Expression Of Beta 2-microglobulin Research Articles

Genetic ablation of adhesion ligands mitigates rejection of allogeneic cellular immunotherapies

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade Tcell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) Tcells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells invitro and invivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy.

Tumor beta2-microglobulin and HLA-A expression is increased by immunotherapy and can predict response to CIT in association with other biomarkers.

Downregulation of MHC class I expression and/or defects in the antigen presentation pathways are commonly reported in human cancers. Numerous studies previously have explored extensively the molecular mechanisms that underlie HLA-class I and Beta2-Microglobulin (B2M) downregulation. However, the techniques presently available to detect expression of MHC class I proteins lack the robustness, specificity and sensitivity needed for systematic integration and analysis in clinical trials. Furthermore, the dynamics of HLA-class I and B2M expression have not been comprehensively studied as a potential biomarker for immunotherapy. Using novel, validated, immunohistochemistry (IHC)-based methods for quantifying B2M and HLA-A in tumor samples from diverse cancer types, we have determined loss of B2M and HLA-A proteins in 336 archived, primary specimens and 329 biopsies from metastatic patients collected during Roche-sponsored Phase 1 clinical trials investigating novel immunotherapy candidates as monotherapy or in combination with CPI. Up to 56% of cases with B2M or HLA-A loss were noted in the investigated tumor types. The frequency of loss was dependent on indication and stage of disease and revealed heterogeneous expression patterns across patients. B2M and HLA-A loss was increased in metastatic lesions compared to primary tumors, indicating selection of MHC class I low clones in metastatic and refractory tumor cells. High on-treatment B2M expression correlated with successful clinical outcome (RECIST), while high baseline B2M did not. A treatment-induced increase of B2M expression was noted in most of the patients with low B2M levels at baseline. The triple biomarker combination of B2M, CD8 and PDL1 strongly improved response prediction to cancer immunotherapy. Our results indicate that B2M and HLA-A loss occurs frequently in tumors and is reversed in most instances following immunotherapy which supports the conclusion that MHC class I loss is not the dominant resistance mechanism to CPI treatment. This investigation reveals a highly dynamic expression of HLA-A and B2M in tumors affected by indication, metastatic status, immunophenotype and immunotherapy treatment. Baseline expression levels of B2M on tumors may be of utility as a constituent of a biomarker panel used for selecting patients for immunotherapy clinical trials.

Immune microenvironment and lymph node yield in colorectal cancer.

Lymph node (LN) harvesting is associated with outcomes in colonic cancer. We sought to interrogate whether a distinctive immune milieu of the primary tumour is associated with LN yield. A total of 926 treatment-naive patients with colorectal adenocarcinoma with more than 12 LNs (LN-high) were compared with patients with 12 or fewer LNs(LN-low). We performed immunohistochemistry and quantification on tissue microarrays for HLA class I/II proteins, beta-2-microglobulin (B2MG), CD8, CD163, LAG3, PD-L1, FoxP3, and BRAF V600E. The LN-high group was comprised of younger patients, longer resections,largertumours, right-sided location, and tumours with deficient mismatch repair (dMMR). The tumour microenvironment showed higher CD8+ cells infiltration and B2MG expression on tumour cells in the LN-high group compared to the LN-low group. The estimated mean disease-specific survival was higher in the LN-high group than LN-low group. On multivariate analysis for prognosis, LN yield, CD8+ cells, extramural venous invasion, perineural invasion, and AJCC stage were independent prognostic factors. Our findings corroborate that higher LN yield is associated with a survival benefit. LN yield is associated with an immune high microenvironment, suggestingthat tumour immune milieu influences the LN yield.

Abstract 2780: Loss of tumor HLA-A and B2M expression and association with biomarkers and clinical outcome

