. Comprehensive sensitivity analyses were used to verify the robustness, heterogeneity, and horizontal pleiotropy of the results.ResultsAfter FDR correction, SCZ had no statistically significant effect on immunophenotypes. It was worth mentioning some phenotypes with unadjusted low P-values, including FSC-A on NKT (β = 0.119, 95% CI = 0.044 ~ 0.194, P = 0.002), DN (CD4-CD8-) NKT %T cell (β = 0.131, 95% CI = 0.054 ~ 0.208, P = 9.03 × 10− 4), and SSC-A on lymphocytes (β = 0.136, 95% CI = 0.059 ~ 0.213, P = 5.43 × 10− 4). The causal effect of SCZ IgD on transitional was estimated to 0.127 (95% CI = 0.051 ~ 0.203, P = 1.09 × 10− 3). SCZ also had a causal effect on IgD+ %B cell (β = 0.130, 95% CI = 0.054 ~ 0.207, P = 8.69 × 10− 4), and DP (CD4+CD8+) %T cell (β = 0.131, 95% CI = 0.054 ~ 0.207, P = 8.05 × 10− 4). Furthermore, four immunophenotypes were identified to be significantly associated with SCZ risk: naive CD4+ %T cell (OR = 0.986, 95% CI = 0.979 ~ 0.992, P = 1.37 × 10− 5), HLA DR on CD14− CD16− (OR = 0.738 (95% CI = 0.642 ~ 0.849, P = 2.00 × 10− 5), CD33dim HLA DR+ CD11b− AC (OR = 0.631, 95% CI = 0.529 ~ 0.753, P = 3.40 × 10− 7) and activated & resting Treg % CD4 Treg (OR = 0.937, 95% CI = 0.906 ~ 0.970, P = 1.96 × 10− 4).ConclusionsOur study has demonstrated the close connection between immune cells and SCZ by genetic means, thus providing guidance for future clinical research.

The mitochondrial protein TSPO in Alzheimer’s disease: relation to the severity of AD pathology and the neuroinflammatory environment

The 18kD translocator protein (TSPO) is used as a positron emission tomography (PET) target to quantify neuroinflammation in patients. In Alzheimer’s disease (AD), the cerebellum is the pseudo-reference region for comparison with the cerebral cortex due to the absence of AD pathology and lower levels of TSPO. However, using the cerebellum as a pseudo-reference region is debated, with other brain regions suggested as more suitable. This paper aimed to establish the neuroinflammatory differences between the temporal cortex and cerebellar cortex, including TSPO expression. Using 60 human post-mortem samples encompassing the spectrum of Braak stages (I–VI), immunostaining for pan-Aβ, hyperphosphorylated (p)Tau, TSPO and microglial proteins Iba1, HLA–DR and MSR-A was performed in the temporal cortex and cerebellum. In the cerebellum, Aβ but not pTau, increased over the course of the disease, in contrast to the temporal cortex, where both proteins were significantly increased. TSPO increased in the temporal cortex, more than twofold in the later stages of AD compared to the early stages, but not in the cerebellum. Conversely, Iba1 increased in the cerebellum, but not in the temporal cortex. TSPO was associated with pTau in the temporal cortex, suggesting that TSPO positive microglia may be reacting to pTau itself and/or neurodegeneration at later stages of AD. Furthermore, the neuroinflammatory microenvironment was examined, using MesoScale Discovery assays, and IL15 only was significantly increased in the temporal cortex. Together this data suggests that the cerebellum maintains a more homeostatic environment compared to the temporal cortex, with a consistent TSPO expression, supporting its use as a pseudo-reference region for quantification in TSPO PET scans.

Impact of HLA eplet mismatch load in immunological outcomes after living donor kidney transplantation

IntroductionHLA eplets mismatches (eMM) have been associated with negative kidney outcomes after transplantation, such as the development of de novo donor-specific antibody (dnDSA), antibody-mediated rejection (ABMR), and early graft loss. This study aimed to evaluate the clinical effects of the HLA eMM load on dnDSA development, ABMR, renal function, allograft survival and graft loss. Material and methodsThis retrospective study involved 159 living donor kidney transplant patients categorized into groups based on antigen HLA mismatches assessed traditionally and HLA eMM load. Patients had followed for at least one year. The EpViX online program was used to evaluate the HLA eMM load. Cox models were constructed to assess the risk of graft loss. Kaplan-Meier survival curves were carried out. The analyses had performed using the R program and p < 0.05 was considered significant. ResultsFrom all 159 patients, 28 (17.6%) lost their allografts. Rejection episodes occurred in 37.1% of patients, 13.6% of whom were ABMR. Patients with rejection episodes had higher HLA-AB (p = 0.032) and HLA-DR (p = 0.008) HLA eMM load, HLA-AB (p = 0.006) and HLA-DR (p = 0.009) antigens mismatches, and higher proportions of the following eMM in the HLA-DR locus: 70R eMM (p = 0.015), 70RE (p = 0.015), 74E (p = 0.015) and 48Q (p = 0.047). In multiple models, the presence of HLA-DR 70qq eMM (HR 3.75, 95% CI 1.47; 9.55) add an increase in creatinine levels at 1-year (HR 3.87, 95% CI 2.30, 6.53) were associated with the risk of graft loss. ConclusionThe HLA eMM load was related to episodes of rejection and allograft loss. The HLA-DR eMM was most strongly associated with a worse immunologic outcome than eMM mismatches for HLA-AB.

