Expression analysis of long non-coding RNAs and their target genes in multiple sclerosis patients.

Multiple sclerosis (MS) is a progressive chronic autoimmune-mediated disease. Recently, long non-coding RNAs (lncRNAs) are characterized to participate in the adjustment of immune responses. Here, we evaluated the expression levels of GSTT1-AS1 and IFNG-AS1 lncRNAs and their targets (TNF and IFNG, respectively) in Iranian MS patients.In this case-control study, 50 relapsing-remitting MS patients and 50 healthy subjects were recruited. Expressions of GSTT1-AS1 and IFNG-AS1 lncRNAs, as well as TNF and IFNG genes, were assessed in their peripheral blood samples by SYBR Green-based Real-time quantitative PCR.Expression levels of GSTT1-AS1 and IFNG-AS1 lncRNAs were both significantly downregulated (p values 0.032 and 0.013, respectively). On the other hand, the expression of TNF and IFNG showed increased levels, however, did not reach statistical significance after our analysis (p > 0.05). Spearman correlation analysis showed that GSTT1-AS1 had a significant positive moderate correlation with IFNG-AS1 (r = 0.541, p < 0.0001), IFNG (r = 0.329, p = 0.001), and TNF (r = 0.204, p = 0.041). Also, IFNG-AS1 revealed the same correlation with IFNG (r = 0.475, p < 0.0001) as well as TNF (r = 0.399, p < 0.0001). Furthermore, GSTT1-AS1 (r = 0.313, p = 0.027) and (IFNG r = 0.478, p < 0.0001) demonstrated a significant positive correlation with age at onset.Briefly, the current study provided for the first time dysregulation of GSTT1-AS1 and IFNG-AS lncRNAs network in MS, which highlights the significant role of epigenetic pathways in this autoimmune disorder. Larger sample size and further investigation assays could shed light on the underlying mechanisms in this area of science.

Preclinical data support leveraging CS1 chimeric antigen receptor T-cell therapy for systemic light chain amyloidosis.

BACKGROUND AIMS: Light chain amyloidosis (AL) is a protein deposition disorder that is a result of a plasma cell dyscrasia, similar to multiple myeloma (MM). Immunotherapy is an attractive approach because of the low burden of disease, but the optimal target for AL is unclear. CS1 and B-cell maturation antigen (BCMA) are two potential targets because they are expressed on normal plasma cells and MM cells. METHODS: We performed a prospective study evaluating bone marrow specimens of 20 patients with plasma cell diseases, 10 with AL and 10 with MM. We evaluated the clonal population of plasma cells for BCMA and CS1 expression. We designed a second-generation CS1 chimeric antigen receptor (CAR) construct, comprising a CS1 antigen-specific scFv, shortened hinge region and CD28 costimulatory domain. Purified central memory T cells were activated and transduced with a lentiviral vector encoding the CS1 CAR. Cytotoxicity was evaluated using 51Cr release assays. Five days after tumor inoculation, NSG mice were injected intravenously with CS1 CAR T cells. RESULTS: Whereas CS1 is present on the plasma cells of AL patients, we found BCMA expression in AL to be markedly low. CS1 CAR T cells were cytotoxic against CS1 positive tumor cells and induced durable tumor regressions in mice. DISCUSSION: Our work represents a novel application of CS1-directed CAR T cells while revealing that BCMA would not be a suitable target. We expect AL to be particularly susceptible to CAR T-cell therapy because of the low tumor burden in the bone marrow.

CD319 (SLAMF7) an alternative marker for detecting plasma cells in the presence of daratumumab or elotuzumab.

BACKGROUND: Daratumumab is an anti-CD38 immunotherapeutic drug that has increasingly been used to treat patients with heavily pre-treated and relapsed/refractory multiple myeloma. In so doing, the detection of CD38 antigen on plasma cells by flow cytometry is impeded. We hypothesized that alternative markers can be used in place or in addition to CD38 when detecting plasma cells post-treated with daratumumab. METHODS: A total of 16 alternative markers were tested using 22 bone marrow aspirates from patients with plasma cell neoplasm. The ability of selected markers to discern plasma cells from other hematopoietic cells were evaluated. The stability of tested markers when stored at 4 or 25°C after T = 0, 24, 48, and 72 h was also established. Finally, selected markers were incorporated into a panel used for monitoring multiple myeloma measurable residual disease to test their utility to identify plasma cells in the presence of daratumumab and/or elotuzumab (anti-CD319) drugs. RESULTS: Out of the 16 tested markers, CD319, CD54, CD229, CD317, and p63 were expressed by >90% of the plasma cells. Only CD319, CD54, and CD229 achieved 100% detection sensitivity. Further analysis showed that CD319 was better than CD229 and CD54 at resolving plasma cells from background hematopoietic cells, with CD54 being the worst (resolution metric, mean ± SD: CD319 [2.04 ± 0.86]; CD229 [1.47 ± 0.45]; and CD54 [1.22 ± 0.60]). CD229 was expressed by >90% of T lymphocytes, whereas CD319 was expressed preferentially by the CD8+ T cells and less frequently in CD4+ T cells. Additionally, CD229 was found on >60% of B and NK cells, as well as minor subsets of monocytes and granulocytes. CD319 was expressed on most NK cells and a minor subset of B cells, granulocytes, and monocytes. Even though CD229 and CD319 were expressed by different leukocyte subsets, their expression levels were highest on plasma cells. The expression of CD138 on plasma cells was significantly lower after storage at 4°C, while the expression levels of CD38, CD229, and CD319 remained stable at 4 or 25°C. Using limiting dilution experiments, the treatment of cells with daratumumab severely impeded the detection of CD38 antigen on plasma cells, whereas elotuzumab treatment did not block detection of CD319 on plasma cells. CONCLUSIONS: CD319 is a suitable alternative to CD38 for identifying plasma cells. Our results showed that a panel used for monitoring multiple myeloma measurable residual disease could be modified by using CD319 alone or in combination with CD38 to detect PCs in daratumumab or elotuzumab treated patients.