Abstract Loss of MHC-I and/or Beta 2 microglobulin (B2M) expression is regarded as a frequent resistance mechanism to immunotherapies. Indeed, downregulation of MHC-I expression and/or defects in the pathways of antigen presentation machinery are commonly reported in human cancers. Despite many reports published, available tools lack the robustness, specificity and sensitivity needed for systematic integration and analysis in clinical trials. In order to explore loss of antigen presentation as one of the major resistance mechanisms to checkpoint inhibitor (CPI) therapy treatment and to enable identification of patients with tumors harboring antigen presentation defects, we have developed immunohistochemistry (IHC)-based robust prototype assays to quantify HLA-A and B2M proteins in patient tissue samples. We have determined the prevalence of loss of HLA-A and B2M in banked primary tumor blocks as well as in 329 tumor metastatic biopsies collected during Roche-sponsored Phase 1 clinical trials investigating novel immunotherapies as single agents or in combination with CPI. Below are the key findings and preliminary interpretations:We confirm a significant prevalence of HLA-A and B2M loss (up to 65%) depending on indication, revealing heterogeneous expression patterns across patients and indications.We describe an increase of HLA-A and B2M loss from primary to metastatic lesions, which may reflect a selection of MHC-I-low/negative clones in spreading cancer cells (and maybe lines of therapies).We show that high on-treatment B2M expression is positively associated with clinical outcome (RECIST), while high baseline B2M expression only shows a trend (but no statistical significance) towards positive association with response. If the former (on-treatment expression) could be seen as a consequence of the drug effect (induction of inflammation, IFN-signaling and subsequent induction of MHC-I expression), the latter (baseline expression) would have been the foundation for enriching patient populations based on MHC-I expression. Based on these data, the absence of strong correlation between baseline expression and response does not support a cross-CIT enrichment approach at this stage.We see a treatment-induced increase of MHC-I expression in most of the patients. Preliminary conclusions could be that 1/MHC-I loss can be rescued in most of the cases we analyzed (few hard losses), 2/Since we only see few responders, MHC-I loss is not the dominant resistance mechanism or the drug(s) did not induce a strong-enough MHC-I expression to rescue it.This investigation reveals a highly dynamic expression of HLA-A and B2M in tumors affected by key variables such as indication, metastatic status, immunophenotype and immunotherapy treatment. We plan to explore MHC-I expression in larger cohorts of CPI-treated patients in order to confirm and broaden our preliminary findings. Citation Format: Bernhard Reis, Sebastian Dziadek, Jan Attig, Nico Graefe, Astrid Heller, Natascha Rieder, Bruno Gomes. Loss of tumor HLA-A and B2M expression and association with biomarkers and clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2780.

Digital spatial profiling to uncover biomarkers of immunotherapy outcomes in head and neck squamous cell carcinoma.

6050 Background: Immunotherapy (ITx) has become the standard of care in the treatment of recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC). However, response rate is limited to 13-18% of patients, underlining the need to identify mechanisms implicated in response or resistance. This study aims to uncover novel biomarkers of ITx outcomes in R/M HNSCC using digital spatial profiling (DSP) technology. Methods: Pre-treatment biopsy samples of 50 ITx-treated R/M HNSCC patients, constructed in the form of tissue microarray (YTMA496), were included in our discovery cohort. Cases underwent DSP with the Human Immuno-Oncology protein panel (NanoString Technologies), comprising 71 photocleavable oligonucleotide-labeled primary antibodies, used for target-protein quantification in 4 distinct molecularly defined compartments: tumor (CK+), leukocyte (CD45+), macrophage (CD68+) and immune stroma (CD45+/CD68+). All markers were explored for associations with progression-free (PFS) and overall survival (OS) using a univariate Cox regression model. Significant markers were validated using an alternative quantitative immunofluorescence method as well as in an independent validation cohort of 29 ITx-treated R/M HNSCC cases (YTMA523). Results: Univariate DSP data analysis revealed high beta2-microglobulin (B2M), LAG-3, CD25 and 4-1BB in tumor, high B2M, CD45 and CD4 in stroma, and low fibronectin in the macrophage compartment, as markers associated with improved PFS. Increased levels of B2M and CD25 in tumor and CD11c in stroma were also correlated with prolonged OS. Focusing on B2M, cases at the top tertile of tumor B2M expression were associated with improved PFS and OS [HR, 0.43; 95% confidence interval (CI), 0.21-0.9; p = 0.034 and HR, 0.41; 95% CI, 0.18-0.90; p = 0.047, respectively], by an orthogonal QIF method. Findings were replicated in our validation cohort for PFS [HR, 0.41; 95% CI, 0.19-0.93; p = 0.034] and showed a similar trend for OS [HR, 0.44; 95% CI, 0.19-1.0; p = 0.074]. B2M-high tumors also had significant enrichment with immune-cell markers (CD3, CD4, CD8, CD11c, CD68 and CD163) and increased immune checkpoint expression (PD-L1, ICOS, TIM-3, LAG-3, IDO1, B7-H3), predominantly in the tumor compartment. Conclusions: Our study indicates that intact, highly functional antigen presentation, sustained by high B2M expression in tumor, confers survival benefit in ITx treated R/M HNSCC patients, an effect driven by increased intra-tumoral immunogenicity.