A Case Report of Aggressive Fumarate Hydrase-deficient Renal Cell Carcinoma With Loss of HLA Antigens.

Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) is a rare RCC subtype, and FH-deficient RCC may be misdiagnosed as another type of RCC, such as type 2 papillary RCC or collecting duct carcinoma. FH and 2-succinocysteine (2SC) are useful diagnostic markers for FH-deficient RCC and can be measured using immunohistochemistry (IHC). A 30-year-old female with 3-month history of fatigue and left-flank mass was diagnosed with a 20×13×10 cm left-side renal mass with massive inferior vena cava (IVC) tumor thrombus that extended into the right atrium. She underwent nephrectomy and IVC thrombectomy, and a pathological diagnosis of type 2 papillary RCC was made. Four months after the surgery, computed tomography scan showed multiple liver metastases not observed immediately after surgery. Systemic treatment with sorafenib was initiated; however, she did not respond and died 3 months after treatment. Subsequent re-review of hematoxylin and eosin-stained sections indicated morphologic characteristics consistent with FH-deficient RCC, and IHC staining was negative for FH but positive for 2SC, indicating a diagnosis of FH-deficient RCC. Further immunological analyses revealed the loss of HLA-class I, b2 microglobulin, and HLA-DR antigens in cancer cells. In addition, a few CD8-positive cytotoxic T cells and CD163-positive tumor-associated macrophages were noted. An immunosuppressive tumor microenvironment that facilitates cancer immune evasion might be associated with the rapid progression and poor prognosis in our patient. Further investigation of the tumor immune microenvironment in patients with FH-deficient RCC is warranted.

Association of HLA class II DR/DQ alleles in children and adolescents with rheumatic heart disease from a tertiary care centre in North India

IntroductionRheumatic fever and RHD constitutes an important public health problem in India. The relatively low attack rate of RF, the high concordance rate for RF in monozygotic twins (19%) compared to dizygotic twins (2.5%), and the high familial incidence of RF suggest the involvement of host genetic factors in susceptibility to RF with consequential progression to RHD. ObjectiveTo study the association of HLA CLASS II DR/DQ alleles in children and adolescents with RHD from a tertiary care center in North India. Methods30 RHD patients and 30 age and sex-matched controls were included in our study and blood samples for HLA typing were processed through LAB Type™ reverse SSO DNA typing method. The assignment of the HLA typing was based on a comparison with already published HLA gene sequences. ResultsThe mean age of RHD patients and matched control groups were 12.97 ± 2.95 and 11.93 ± 3.23, respectively. In the cases and control group, males accounted for 63.3% and 50% of the patients respectively. A significant difference was found between the cases and controls for HLA DR∗ 15 (p-value 0.002), HLA DR∗ B4 (p-value 0.045), HLA DR∗ B5 (p-value 0.017), and HLA DQB1∗ 02 (p-value 0.005). ConclusionOur study suggests that HLA class II haplotypes may provide insight into the molecular mechanism of RHD and be a useful tool in predicting the clinical outcome in RF patients, thereby affording new means of intervention or vaccine design. Larger studies are needed to address this in our population.

P-512 Cell-mediated immunity as a biomarker of predisposition in recurrent miscarriage.

Abstract Study question Are alterations in the peripheral cellular immune system a potential risk factor for recurrent miscarriage? Summary answer Percentage alteration of some circulating immune cell subpopulations could be used as biomarkers or potential therapeutic targets in recurrent miscarriage. What is known already About 50% of patients with recurrent miscarriage (RM) have no known cause of the disease. The maternal immune system plays an important role in maternal-fetal tolerance, so an imbalance in the maternal immune system could trigger reproductive failure in women. Antiphospholipid syndrome is the only immunological alteration with sufficient evidence of association with RM, however, there are many other immunological parameters under investigation that appear to mediate the maternal-fetal relationship such as KIR receptors and HLA-C. Other authors are focusing their studies on reproductive immunology at the cellular level, looking at the role of different lymphocyte subpopulations in RM. Study design, size, duration Prospective observational analytical study (cases and controls) conducted in the Immunology Department of the Hospital General Universitario Gregorio Marañón (Madrid, Spain) from 2019 to 2022. It included a group of 49 non-pregnant women with a clinical history of recurrent miscarriages of unknown cause, and two control groups, one with 40 women of childbearing age who had at least one live newborn and another with 60 women of childbearing age who had no history of pregnancy. Participants/materials, setting, methods The inclusion criteria for the study were: two or more gestational losses (&amp;lt; 22 weeks of pregnancy). Patients with severe uterine pathologies, genetic disorders, thrombophilias, obesity, infections, coeliac disease without gluten restriction, uncontrolled endocrine disorders, immunomodulatory treatment, autoimmune disorders and documented RM were excluded from the study. An immunophenotypic study of T, B, NK and NKT lymphocyte subpopulations and monocytic series was performed. Cell characterization was carried out by 6-colour flow cytometry in peripheral blood. Main results and the role of chance When comparing the cellular profile of RM patients with the profile of both control groups, we observed alterations in the percentage levels of CD3+ T cells, CD8+ T cells, NKT cells and non-switch and class switch memory B cells in the patient group, suggesting the presence of a predominantly pro-inflammatory peripheral cellular immune profile in RM patients. In contrast to previous studies by our research group, we did not observe significant differences between the percentage levels of NK cells and activated CD8+ T cells (HLA DR+) in the patient and control groups, which may be explained by the high and exhaustive patient selection in this study. After association study of these alterations with RM, we identified levels of NKT cells &amp;gt; 5%, CD8+ T cells &amp;gt; 26.50%, non-switch memory B cells &amp;gt; 22% and class switch memory B cells &amp;gt; 32% as new biomarkers of RM risk. In addition, the combination of at least two of these new biomarkers was found to significantly increase the likelihood of developing the disease, suggesting that RM is a complex pathology in which multiple factors may influence the pathogenesis of the disease. Limitations, reasons for caution Among the limitations of our study, we can highlight the collection of control group variables from databases developed in previous studies, in addition to a small sample size. Wider implications of the findings The determination of these immunological biomarkers in RM allows us to evaluate the role of immunomodulatory therapies on these biomarkers in order to evaluate possible new treatments to help prevent immune-mediated reproductive failure, thus contributing to a more personalized medicine. Trial registration number Not applicable

