68 research outputs found
Evaluating STAT5 Phosphorylation as a Mean to Assess T Cell Proliferation
Here we present a simple and sensitive flow cytometricâbased assay to assess T cell proliferation. Given the critical role STAT5A phosphorylation in T cell proliferation, we decided to evaluate phosphorylation of STAT5A as an indicator of T cell proliferation. We determined pSTAT5A in T cell treated with either CD3/CD28 or PHA. After stimulation, T cells from adult healthy donors displayed a strong long-lasting phosphorylation of STAT5A, reaching a peak value after 24 h. The median fluorescence intensity (MFI) of pSTAT5A increased from 112 ± 17 to 512 ± 278 (CD3/CD28) (24 h) and to 413 ± 123 (PHA) (24 h), the IL-2 receptor-α (CD25) expression was greatly enhanced and after 72 h T cell proliferation amounted to 52.3 ± 10.3% (CD3/CD28) and to 48.4 ± 9.7% (PHA). Treatment with specific JAK3 and STAT5 inhibitors resulted in a complete blockage of phosphorylation of STAT5A, CD25 expression, and suppression of T cell proliferation. Compared with currently available methods, STAT5A phosphorylation is well-suited to predict T cell proliferation. Moreover, the method presented here is not very time consuming (several hours) and delivers functional information from which conclusions about T cell proliferation can be drawn
Taking Lessons from CAR-T Cells and Going Beyond: Tailoring Design and Signaling for CAR-NK Cells in Cancer Therapy
Cancer immunotherapies utilize the capabilities of the immune system to efficiently target
malignant cells. In recent years, chimeric antigen receptor (CAR) equipped T cells showed
promising results against B cell lymphomas. Autologous CAR-T cells require patientspecific
manufacturing and thus extensive production facilities, resulting in high priced
therapies. Along with potentially severe side effects, these are the major drawbacks of
CAR-T cells therapies. Natural Killer (NK) cells pose an alternative for CAR equipped
immune cells. Since NK cells can be safely transferred from healthy donors to cancer
patients, they present a suitable platform for an allogeneic âoff-the-shelfâ immunotherapy.
However, administration of activated NK cells in cancer therapy has until now shown poor
anti-cancer responses, especially in solid tumors. Genetic modifications such as CARs
promise to enhance recognition of tumor cells, thereby increasing anti-tumor effects and
improving clinical efficacy. Although the cell biology of T and NK cells deviates in many
aspects, the development of CAR-NK cells frequently follows within the footsteps of CART
cells, meaning that T cell technologies are simply adopted to NK cells. In this review, we
underline the unique properties of NK cells and their potential in CAR therapies. First, we
summarize the characteristics of NK cell biology with a focus on signaling, a fine-tuned
interaction of activating and inhibitory receptors. We then discuss why tailored NK cellspecific
CAR designs promise superior efficacy compared to designs developed for T
cells. We summarize current findings and developments in the CAR-NK landscape:
different CAR formats and modifications to optimize signaling, to target a broader pool of
antigens or to increase in vivo persistence. Finally, we address challenges beyond NK cell
engineering, including expansion and manufacturing, that need to be addressed to pave
the way for CAR-NK therapies from the bench to the clinics
From Cancer to Immune-Mediated Diseases and Tolerance Induction: Lessons Learned From Immune Oncology and Classical Anti-cancer Treatment
Success in cancer treatment over the last four decades has ranged from improvements
in classical drug therapy to immune oncology. Anti-cancer drugs have also often proven
beneficial for the treatment of inflammatory and autoimmune diseases. In this review,
we report on challenging examples that bridge between treatment of cancer and
immune-mediated diseases, addressing mechanisms and experimental models as well
as clinical investigations. Patient-derived tumor xenograft (PDX) (humanized) mouse
models represent useful tools for preclinical evaluation of new therapies and biomarker
identification. However, new developments using human ex vivo approaches modeling
cancer, for example in microfluidic human organs-on-chips, promise to identify key
molecular, cellular and immunological features of human cancer progression in a fully
human setting. Classical drugs which bridge the gap, for instance, include cytotoxic
drugs, proteasome inhibitors, PI3K/mTOR inhibitors and metabolic inhibitors. Biologicals
developed for cancer therapy have also shown efficacy in the treatment of autoimmune
diseases. In immune oncology, redirected chimeric antigen receptor (CAR) T cells have
achieved spectacular remissions in refractory B cell leukemia and lymphoma and are
currently under development for tolerance induction using cell-based therapies such as
CAR Tregs or NK cells. Finally, a brief outline will be given of the lessons learned from
bridging cancer and autoimmune diseases as well as tolerance induction
Flow cytometric measurement of STAT5 phosphorylation in cytomegalovirus-stimulated T cells
Cytomegalovirus (CMV)-specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. Given the critical role STAT5A phosphorylation (pSTAT5A) in T cell proliferation, this study presents a simple and sensitive flow cytometric-based pSTAT5A assay to quickly identify CMV-specific T cell proliferation. We determined pSTAT5A in T cells treated with CMV-specific peptide mix (pp65â+âIE1 peptides) from 20 healthy adult subjects and three immunodeficient patients with CARMIL-2 mutation. After stimulation, the percentage of pSTAT5A+ T cells in CMV-seropositive (CMV+) subjects significantly increased from 3.0%â±â1.9% (unstimulated) to 11.4%â±â5.9% (stimulated) for 24âh. After 7âdays of stimulation, the percentage of expanded T cells amounted to 26%â±â17.2%. Conversely, the percentage of pSTAT5A+ T cells and T cell proliferation from CMV-seronegative (CMVâ) subjects hardly changed (from 3.0%â±â1.3% to 3.7%â±â1.8% and from 4.3%â±â2.1% to 5.7%â±â1.7%, respectively). We analyzed the correlation between the percentage of pSTAT5A+ T cells versus (1) CMV-IgG concentrations versus (2) the percentage of expanded T cells and versus (3) the percentage of initial CMV-specific T cells. In immunodeficient patients with CARMIL-2 mutation, CMV-specific pSTAT5A and T cell proliferation were completely deficient. In conclusion, flow cytometric-based pSTAT5A assay represents an appropriate tool to quickly identify CMV-specific T cell proliferation and helps to understand dysfunctions in controlling other pathogens. Flow cytometric-based pSTAT5A assay may be a useful test in clinical practice and merits further validation in large studies
Sequential anti-cytomegalovirus response monitoring may allow prediction of cytomegalovirus reactivation after allogeneic stem cell transplantation
Background: Reconstitution of cytomegalovirus-specific CD3+CD8+ T cells (CMV-CTLs) after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary to bring cytomegalovirus (CMV) reactivation under control. However, the parameters determining protective CMV-CTL reconstitution remain unclear to date.