Lynch syndrome-associated epithelial ovarian cancer and its immunological profile

IntroductionLynch syndrome is a multi-tumor syndrome characterized by mismatch repair deficiency (MMR-d), microsatellite instability (MSI), and increased tumor-infiltrating lymphocytes (TILs) making these tumors candidates for treatment with immune checkpoint inhibitors. However, response may depend on tumor-induced immune evasion mechanisms, e.g. loss of Beta-2-Microglobulin (B2M) or upregulation of programmed death protein ligand 1 (PD-L1). We investigated the immune response and B2M and PD-L1 expression in Lynch syndrome-associated ovarian cancers. MethodsWe successfully analyzed 30 Lynch syndrome-associated epithelial ovarian cancers collected through the Danish Hereditary Non-Polyposis Colorectal Cancer (HNPCC) register. MMR-d, MSI, immune response (CD3, CD8, and CD68), and immune evasion mechanisms (B2M and PD-L1) were investigated. Statistical associations between these markers were evaluated in addition to survival in relation to B2M/PD-L1. ResultsOf the 29 evaluable tumors, 27 were MMR-d (93.1%). Likewise of 26 evaluable tumors, 14 were MSI (53.8%). MMR-d/MMR-proficiency associated with MSI/MSS in 60.0%. Half of the ovarian tumors presented with high levels of TILs. Loss of B2M expression was observed in 46.7% of the tumors, while expression of PD-L1 was seen in 28.0% of the cases. There was no association between B2M/PD-L1 and MSI/TILs/survival. Loss of B2M was often seen in tumors with low TILs (p = 0.056 or p = 0.059 for CD3 and CD8 positive cells, respectively). ConclusionMMR-d, MSI, and TILs are also seen in Lynch syndrome-associated ovarian cancers making these potential candidates for checkpoint-based immunotherapy. The clinical impact from immune evasion through loss of B2M needs to be investigated further in larger cohorts.

Beta-2-microglobulin Mutations Are Linked to a Distinct Metastatic Pattern and a Favorable Outcome in Microsatellite-Unstable Stage IV Gastrointestinal Cancers.

Immune checkpoint blockade (ICB) shows remarkable clinical effects in patients with metastatic microsatellite-unstable (MSI) cancer. However, markers identifying potential non-responders are missing. We examined the prevalence of Beta-2-microglobulin (B2M) mutations, a common immune evasion mechanism, in stage IV MSI gastrointestinal cancer and its influence on metastatic pattern and patients’ survival under ICB. Twenty-five patients with metastatic, MSI gastrointestinal adenocarcinoma were included. Eighteen patients received ICB with pembrolizumab and one patient with nivolumab/ipilimumab. Sequencing was performed to determine B2M mutation status. B2M mutations and loss of B2M expression were detected in 6 out of 25 stage IV MSI cancers. B2M mutations were strongly associated with exclusively peritoneal/peritoneal and lymph node metastases (p=0.0055). However, no significant differences in therapy response (25% vs. 46.6%, p>0.99) and survival (median PFS: 19.5 vs 33.0 months, p=0.74; median OS 39 months vs. not reached, p>0.99) were observed between B2M-mutant and B2M-wild type tumor patients. Among metastatic MSI GI cancers, B2M-mutant tumors represent a biologically distinct disease with distinct metastatic patterns. To assess ICB response in B2M-mutant MSI cancer patients, future studies need to account for the fact that baseline survival of patients with B2M-mutant MSI cancer may be longer than of patients with B2M-wild type MSI cancer.

Impact of beta-2 microglobulin expression on the survival of glioma patients via modulating the tumor immune microenvironment.