A paired-kidney allocation study found superior survival with HLA-DR compatible kidney transplants in the Eurotransplant Senior Program

The Eurotransplant Senior Program (ESP) has expedited the chance for elderly patients with kidney failure to receive a timely transplant. This current study evaluated survival parameters of kidneys donated after brain death with or without matching for HLA-DR antigens. This cohort study evaluated the period within ESP with paired allocation of kidneys from 675 donors 65 years and older to transplant candidates 65 years and older, the first 341 patients within the Eurotransplant Senior DR-compatible Program and 334 contralateral kidneys without (ESP) HLA-DR antigen matching. We used Kaplan-Meier estimates and competing risk analysis to assess all cause mortality and kidney graft failure, respectively. The log-rank test and Cox proportional hazards regression were used for comparisons. Within ESP, matching for HLA-DR antigens was associated with a significantly lower five-year risk of mortality (hazard ratio 0.71; 95% confidence interval 0.53-0.95) and significantly lower cause-specific hazards for kidney graft failure and return to dialysis at one year (0.55; 0.35-0.87) and five years (0.73; 0.53-0.99) post-transplant. Allocation based on HLA-DR matching resulted in longer cold ischemia (mean difference 1.00 hours; 95% confidence interval: 0.32-1.68) and kidney offers with a significantly shorter median dialysis vintage of 2.4 versus 4.1 yrs. in ESP without matching. Thus, our allocation based on HLA-DR matching improved five-year patient and kidney allograft survival. Hence, our paired allocation study suggests a superior outcome of HLA-DR matching in the context of old-for-old kidney transplantation.

Biostatistical Analysis of Relation between HLA-A/B and HLA-DR Eplet Mismatches and Renal Graft Outcome

Abstract: Despite all the clinical interventions that happened during the last 2 decades, the unavailability of a potent donor for renal transplantation remains a concern in the field of transplantation immunology. Recent studies promise the establishment of an effective clinical method to find a potent renal transplant donor, in the near future. Combining the use of molecular techniques with biostatistical methods can be a great tool for clinicians in choosing a potent donor. The present study was conducted from a cohort of 1144 (n=572 pair) renal transplant recipients and their donors to analyse eplet matches and mismatches. Initial observation of increased serum creatinine level which ranged from 2.0 to 21.0 mg/dL in patients treated for end-stage renal disease implied a single event of graft rejection. The statistical correlation between eplet mismatches and graft survival was observed to be significant with P value 0.003. A value of -0.215 at 0.01 significance showed the positive linear correlation between the increased numbers of HLA DR eplet mismatches towards chronic renal graft rejection. The correlation coefficient of -0.888 observed between the number of HLA DR eplet mismatches and days of graft survival clearly implies the effect of increased HLA DR eplet mismatch delayed graft rejection. The study gives an idea on the minimization of class II HLA DR eplet mismatches to decreased frequency of graft rejection as indicated by increased survival rate.

A cell-free antigen processing system informs HIV-1 epitope selection and vaccine design.

Distinct CD4+ T cell epitopes have been associated with spontaneous control of HIV-1 replication, but analysis of antigen-dependent factors that influence epitope selection is lacking. To examine these factors, we used a cell-free antigen processing system that incorporates soluble HLA-DR (DR1), HLA-DM (DM), cathepsins, and full-length protein antigens for epitope identification by LC-MS/MS. HIV-1 Gag, Pol, Env, Vif, Tat, Rev, and Nef were examined using this system. We identified 35 novel epitopes, including glycopeptides. Epitopes from smaller HIV-1 proteins mapped to regions of low protein stability and higher solvent accessibility. HIV-1 antigens associated with limited CD4+ T cell responses were processed efficiently, while some protective epitopes were inefficiently processed. 55% of epitopes obtained from cell-free processing induced memory CD4+ T cell responses in HIV-1+ donors, including eight of 19 novel epitopes tested. Thus, an in vitro processing system utilizing the components of Class II processing reveals factors influencing epitope selection of HIV-1 and represents an approach to understanding epitope selection from non-HIV-1 antigens.

Peripheral Mononuclear Cells Surface Markers Evaluation in Different Stages of Hepatocellular Carcinoma; in a Trial for Early and Accurate Diagnosis in Patients with Post-Hepatitis Liver Cirrhosis and Unremarkable Raised AFP.