Immunological exhaustion and functional profile of CD8+ T lymphocytes as cellular biomarkers of therapeutic efficacy in chronic Chagas disease patients.

The lack of useful tools for detection the impact of treatment during the follow-up of chronic Chagas disease treated patients difficult the adequate care to the affected population. The objective of this study was to evaluate the functional response of CD8+ T lymphocyte population, critical for the control of Trypanosoma cruzi infection, as a possible cellular biomarker of treated Chagas disease patients. Thus, we analyzed the antigen-specific CD8+ T-cell response before and after benznidazole treatment in asymptomatic (indeterminate) and cardiac chronic Chagas disease patients. A marked dysfunctional process of the CD8+ T cell population was found in patients with an advanced pathology. Thus, the cardiac patients have a higher co-expression of inhibitory receptors and a lower antigen-specific multifunctional capacity compared with that of asymptomatic patients. Remarkably, benznidazole treatment partially reverses this functional exhaustion process of CD8+ T cells in both asymptomatic and cardiac Chagas disease patients. Thus, the co-expression of inhibitory molecules tends to be reduced after benznidazole treatment, mainly in asymptomatic patients, finding a significant drop in the expression of inhibitory receptors such as PD-1 and 2B4. In addition, the multifunctional antigen-specific response of CD8+ T cells is enhanced after treatment in chronic patients. An increase in the subset of cells with cytotoxic capacity and production of the IFN-γ cytokine was also observed in both treated asymptomatic and cardiac chronic Chagas disease patients. The results derived from this study show the improvement of the functional capacity of CD8+ T cells after treatment which could be have a positive effect on parasitic control. In addition, the phenotypic and functional profile of the CD8+ T cells described could serve as a tool for monitoring the impact of benznidazole treatment.

Proteomic markers with prognostic impact on outcome of chronic lymphocytic leukemia patients under chemo-immunotherapy: results from the HOVON 109 study.

Despite recent identification of several prognostic markers, there is still a need for new prognostic parameters able to predict clinical outcome in chronic lymphocytic leukemia (CLL) patients. Here, we aimed to validate the prognostic ability of known (proteomic) markers measured pretreatment and to search for new proteomic markers that might be related to treatment response in CLL. To this end, baseline serum samples of 51 CLL patients treated with chemo-immunotherapy were analyzed for 360 proteomic markers, using Olink technology. Median event-free survival (EFS) was 23 months (range: 1.25-60.9). Patients with high levels of sCD23 (>11.27, p = 0.026), sCD27 (>11.03, p = 0.04), SPINT1 (>1.6, p = 0.001), and LY9 (>8.22, p = 0.0003) had a shorter EFS than those with marker levels below the median. The effect of sCD23 on EFS differed between immunoglobulin heavy chain variable gene-mutated and unmutated patients, with the shortest EFS for unmutated CLL patients with sCD23 levels above the median. Taken together, our results validate the prognostic impact of sCD23 and highlight SPINT1 and LY9 as possible promising markers for treatment response in CLL patients.

Development and Validation of Electrochemiluminescence Assays to Measure Free and Total sSLAMF7 in Human Serum in the Absence and Presence of Elotuzumab.

Elotuzumab is a first in class humanized IgG1 monoclonal antibody for the treatment of multiple myeloma (MM). Elotuzumab targets the glycoprotein signaling lymphocyte activation molecule family 7 (SLAMF7, also described as CS1 or CRACC) which is expressed on the surface of myeloma cells and a subset of immune cells, including natural killer cells. A soluble version of SLAMF7 (sSLAMF7) has also been reported in MM patients but has not been evaluated as a potential biomarker following therapeutic intervention. In order to measure serum levels of sSLAMF7, two immunoassays were developed to monitor changes in circulating sSLAMF7 before and after elotuzumab treatment. Free (drug-unbound) and total (drug-bound and unbound) electrochemiluminescence (ECL) ELISA assays were developed and validated following a fit for purpose (FFP) methodology. Both assays met analytical acceptance criteria for precision, drug interference, dilution linearity, spike recovery, parallelism, and stability. Both exhibited the range and sensitivity necessary to measure clinical samples with an LLOQ of 51.2 pg/mL and ULOQs of 160 (free) and 800 ng/mL (total). Previously described assays were unable to detect sSLAMF7 in healthy individuals. However, due to the increased sensitivity of these new assays, low but measurable sSLAMF7 levels were detected in all normal healthy sera evaluated and were significantly elevated in MM patients. Cohort statistics revealed a significant increase of circulating sSLAMF7 in MM patients versus normal controls and both significant decreases in free and increases in total levels of protein post-elotuzumab treatment.