AimsHigh immune cell infiltration in gliomas establishes an immunosuppressive tumor microenvironment, which in turn promotes resistance to immunotherapy. Hence, it is important to identify novel targets associated with high immune cell infiltration in gliomas. Our previous study showed that serum levels of beta‐2 microglobulin (B2M) in lower‐grade glioma patients were lower than those in glioblastoma patients. In the present study, we focused on exploring the roles of B2M in glioma immune infiltration.MethodsA large cohort of patients with gliomas from the TCGA, CGGA, and Gravendeel databases was included to explore differential expression patterns and potential roles of B2M in gliomas. A total of 103 glioma tissue samples were collected to determine the distributions of B2M protein levels by immunofluorescent assays. Kaplan‐Meier survival analysis and meta‐analysis were used for survival analysis. GO(Gene‐ontology) enrichment analysis, co‐expression analysis, KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway analysis, and immune infiltration analysis were performed to explore roles and related mechanisms of B2M in glioma.ResultsWe found that both B2M mRNA and protein levels were abnormally upregulated in glioma samples compared with those from normal brain tissue. B2M expression was correlated with tumor grade and was downregulated in IDH1 mutant samples. Furthermore, B2M was a moderately sensitive indicator for predicting the mesenchymal molecular subtype of gliomas. Interestingly, glioma patients with lower B2M expression had remarkably longer survival times than those with higher B2M expression. Moreover, meta‐analysis showed that B2M was an independent predictive marker in glioma patients. The results of GO enrichment analysis revealed that B2M contributed to immune cell infiltration in glioma patients. In addition, results of KEGG pathway analysis and co‐expression analysis suggested that B2M may mediate glioma immune infiltration via chemokines.ConclusionsWe conclude that B2M levels are critical for the survival times of glioma patients, at least in part due to mediating high immune infiltration.

Forecast and clinical-diagnostic significance of increased expression of beta-2microglobulin in the urine in the process of nephropathy formation in essential hypertension

Introduction. At present, it is obvious that the prognosis for the life of patients with essential hypertension, according to the cardiovascular continuum, is clearly determined primarily by the involvement in the pathological process of target organs and the severity of their structural and functional changes.The aim of the study is to identify the clinical-diagnostic and prognostic value of B2-MGU as a marker of kidney damage in essential hypertension.Materials and methods. To achieve this goal, 46 patients with essential hypertension (45.65% women and 54.35% men) aged from 28 to 71 years (mean age was 51.88 ± 2.17 years), I-II stage of the disease with level of hypertension I-III degree, varying degrees of risk, who did not receive systematic adequate antihypertensive therapy and in which as a result of comprehensive clinical, laboratory and instrumental studies there were no evidence of clinically significant chronic kidney disease. The control group consisted of 17 healthy individuals.Results. In patients with essential hypertension is forming hypertensive nephropathy, which is characterized by statistically significant excessive excretion of beta-2-microglobulin in the urine compared with almost healthy individuals. In patients with increased excretion of beta-2-microglobulin violations of intrarenal hemodynamics were registered significantly more often, violations of functional renal reserve, violations of vascular resistance of renal vessels, in a significantly higher percentage there were pathological changes in circadian rhythm of blood pressure and renal filtration function, the prevalence of concentric and eccentric hypertrophy, proteinuria, which indicates an important clinical, diagnostic and prognostic value of hyper-beta-2-microglobulinuria.Conclusions. The indicator beta-2-microglobulinuria can be used as an adequate marker of hypertensive nephropathy, as having high sensitivity, specificity, positive and negative prognosis.

Quantification of Neonatal Fc Receptor and Beta-2 Microglobulin in Human Liver Tissues by Ultraperformance Liquid Chromatography-Multiple Reaction Monitoring-based Targeted Quantitative Proteomics for Applications in Biotherapeutic Physiologically-based Pharmacokinetic Models.

Neonatal Fc receptor (FcRn) and beta-2 microglobulin (β2M) play an important role in transporting maternal IgG to fetuses, maintaining the homeostasis of IgG and albumin in human body, and prolonging the half-life of IgG- or albumin-based biotherapeutics. Little is known about the influence of age, gender and race, and interindividual variability of human FcRn and β2M on the protein level. In this study, an ultraperformance liquid chromatography-multiple reaction monitoring mass spectrometry-based targeted quantitative proteomic method was developed and optimized for the quantification of human FcRn and β2M. Among the 39 human livers studied (age 13-80 years), the mean (±S.D.) concentrations of FcRn and β2M were 147 (±39) and 1250 (±460) pmol/g of liver tissue, respectively. A four-fold interindividual variability (63-243 pmol/g of liver tissue) was observed for the hepatic FcRn concentration. A moderate correlation was found between the hepatic β2M and FcRn expression levels. Influences of age, gender, and race on the hepatic expression of FcRn and β2M were evaluated. The findings from this study may aid the development of physiologically-based pharmacokinetic models that incorporate empirical FcRn tissue concentrations and interindividual variabilities, and the development of personalized dosing of biopharmaceuticals. SIGNIFICANCE STATEMENT: This is the first study to evaluate the influence of age, gender, and race on the expression of neonatal Fc receptor (FcRn) and beta-2 microglobulin (β2M) and their interindividual variability in human livers. This study describes a validated ultraperformance liquid chromatography-multiple reaction monitoring-based targeted quantitative proteomic method for quantifying human FcRn and β2M in biological tissues. Results from this study may aid current development of physiologically-based pharmacokinetic models for biotherapeutics, where FcRn plays a significant role in clearance mechanism, and its expression level and interindividual variability are largely unknown.