HCC is frequently diagnosed late, when only palliative treatment is available. So, we try to use different immunological markers to identify early HCC in patients with unremarkable raised AFP. This study was conducted on 112 participants divided into two equal groups: Group I, 56 patients with liver cirrhosis and different stages of HCC; Group II, 56 patients with liver cirrhosis. The diagnosis of HCC was based on AASLD guidelines. TNM and BCLC classification systems are used for staging of HCC. A significant reduction in the median percentage of lymphocyte subset (CD3+, CD4+, CD8+, CD19+) and NK cell percentage (CD56+) has been detected in HCC patients (all P < 0.001). In the HCC group the median monocyte subpopulations CD14+ CD16- Classical, CD14++ CD16+ Intermediate, and CD14-+ CD16++ Non-Classical were 11.7, 4.0, and 3.5, respectively, with marked reduction compared with liver cirrhosis group (all P < 0.001). Patients with advanced stages (BCLC C and D) were more likely to have significantly higher median CD33+ than patients with early stages (BCLC A and B) (P = 0.05); also, the median levels of HLA DR+ lymphocytes % in the HCC case group were 21.8 in patients with advanced disease (BCLC C and D) and 13.1 in patients with early stages of the disease (P = 0.04). Patients with late stage (TNM III) were more likely to have significantly higher median CD14+ CD16- Classical monocyte subset, CD36+ HLA DR+, and CD36+ CD16- than patients with early stages (TNM I and II). Patients with HCC with unremarkable raised AFP showed marked reduction in lymphocytes, natural killer cells, and all monocyte subpopulations. In addition, patients with advanced HCC showed increased CD33+ and HLA DR+ lymphocytes %, CD14+ CD16- Classical monocyte subset, CD36+ HLA DR+, and CD36+ CD16- compared with patients with early stages of HCC.

Abstract PD5-04: PD5-04 Characterizing the HER2-/Immune-/DNA repair (DRD-) response predictive breast cancer subtype: the hunt for new protein targets in a high-needs population with low response to all I-SPY2 agents

Abstract Background: In previous work we leveraged the I-SPY2 trial to create treatment response predictive subtypes (RPS) incorporating tumor biology beyond clinical HR/HER2, to better predict drug responses in an expanded treatment landscape that includes platinum agents, dual HER2-targeting regimens and immunotherapy [1]. We showed that best performing schemas incorporate Immune, DRD and HER2/Luminal phenotypes, and that treatment allocation based on these would increase the overall pCR rate to 63% from 51% using HR/HER2-based treatment selection. The RPS schema has been selected for prospective evaluation in I-SPY2. Using the RPS, one would prioritize platinum-based therapy for HER2-/Immune-/DRD+, immunotherapy for HER2-/Immune+, and dual-anti-HER2 for HER2+ that are not luminal. HER2+/Luminal patients have low response rates to dual-anti-HER2 therapy but may respond better to anti-AKT. However, there is still a considerable ‘biomarker-negative’ group of resistant cancers (HER2-/Immune-/DRD-) with very low pCR rates to all tested agents, that require a new therapeutic approach. Here we characterize the protein signaling architecture of these tumors to identify new target candidates. Methods: 987 I-SPY 2 patients from 10 arms of the trial were considered for this analysis. All have gene expression, pCR and RPS; 944 have distant recurrence free survival (DRFS) data; and 736 have reverse phase protein array (RPPA) data from laser capture microdissected tumor epithelium. These data – known collectively as the I-SPY2-990 mRNA/RPPA Data Resource - were recently made public on NCBI’s Gene Expression Omnibus [GEO: GSE196096]. We focus on HER2-/Immune-/DRD- tumors, applying Wilcoxon and t-tests to identify phosphoproteins that differ between HR+HER2-/Immune-/DRD- and other HR+HER2- tumors; and between TN/Immune-/DRD- and other TNs. The Benjamini-Hochberg (BH) method is used to adjust p-values for multiple hypothesis testing. In addition, the Kaplan-Meier method is used to estimate DRFS. Results: 201/736 I-SPY 2 patients with RPPA data are classified HER2-/Immune-/DRD- (HR+HER2-: n=138; TN: n=63). Of these, 8.5% (17/201) achieved pCR. Non-responding HER2-/Immune-DRD- had worse outcomes than responders (~75% vs. ~95% DRFS at 5 years). 60/139 phospho-proteins differ significantly between HR+HER2-/Immune-/DRD- and other HR+HER2- tumors (n=122). These tumors are relatively ‘cold’, in that 90% (54/60) of the phosphoprotein activities characterizing this group are at lower levels than in the overall HR+HER2- population; including immune (e.g. pPDL1, pJAK/STAT) and proliferation (e.g., Ki67, CyclinB1, pAURK) endpoints. Phosphoproteins showing higher levels in this subset include ERBB2 (BH p=1.7E-06), Cyclin D1 (BH p=1.4E-05), pAR (BH p=1.4E-05), and ER (BH p=3E-04). Within the TN subset, only 3/139 phospho-proteins differed significantly between TN/Immune-/DRD- and other TN tumors (n=189). These were all immune-related (pPDL1, pSTAT1, and HLA DR), with lower expression in the TN/Immune-/DRD- group. Conclusion: HR+HER2- and TN patients who are Immune-Low and DRD-Low have very low pCR rates to all tested therapeutics in I-SPY2 including standard chemotherapy, platinum, and immunotherapy. Senolytics (possibly targeting Cyclin D1), HER2low agents, and AR modulators may overcome resistance in HR+HER2-/Immune-/DRD-, whereas an immune activator beyond checkpoint inhibition is suggested for TN/Immune-/DRD- patients. [1] Wolf et. al., Redefining Breast Cancer Subtypes to Guide Treatment Prioritization and Maximize Response: Predictive Biomarkers across 10 Cancer Therapies. Cancer Cell 2022 Citation Format: Denise M. Wolf, Christina Yau, Julia Wulfkuhle, Rosa I. Gallagher, Lamorna A. Brown Swigart, Gillian L. Hirst, Jean-Philippe Coppe, Mark Jesus M. Magbanua, Rosalyn Sayaman, I-SPY2 Investigators, Laura Sit, Nola M. Hylton, Angela DeMichele, Donald A. Berry, Lajos Pusztai, Douglas Yee, Laura J. Esserman, Emanuel F. Petricoin, Laura Van ’t Veer. PD5-04 Characterizing the HER2-/Immune-/DNA repair (DRD-) response predictive breast cancer subtype: the hunt for new protein targets in a high-needs population with low response to all I-SPY2 agents [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-04.