Abundant CD8+ tumor infiltrating lymphocytes and beta-2-microglobulin are associated with better outcome and response to interleukin-2 therapy in advanced stage clear cell renal cell carcinoma

Studies assessing tumor-infiltrating lymphocytes (TILs) in clear cell renal cell carcinoma (ccRCC) and clinical outcomes have mixed results. Given fundamental interaction of MHC class I with CD8+ T-cells, we hypothesized that expression of MHC class I associated protein, beta-2-microglobulin (B2M), may be an important immunologic marker in RCC. We sought to understand potential implications of CD8 + TILs and tumor B2M expression on overall survival and response to high-dose interleukin-2 (IL-2) therapy, in a cohort of patients with high-stage (clinical stage III and IV) ccRCC. Four tumor regions from 56 patients with ccRCC were retrospectively assessed immunohistochemically. At a median follow-up time of 33 months, 22 (39%) patients had died of disease, 23 (41%) were alive disease, and 11 (20%) had no evidence of disease. Tumors with high CD8 + TILs had a significantly lower death rate [hazard ratio (HR): 0.33, p = 0.02]. CD8 + TILs correlated with B2M expression (p = 0.007). On multivariable analyses, patients with both high B2M and CD8 + TILs had lower death rate (HR: 0.27, p = 0.03). Within the subgroup treated with IL-2 (n = 27, 48%), tumors with high CD8 + TILs were more likely to respond to IL-2 therapy [coefficient (coef): 1.6, p = 0.05]. On multivariable analyses, tumors with a combination of both high B2M expression and high CD8 + TILs also showed trend to responding to IL-2 therapy (coef: 2.5, p = 0.06). In conclusion, abundant CD8+ TILs and high tumor expression of beta-2-microglobulin were good prognostic indicators associated with longer survival in patients with high-stage ccRCC. Abundant CD8+ TILs may predict response to IL-2 therapy.

B2M Gene Expression Reflects an Immunologically Active Tumor Microenvironment in DLBCL

Introduction Interactions between tumor cells and the immune system play a critical role in regulating tumor development. Immune-based therapies have shown variable responses in diffuse large B-cell lymphoma (DLBCL) which suggests that the immune cell composition of the tumor microenvironment (TME) influences response to these agents. However, the key determinants of the immune TME are poorly understood. We have recently shown that indolent lymphomas can be stratified as immunologically 'hot' or 'cold' (Tobin et al J. Clin Oncol, in press) and sought to test for this in DLBCL. Also, given that beta-2-microglobulin (B2M) has a key role in antigen presentation and its loss is a frequent immune evasion mechanism in DLBCL, we determined the effect of B2M expression on the nature and magnitude of immune infiltration in the tumor microenvironment of DLBCL. Methods Ninety-seven de novo systemic DLBCL FFPE biopsies underwent targeted exon re-sequencing of B2M (Illumina) in addition to quantitative gene expression of Β2M, immune effector (CD4, CD8, CD56, CD137), immunosuppressive macrophage markers (CD68, CD163) and immune checkpoints (TIM3, LAG3, PD1 and PDL1) (Nanostring Technologies). B2M promoter methylation by mass array, immunohistochemistry for the above markers and high throughput T-cell receptor β sequencing (Adaptive Biotechnologies) were performed on a subset of cases. Genomic findings were validated in a whole exome and transcriptome cohort of approximately 1000 DLBCL samples (Reddy et al Cell 2017). Results Β2MMut were detected in 14/97 (14.4%) samples and had lower Β2M gene expression compared to Β2MWT (p = 0.0094) and all of these showed B2M protein loss. However, 29/40 (72%) of B2MWT samples tested also had B2M protein loss. There was no differential methylation of B2M promoter regions observed compared to lymph node controls. Results indicate that mechanisms other than mutation and methylation status contribute to loss of B2M surface expression and that a more comprehensive assessment of B2M expression within tumor tissues is achieved by B2M digital gene quantification. Consistent with this, there were no significant differences in expression of intra-tumoral immune markers between B2MMut and B2MWT tissues, whereas gene expression of B2M was significantly associated and positively correlated with the gene expression of CD4, CD8, CD56, CD137, CD68, CD163, PD1, PDL1, LAG3 and TIM3 (all p <0.01) and with the protein expression of CD8, CD56, CD137, PDL1 and LAG3 (all p <0.01), irrespective of their classification as an immune-effector, immune-checkpoint or macrophage markers. These observations are consistent with a co-ordinately regulated immune response, indicating an adaptive immune-checkpoint response to regulate immune-effector activation. In keeping with this, the housekeeper genes did not correlate with immune gene expression indicating that high or low co-ordinate expression of immune genes was not reflecting tissue RNA quality or quantity (Fig 1). Next, we tested the discovery cohort for relationships with the TCR repertoire. High B2M gene expression was significantly associated with reduced TCR diversity (p = 0.0101) compared to low B2M gene expression, suggesting that clonal T cell expansions are more likely with intact antigen presentation. The validation cohort also demonstrated that B2M gene expression correlated with immune cell infiltration and additionally showed that B2M positively correlated with the gene expression of HLA Class I//II molecules and a range of regulatory, transport and assembly molecules involved in the antigen presentation machinery pathway. However, no differential survival benefit was observed in patients with high versus low B2M. Conclusions In summary, digital gene expression is a robust measure of B2M quantification in the TME. Our data show that high B2M gene expression reflects an immunologically active or 'hot' tumor microenvironment in DLBCL characterised by higher levels of immune cell infiltration. These findings indicate that B2M gene expression level could be used as a biomarker of an active intra-tumoral immune response in DLBCL. Further studies are required to determine if B2M gene expression may have a role in stratifying the selection of patients in whom immune-based therapies are more likely to be effective. Disclosures Gould: NovoNordisk: Other: Travel funding - domestic flights to attend education, May 2018. Keane:MSD: Consultancy; BMS: Research Funding; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy, Other: Travel Grant. Hertzberg:Takeda: Consultancy, Honoraria; MSD: Consultancy; Roche: Consultancy, Honoraria. Gandhi:Gilead: Honoraria, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

A strategy to protect off-the-shelf cell therapy products using virus-specific T-cells engineered to eliminate alloreactive T-cells

BackgroundThe use of “off-the-shelf” cellular therapy products derived from healthy donors addresses many of the challenges associated with customized cell products. However, the potential of allogeneic cell products to produce graft-versus-host disease (GVHD), and their likely rejection by host alloreactive T-cells are major barriers to their clinical safety and efficacy. We have developed a molecule that when expressed in T-cells, can eliminate alloreactive T-cells and hence can be used to protect cell therapy products from allospecific rejection. Further, expression of this molecule in virus-specific T-cells (VSTs) should virtually eliminate the potential for recipients to develop GVHD.MethodsTo generate a molecule that can mediate killing of cognate alloreactive T-cells, we fused beta-2 microglobulin (B2M), a universal component of all human leukocyte antigen (HLA) class I molecules, to the cytolytic endodomain of the T cell receptor ζ chain, to create a chimeric HLA accessory receptor (CHAR). To determine if CHAR-modified human VSTs could eliminate alloreactive T-cells, we co-cultured them with allogeneic peripheral blood mononuclear cells (PBMC), and assessed proliferation of PBMC-derived alloreactive T-cells and the survival of CHAR-modified VSTs by flow cytometry.ResultsThe CHAR was able to transport HLA molecules to the cell surface of Daudi cells, that lack HLA class I expression due to defective B2M expression, illustrating its ability to complex with human HLA class I molecules. Furthermore, VSTs expressing CHAR were protected from allospecific elimination in co-cultures with allogeneic PBMCs compared to unmodified VSTs, and mediated killing of alloreactive T-cells. Unexpectedly, CHAR-modified VSTs eliminated not only alloreactive HLA class I restricted CD8 T-cells, but also alloreactive CD4 T-cells. This beneficial effect resulted from non-specific elimination of activated T-cells. Of note, we confirmed that CHAR-modified VSTs did not affect pathogen-specific T-cells which are essential for protective immunity.ConclusionsHuman T-cells can be genetically modified to eliminate alloreactive T-cells, providing a unique strategy to protect off-the-shelf cell therapy products. Allogeneic cell therapies have already proved effective in treating viral infections in the stem cell transplant setting, and have potential in other fields such as regenerative medicine. A strategy to prevent allograft rejection would greatly increase their efficacy and commercial viability.