Study of the action of peripheral blood T-lymphocytes on renal cell carcinoma cells in model systems

Background. The introduction of immunotherapy based on immune checkpoint inhibitors has significantly improve the effectiveness of kidney cancer treatment. Nevertheless, not all patients respond to such treatment and there are no reliable predictive markers. Therefore, the development of a model system for assessing the cellular immune response to a tumor seems to be an urgent task.Aim. Development of a model to assess the T-cell immune response was the focus of this study.Materials and methods. Primary tumor cell culture and peripheral blood T-cell fraction were obtained under standard sterile conditions. T-cell activation were perform via anti-CD3 and anti-CD28 antibodies. The cell index was assessed using the RTCA xCELLigence biosensor technology (ACEA Biosciences, USA).Results. Tumor and T-cells from the same patient were cultured together to assess the growth rate of the tumor cell population. Measurements were taken at 30-minute intervals. The duration of observation was 24 hours. It has been shown that non-activated T-cells do not affect the proliferative properties of cultured cancer cells. On the contrary, activated T-cells suppressed the proliferative properties of cancer cells, which was associated with an increase in the proportion of Т-cells carrying the HLA-DR receptor (CD3+HLA–DR+) because of activation. Tumor-specific T-cell activity can lead to three consequences: lack of effect, partial suppression of proliferative properties, and complete death of tumor cells. In the latter case, the absence of such cells was determined by flow cytometry.Conclusion. The developed approach makes it possible to evaluate the cytotoxic properties of T cells in relation to tumor cells in a particular patient. The advantage of this method is that the measurement can be carried out in the presence of immune checkpoint inhibitors. The proposed method may be useful for evaluating the treatment regimen within the framework of personalized therapy.

Role of the autoantibodies and IL-1 cytokine in the pathogenesis diabetes mellitus

Diabetes mellitus is a complex multifactorial and heterogeneous syndrome characterized by hyperglycemia resulting from inadequate insulin secretion and/or insulin action. T lymphocytes and macrophages appear to play an important role in mediating ß-cell damage and causing Type 1 diabetes.A case-control study is done during the period from Feb., 2011, through the end of Feb. 2012. The study enrolled 90 diabetic patients divided into three groups Type 1, Type2 and LADA ( 30 patients in each group) who attended in Al Zahraa Teaching Hospital in Al-Kut city &amp; 25 non diabetic as a control to determine the prevalence of islet cell autoantibodies, glutamic acid decarboxylase autoantibodies (ICAs&amp; GADAs) and measured (IL-1Α α , IL-4, IL17, TNF α, IFN γ) as well as the frequency of HLA DR was measured in diabetic patients and non-diabetic control with no family history of diabetes. Serological tests for GADA &amp; ICA (by using ELISA) have been done for all sera of the study groups. Dual set ELISA technique is used for estimating the cytokines levels in the sera of all study groups while DNA extracted from whole blood from all study groups and these DNA were used to detected the HLA DR allele by using HISTO SPOT SSO system.According to the results of this study, Type 1 diabetic patients had higher frequency of GADA 65 autoantibodies compared with other study groups, as eighteen of them (60%) showed GADA positive in comparison to 6.6 % in type 2 DM, 56.7% in LADA and 0% in control group. Furthermore, twenty of type 1 diabetic patients which represent (66.7%) were ICA positive in comparison to 33.33% in type 2 DM, 70% in LADA and 4% in control group. Statistical analysis showed highly significant difference.Analysis of the serum sample showed that IL-1 α levels has been elevated significantly in the sera of Type 1(635.8± 685 pg./ml), type 2 (625.5± 55.86 pg./ml) and LADA (383.2± 533.5 pg./ml) diabetics patients positive autoantibodies in comparison with other groups type 1, type 2 and LADA diabetics patients negative autoantibodies and healthy control (60±35.21 pg. /ml)

Impact of the flame retardant 2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) in THP-1 macrophage-like cell function via small extracellular vesicles.

2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most widespread environmental brominated flame-retardant congeners which has also been detected in animal and human tissues. Several studies have reported the effects of PBDEs on different health issues, including neurobehavioral and developmental disorders, reproductive health, and alterations of thyroid function. Much less is known about its immunotoxicity. The aim of our study was to investigate the effects that treatment of THP-1 macrophage-like cells with PBDE-47 could have on the content of small extracellular vesicles' (sEVs) microRNA (miRNA) cargo and their downstream effects on bystander macrophages. To achieve this, we purified sEVs from PBDE-47 treated M(LPS) THP-1 macrophage-like cells (sEVsPBDE+LPS) by means of ultra-centrifugation and characterized their miRNA cargo by microarray analysis detecting the modulation of 18 miRNAs. Furthermore, resting THP-1 derived M(0) macrophage-like cells were cultured with sEVsPBDE+LPS, showing that the treatment reshaped the miRNA profiles of 12 intracellular miRNAs. This dataset was studied in silico, identifying the biological pathways affected by these target genes. This analysis identified 12 pathways all involved in the maturation and polarization of macrophages. Therefore, to evaluate whether sEVsPBDE+LPS can have some immunomodulatory activity, naïve M(0) THP-1 macrophage-like cells cultured with purified sEVsPBDE+LPS were studied for IL-6, TNF-α and TGF-β mRNAs expression and immune stained with the HLA-DR, CD80, CCR7, CD38 and CD209 antigens and analyzed by flow cytometry. This analysis showed that the PBDE-47 treatment does not induce the expression of specific M1 and M2 cytokine markers of differentiation and may have impaired the ability to make immunological synapses and present antigens, down-regulating the expression of HLA-DR and CD209 antigens. Overall, our study supports the model that perturbation of miRNA cargo by PBDE-47 treatment contributes to the rewiring of cellular regulatory pathways capable of inducing perturbation of differentiation markers on naïve resting M(0) THP-1 macrophage-like cells.

A study of leukocyte surface antigen CD64, and monocyte surface antigen HLA DR as a marker of sepsis in preterm and full-term neonates

A study of leukocyte surface antigen CD64, and monocyte surface antigen HLA DR as a marker of sepsis in preterm and full-term neonates

CD34 negative HLA-DR negative acute myeloid leukaemia: A higher association with NPM1 and FLT3-ITD mutations.

CD34 and HLA-DR negativity is often used as a characteristic immunophenotypic feature of acute promyelocytic leukaemia (APL) that differentiates APL from other subtypes of acute myeloid leukaemia (AML). However, other subtypes of AML, without expression of CD34 and HLA-DR antigens, have also been reported. We analysed the HLA-DR negative de novo non-APL AML cases by dividing HLA-DR negative non-APL group into 2 sub-groups based on CD34 expression and compared the characteristics of CD34 negative HLA-DR negative with CD34 positive HLA-DR negative non-APL AML cases with respect to morphologic, immunophenotypic, molecular and clinical parameters. There were 70 cases (8.54%) which were CD34 negative HLA-DR negative and 52 cases (6.34%) were CD34 positive HLA-DR negative. The median age at diagnosis was higher in CD34 negative HLA-DR negative AML than in CD34 positive HLA-DR negative AML group (38 years vs. 12 years, p < 0.001). DIC rate was higher in CD34 negative HLA-DR negative group than the other group (p < 0.001). Median total leucocyte count was higher with higher blast count in peripheral blood and bone marrow in CD34 negative HLA-DR negative AML cases than the other group (p < 0.05). CD34 negative HLA-DR negative AML was more associated with normal karyotype (96.2% vs. 38.5%; p < 0.001), NPM1 mutation (67.8% vs. 8.3%; p < 0.001) and FLT-ITD mutation (37.3% vs. 13.9%; p < 0.05). In CD34 negative HLA-DR negative group, 16 cases had co-occurrence of NPM1 and FLT3-ITD mutations, whereas no case of CD34 positive HLA-DR negative group had such dual mutation positivity. There was poor median overall survival [3.8months (95%CI: 2.3-7.8months) vs. 20.4months (95% CI: 12.8-25.7months); p=0.0148] in CD34 positive HLA-DR negative AML than CD34 negative HLA-DR negative AML cases. We found that the CD34 negative HLADR negative non APL AML is highly associated with NPM1 and FLT3-ITD mutation, older age at diagnosis, DIC, higher total leucocyte count, higher blast counts and normal karyotype in comparison to CD34 positive HLA-DR negative AML group. Co-occurrence of NPM1 and FLT3-ITD mutation was also exclusively seen in CD34 negative HLA-DR negative group. There was poor overall survival in CD34 positive HLA-DR negative AML than CD34 negative HLA-DR negative AML cases.

Skewed fate and hematopoiesis of CD34+ HSPCs in umbilical cord blood amid the COVID-19 pandemic.

Umbilical cord blood (UCB) is an irreplaceable source for hematopoietic stem progenitor cells (HSPCs). However, the effects of SARS-CoV-2 infection and COVID-19 vaccination on UCB phenotype, specifically the HSPCs therein, are currently unknown. We thus evaluated any effects of SARS-CoV-2 infection and/or COVID-19 vaccination from the mother on the fate and functionalities of HSPCs in the UCB. The numbers and frequencies of HSPCs in the UCB decreased significantly in donors with previous SARS-CoV-2 infection and more so with COVID-19 vaccination via the induction of apoptosis, likely mediated by IFN-γ-dependent pathways. Two independent hematopoiesis assays, a colony forming unit assay and a mouse humanization assay, revealed skewed hematopoiesis of HSPCs obtained from donors delivered from mothers with SARS-CoV-2 infection history. These results indicate that SARS-CoV-2 infection and COVID-19 vaccination impair the functionalities and survivability of HSPCs in the UCB, which would make unprecedented concerns on the future of HSPC-based therapies.