Targeted gene silencing in human embryonic stem cells using cell-penetrating peptide PepFect 14

BackgroundHuman embryonic stem (hES) cells serve as an invaluable tool for research and future medicine, but their transfection often leads to unwanted side effects as the method itself may induce differentiation. On the other hand, RNA interference (RNAi)-based targeted gene silencing is a quick, cost-effective, and easy-to-perform method to address questions regarding the function of genes, especially when hypomorphic knockdowns are needed. Therefore, effective transfection method with minimal side effects is essential for applying RNAi to hES cells. Here, we report a highly promising approach for targeted gene silencing in hES cells with siRNA complexed with cell-penetrating peptide PepFect 14 (PF14). This strategy provides researchers with efficient tool for unraveling the functions of genes or addressing the differentiation of pluripotent stem cells.MethodsWe present a method for delivery of siRNA into hES cells with cell-penetrating peptide PF14. Accordingly, hES cells were transfected in ROCK inhibitor containing medium for 24 h right after EDTA passaging as small cell clumps. Fluorescently labeled siRNA and siRNAs targeting OCT4 or beta-2-microglobulin (B2M) mRNA sequences were used to evaluate the efficiency of transfection and silencing. Analyses were performed at various time points by flow cytometry, RT-qPCR, and immunofluorescence microscopy.ResultsEffective downregulation of OCT4 in 70% of treated hES cells at protein level was achieved, along with 90% reduction at mRNA level in bulk population of cells. The applicability of this low-cost and easy-to-perform method was confirmed by inducing silencing of another target not associated with hES cell pluripotency (B2M). Furthermore, we discovered that downregulation of OCT4 induces neuroectodermal differentiation accompanied by reduced expression of B2M during early stage of this lineage.ConclusionsThe results demonstrate PF14 as a promising tool for studying gene function and regulatory networks in hES cells by using RNAi.

Expression and clinical significance of antigen presentation components beta-2 microglobulin, HLA class I heavy chains, and HLA class II in non-small cell lung cancer (NSCLC).

12015Background: Defective antigen presenting machinery (APM) limits tumor recognition by cognate T cells mediating resistance to immune checkpoint inhibitors (ICIs). The expression frequency, biol...

Recovery HLA-A2 and Beta2-microglobulin Expression in Tumor Cells Using Viral Vectors

Background: Tumor elimination and the success of cancer immunotherapy depend on the proper expression of HLA class I complex (HLA-I) required for the presentation of tumor-associated peptides to cytotoxic T-lymphocytes. Tumors escape immune attack by losing HLA-I expression, often due to irreversible genetic/chromosomal alterations, including mutations in beta2-microglobulin (B2M) or lack of HLA-A2 allele due to a haplotype loss. The introduction of these genes and re-expression of the missing HLA-I specificity on the tumor cell surface is an attractive strategy to induce tumor rejection by T-lymphocytes. Methods: Using genomic HLA-I typing and gene sequencing we determined HLA-I phenotypes and alterations in different human tumor cell lines previously characterized in our laboratory. We used adeno- and adeno-associated viruses to reconstitute/up-regulate HLA-A2 or/and B2M expression in these cells in vitro. Using flow cytometry and immunocytochemistry we evaluated levels and patterns of HLA-I expression in these cells. Methods: Using genomic HLA-I typing and gene sequencing we determined HLA-I phenotypes and alterations in different human tumor cell lines previously characterized in our laboratory. We used adeno- and adeno-associated viruses to reconstitute/up-regulate HLA-A2 or/and B2M expression in these cells in vitro. Using flow cytometry and immunocytochemistry we evaluated levels and patterns of HLA-I expression in these cells. Conclusion: Using gene therapy, it is possible to recover normal B2M and HLA-A2 gene expression caused by structural “hard” alteration and to induce co-expression of both genes in cells naturally lacking HLA-A2 allele. In addition, we demonstrated that transfected tumor cells ae able to express seven HLA-I alleles. The recovery of the missing HLA-I molecules frequently observed in tumor cells using adeno and adeno-associated viruses can be a useful strategy to circumvent cancer immune escape and increase tumor rejection.

Efficient generation of B2m-null pigs via injection of zygote with TALENs.

Donor major histocompatibility complex class I (MHC I) molecules are the main targets of the host immune response after organ allotransplantation. Whether and how MHC I-deficiency of pig donor tissues affects rejection after xenotransplantation has not been assessed. Beta2-microglobulin (B2M) is indispensable for the assembly of MHC I receptors and therefore provides an effective target to disrupt cell surface MHC I expression. Here, we report the one-step generation of mutant pigs with targeted disruptions in B2m by injection of porcine zygotes with B2m exon 2-specific TALENs. After germline transmission of mutant B2m alleles, we obtained F1 pigs with biallelic B2m frameshift mutations. F1 pigs lacked detectable B2M expression in tissues derived from the three germ layers, and their lymphocytes were devoid of MHC I surface receptors. Skin grafts from B2M deficient pigs exhibited remarkably prolonged survival on xenogeneic wounds compared to tissues of non-mutant littermates. Mutant founder pigs with bi-allelic disruption in B2m and B2M deficient F1 offspring did not display visible abnormalities, suggesting that pigs are tolerant to B2M deficiency. In summary, we show the efficient generation of pigs with germline mutations in B2m, and demonstrate a beneficial effect of donor MHC I-deficiency on xenotransplantation.