Fast-Track Measurable Residual Disease Detection By Multiparametric Flow Cytometry in Acute Myeloid Leukemia before Allogeneic Hematopoietic Cell Transplantation

Introduction In acute myeloid leukemia (AML), measurable residual disease (MRD) becomes more and more important for individualized treatment decisions. The MRD status at the time of allogeneic hematopoietic cell transplantation (alloHCT) provides prognostic information beyond baseline risk features. Peri-transplant treatment strategies and conditioning intensity can be stratified according MRD-levels. However, therapeutic interventions (e.g. conditioning intensity) usually require the prompt availability of reliable information on the MRD status. We assessed the prognostic value of a standardized, reproducible, and fast turnover multiparametric flow cytometry (MFC) approach that has been validated at the end of intensive induction therapy to monitor MRD before alloHCT (Röhnert, Leukemia 2022). This approach allows for the simultaneous detection of MRD by the leukemia-associated immunophenotype (LAIP) and the different from normal (DfN) method. Methods Between October 2016 and March 2020, bone marrow (BM) aspirates of patients (pts) with AML were analyzed for the presence of MRD immediately before the start of conditioning. therapy with the aim of analyzing to predictive value of this fast-track parameter. Pts receiving sequential regimens (re-induction chemotherapy followed by reduced-intensity conditioning) were excluded. Briefly, the LAIP-based DfN analysis is based on a hierarchical gating strategy with fixed gates and encompasses the antigens CD34, CD117, HLA-DR (backbone markers), CD13, CD33 (myeloid markers), CD7 and CD56 (lymphoid markers). Within less than 5 minutes, the analysis checks for the presence of the aberrant categories i) deficiency of myeloid markers and ii) cross-lineage expression of lymphoid markers. Reference values for 32 immunophenotypically defined populations were established on BM aspirates of 90 leukemia free controls. An aberrant category already detectable at diagnosis on ≥10% of myeloid progenitors/ monocytes was termed aberrant LAIP (aLAIP) and a de-novo appearance (after chemotherapy) post-treatment DfN (ptDfN). In conclusion this approach is very fast and provides high reproducibility. Results We identified 58 pts with a median follow-up of 33.3 months after alloHCT (Table 1 with pts characteristics). Response assessment was performed in median 8 days before alloHCT. Most of the patients were in morphologic complete remission (CR n=37). A subset of 21 patients had refractory disease with a median BM blast count of 6.5%. Samples at AML diagnosis were available for 48/58 (83%) pts, of whom 34/48 (71%) showed at least one aLAIP at initial diagnosis. Before start of conditioning, 44/58 (76%) patients were MRDpos. The proportion of MRDpos in responders was lower compared to non-responders: 24/37 (65%) vs. 20/21 (95%) (p=0.0106). MRDpos was designated by aLAIPonly, ptDfNonly and aLAIP+ptDfN in 18%, 48% and 34% of cases, respectively. Noteworthy, 8 of 21 pts classified MRDpos by ptDfNonly had no sample available at diagnosis and therefore could not be assessed for aLAIP persistence. Amongst 37 pts in CR before alloHCT, 12/24 (50%) of MRDpos pts relapsed compared to 3/13 (23%) of MRDneg pts. TRM in these patients did not differ (13% vs. 8%). In a univariable Cox regression analysis, the 1-year Overall survival (OS) rate of MRDpos cohort was significantly shorter compared to MRDneg pts (64% vs. 93%, Hazard ratio (HR) 10.5, p=0.004). MRD was detected by ptDfNonly in 23/44 (52%) of MRDpos pts. The OS of these pts was comparable to MRDpos by aLAIP with or without ptDfN (Figure 1). As expected, the MRDpos cohort was enriched for non-responders. However, when the analysis was restricted to pts in CR (n=37), MRD status preserved its prognostic significance (HR 7.9, p=0.019). And even in the subpopulation of pts in first CR, MRDpos was associated with a significantly reduced OS compared to MRDneg (HR 6.3, p=0.043). Interestingly, a single non-responder was classified as MRDneg and showed a long-term OS. Conclusions Our fast-track LAIP-based DfN approach provides relevant prognostic information prior to conditioning for allo HCT. On top of the classical LAIP approach, the ptDfN analysis gives additional prognostic information. The LAIP-based DfN approach might be a suitable tool for fast-track and reliable MRD assessment before starting the preparative regimen for alloHCT and might help to guide potential treatment modifications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The High Mobility Group A1 Chromatin Regulator Drives Immune Evasion during MPN Progression By Repressing Genes Involved in Antigen Presentation

Introduction: Myeloproliferative Neoplasms (MPN) are blood diseases caused by mutations activating JAK2 in hematopoietic stem cells (HSCs) which lead to clonal expansion and overproduction of myeloid lineages. MPN patients are at increased risk for transformation to bone marrow fibrosis (myelofibrosis) and acute myeloid leukemia (AML), both of which are associated with poor clinical outcomes. Importantly, targetable mechanisms to prevent progression remain elusive. The High Mobility Group A1 (HMGA1) gene encodes chromatin regulators which are enriched in hematopoietic stem cells and aberrantly overexpressed in MPN with highest levels after transformation to myelofibrosis and acute leukemia (Li & Kim et al Blood 2022, Xian et al Nature Commun 2017). We recently discovered that HMGA1 is required for progression to leukemia in murine models of JAK2V617F MPN leukemia. Further, loss of just a single Hmga1 allele prevents progression to myelofibrosis in JAK2V617F transgenic mice. Emerging evidence suggests that immune escape represents a fundamental pathway involved in progression in diverse tumors. We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by dysregulating gene networks involved in immune evasion, and, 2) identify mediators of immune escape that could be disrupted in therapy. Methods: To identify gene networks regulated by HMGA1 in JAK2V617F MPN, we performed multi-omics sequencing analyses, including RNAseq, chromatin immunoprecipitation (ChIPseq), and assays of chromatin accessibility (ATACseq) from JAK2V617Fmutant AML cell lines (DAMI, SET-2) + HMGA1 depletion. HMGA1 gene expression was inactivated using CRISPR/Cas9 or short hairpin RNAs (shRNA) targeting 2 different sequences. We used gene set enrichment analysis to dissect molecular mechanisms underlying HMGA1. Transcriptional networks were validated at the level of mRNA and protein via quantitative RT-PCR and flow cytometry. To identify drugs to disrupt HMGA1 immune evasion networks, we applied the Broad Institute Connectivity Map (CMAP) and cytotoxicity assays. To validate these pathways in human MPN, we assessed gene expression in independent MPN patient cohorts. Results: To elucidate transcriptional networks governed by HMGA1 during MPN progression to leukemia, we integrated RNAseq, ChIPseq, and ATACseq in JAK2V617F mutant AML cells (DAMI, SET-2) which revealed that HMGA1 represses genes involved in antigen presentation (Interferon γ response networks), including genes encoding major histocompatibility complex (MHC) class I and II antigens. Silencing HMGA1 via CRISPR or shRNA results in up-regulation of MHC class I and II antigen genes, with greatest induction of MHC class II genes HLA-DRA/DRB1. Similarly, HMGA1 depletion increases cell surface expression of HLA-DR antigens by flow cytometry. Further, inhibiting HMGA1 in JAK2V617Fmutant AML cells results in up-regulating the class II major histocompatibility complex transactivator (CIITA) and invariant chain (CD74), which function in inducing MHC class II gene expression and chaperoning structure assembly, respectively. To identify underlying mechanisms, we intersected ChIPseq with ATACseq, which revealed HMGA1 occupancy downstream of the HLA-DRB1 locus, including regions at +6.27kb, +6.7kb, +14kb, and +15kb from the transcription start site. Further, preliminary results suggest that HMGA1 recruits repressive histone marks to repress HLA-DRB1 at these sites. To target HMGA1 networks, we queried CMAP, which identified the histone deacytelyase inhibitor (HDACi), entinostat. Strikingly, entinostat not only up-regulates MHC class II genes and antigens, but is also cytotoxic in JAK2V617F mutant AML cells. Finally, HMGA1 depletion enhances sensitivity to entinostat, which synergizes with JAK inhibitor, Ruxolitinib, to further induce MHC gene expression. Conclusions: We discovered a novel epigenetic program whereby HMGA1 drives immune evasion during MPN progression by binding to chromatin and modulating transcriptional networks to repress MHC antigen presentation. Most importantly, HMGA1 immune evasion networks are dysregulated in human MPN after progression to AML. Together, our studies reveal a new paradigm whereby HMGA1 down-regulates MHC antigens during MPN progression, suggesting that targeting HMGA1 networks with entinostat could activate an immune attack and prevent MPN progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DDEL-13. ULTRASOUND-ENHANCED DELIVERY OF LIPOSOMAL DOXORUBICIN ACROSS THE BLOOD BRAIN BARRIER INDUCES AN IFN-GPHENOTYPE IN MICROGLIA, MACROPHAGES, AND T CELLS AND IMPROVES RESPONSE TO PD-1 BLOCKADE IN GLIOMAS

Abstract INTRODUCTION Given the limited drug penetration across the blood-brain barrier (BBB), the therapeutic potential of new and existing therapies has not been fully exploited for the benefit of glioblastoma (GBM) patients. METHODS Here we employed a novel drug delivery technology based on low-intensity pulsed ultrasound combined with intravenous microbubbles (LIPU/MB) that temporarily opens the BBB to deliver liposomal doxorubicin (DOX) and anti-PD-1 therapy (aPD-1) in mouse glioma models and 3 recurrent GBM patients. Immunological variables were evaluated in tumor and immune cells as well as efficacy in glioma-bearing mice treated with DOX delivered by LIPU. These included measurement of HLA ABC and HLA DR protein expression by tumor cells, microglia, and macrophages and IFN-g production by glioma-associated microglia and macrophages in mouse and human tumors. We also assessed efficacy of LIPU/MB enhanced combination therapy in glioma-bearing mice. RESULTS Upregulation of HLA ABC and HLA DR was observed in GBM cell lines at low concentrations of DOX. Tumor cells from GBM patients treated with DOX, aPD-1 and LIPU/MB showed increased expression of HLA ABC and HLA DR compared to paired pretreatment samples. In both mice and humans, LIPU/MB liposomal DOX increased absolute brain drug concentrations and elicited a specific IFN-g phenotype and MHC I expression in glioma-associated microglia and macrophages in mice and humans. Furthermore, LIPU/MB-mediated BBB opening increased brain concentrations of aPD-1 in mice and in peritumoral regions of GBM patients. Combined treatment with liposomal DOX and aPD-1 delivered with LIPU/MB resulted in long-term survival of glioma-bearing mice that relied on the activity of CD8+ T cells for its efficacy. CONCLUSIONS Overall, this translational study demonstrates the utility of LIPU/MB to stimulate intracranial immune responses in the context of treatment with DOX and aPD-1 for gliomas.