Urinary expression of beta-2 microglobulin and osteopontin increases in salt-induced hypertensive rats

Urinary expression of beta-2 microglobulin and osteopontin increases in salt-induced hypertensive rats

Human epithelial-type ovarian tumour marker beta-2-microglobulin is regulated by the TGF-β signaling pathway.

BackgroundBeta-2-microglobulin (B2M), a light chain subunit of the major histocompatibility complex (MHC) class I complex, has been implicated in tumorigenesis. However, whether it is expressed in different epithelial-type ovarian tumours remains unknown. This study was performed to examine the expression of B2M in different histopathological types of ovarian tumours, to explore the function of B2M in ovarian cancer (OC) cells and to investigate the mechanisms underlying the regulation of B2M by the TGF-β signaling pathway.MethodsB2M expression in normal ovarian tissues and epithelia-type ovarian tumours was detected by immunohistochemistry and Western blot, followed by the analysis of association with clinical features. OC cells were transfected with B2M-siRNA and cell proliferation, migration and invasion were determined by WST-1 assay, wound healing assay and Transwell invasion assay, respectively. The regulation of B2M by the TGF-β signaling pathway in OC cells was examined by Western blot, ELISA and qRT-PCR.ResultsWe found that B2M was overexpressed in ovarian borderline and malignant tumours compared with benign tumours and normal controls, but was not associated with age, tumour size, lymph node metastasis and clinical stage. Knocking down of B2M led to a decrease in OC cell proliferation, migration and invasion. The expression of B2M was downregulated by TGF-β1 in OC cells, which was abolished in the presence of the inhibitor of TGF-β type I receptor.ConclusionOur findings suggest that B2M is a potential tissue biomarker and therapeutic target of borderline and malignant ovarian tumours and the dysregulation of B2M in these tumours may be mediated by the TGF-β signaling pathway.

Beta-2-microglobulin (B2M) expression in the urinary sediment correlates with clinical markers of kidney disease in patients with type 1 diabetes

PurposeAfter observing variation in the expression of the housekeeping gene B2M in cells of the urinary sediment during a study of candidate genes potentially involved in diabetic kidney disease (DKD), we hypothesized that B2M mRNA expression in the urinary sediment could reflect the presence of DKD. MethodsqPCR was used to quantify B2M mRNA expression in cells of the urinary sediment of 51 type 1 diabetes (T1D) patients (61% women, 33.5 [27.0–39.7] years old, with diabetes duration of 21.0 [15.0–28.0] years and HbA1c of 8.2% [7.3–8.9]; median [interquartile interval]) sorted according to the diabetic nephropathy (DN) stages; 8 focal segmental glomerulosclerosis (FSGS) patients and 10 healthy controls. B2M mRNA expression was also evaluated in human embryonic kidney epithelium-like (HEK-293) cells exposed to 25mM glucose and to albumin in order to mimic, respectively, a diabetic and a proteinuric milieu. ResultsNo differences were found in B2M mRNA expression among healthy controls, FSGS and T1D patients. Nonetheless B2M mRNA expression was higher in the group composed by T1D patients with incipient or overt DN combined with FSGS patients versus T1D patients without DN combined with healthy controls (P=0.0007). B2M mRNA expression was higher in T1D patients with incipient or overt DN versus without DN (P=0.03). B2M mRNA expression positively correlated with albuminuria in the overall T1D population (r=0.43; P=0.01) and negatively correlated with estimated glomerular filtration rate in male T1D patients (r=− 0.57; P=0.01). Increased B2M expression was observed in HEK-293 cells exposed to 25mM glucose and to albumin. ConclusionsΒ2M mRNA expression in cells of the urinary sediment is higher in T1D patients with DKD and in patients with FSGS in comparison to healthy subjects, maybe reflecting a tubulointerstitial injury promoted by albumin. Given the proinflammatory nature of B2M, we suggest that this protein contributes to diabetic (and possibly, to non-diabetic) tubulopathy.