Design and Methods: In a prospective tri-center study, CMV-CTL reconstitution was analyzed in the peripheral blood from 278 patients during the year following HSCT using 7 commercially available tetrameric HLA-CMV epitope complexes. All patients included could be monitored with at least CMV-specific tetramer.
Results: CMV-CTL reconstitution was detected in 198 patients (71%) after allogeneic HSCT. Most importantly, reconstitution with 1 CMV-CTL per ”l blood between day +50 and day +75 post-HSCT discriminated between patients with and without CMV reactivation in the R+/D+ patient group, independent of the CMV-epitope recognized. In addition, CMV-CTLs expanded more daramtaically in patients experiencing only one CMV-reactivation than those without or those with multiple CMV reactivations. Monitoring using at least 2 tetramers was possible in 63% (n = 176) of the patients. The combinations of particular HLA molecules influenced the numbers of CMV-CTLs detected. The highest CMV-CTL count obtained for an individual tetramer also changed over time in 11% of these patients (n = 19) resulting in higher levels of HLA-B*0801 (IE-1) recognizing CMV-CTLs in 14 patients.
Conclusions: Our results indicate that 1 CMV-CTL per ”l blood between day +50 to +75 marks the beginning of an immune response against CMV in the R+/D+ group. Detection of CMV-CTL expansion thereafter indicates successful resolution of the CMV reactivation. Thus, sequential monitoring of CMV-CTL reconstitution can be used to predict patients at risk for recurrent CMV reactivation
A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes
Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems
Detection of Immune Checkpoint Receptors â A Current Challenge in Clinical Flow Cytometry
Immunological therapy principles are increasingly determining modern medicine. They are
used to treat diseases of the immune system, for tumors, but also for infections,
neurological diseases, and many others. Most of these therapies base on antibodies,
but small molecules, soluble receptors or cells and modified cells are also used. The
development of immune checkpoint inhibitors is amazingly fast. T-cell directed antibody
therapies against PD-1 or CTLA-4 are already firmly established in the clinic. Further
targets are constantly being added and it is becoming increasingly clear that their
expression is not only relevant on T cells. Furthermore, we do not yet have any
experience with the long-term systemic effects of the treatment. Flow cytometry can be
used for diagnosis, monitoring, and detection of side effects. In this review, we focus on
checkpoint molecules as target molecules and functional markers of cells of the innate and
acquired immune system. However, for most of the interesting and potentially relevant
parameters, there are still no test kits suitable for routine use. Here we give an overview of
the detection of checkpoint molecules on immune cells in the peripheral blood and show
examples of a possible design of antibody panels
Mesenchymal stromal cells for treatment of steroid-refractory GvHD : a review of the literature and two pediatric cases
Severe acute graft versus host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation. Human mesenchymal stromal cells (MSCs) play an important role in endogenous tissue repair and possess strong immune-modulatory properties making them a promising tool for the treatment of steroid-refractory GvHD. To date, a few reports exist on the use of MSCs in treatment of GvHD in children indicating that children tend to respond better than adults, albeit with heterogeneous results. We here present a review of the literature and the clinical course of two instructive pediatric patients with acute steroid-refractory GvHD after haploidentical stem cell transplantation, which exemplify the beneficial effects of third-party transplanted MSCs in treatment of acute steroid-refractory GvHD. Moreover, we provide a meta-analysis of clinical studies addressing the outcome of patients with steroid-refractory GvHD and treatment with MSCs in adults and in children (n = 183; 122 adults, 61 children). Our meta-analysis demonstrates that the overall response-rate is high (73.8%) and confirms, for the first time, that children indeed respond better to treatment of GvHD with MSCs than adults (complete response 57.4% vs. 45.1%, respectively). These data emphasize the significance of this therapeutic approach especially in children and indicate that future prospective studies are needed to assess the reasons for the observed differential response-rates in pediatric and adult patients. Additional file 1: MSCs expansion and release criteria.his file contains a detailed description of the MSCs expansion and release criteria for Case A and Case B
Functionality and Cell Senescence of CD4/ CD8-Selected CD20 CAR T Cells Manufactured Using the Automated CliniMACS ProdigyÂź Platform
Clinical studies using autologous CAR T cells have achieved
spectacular remissions in refractory CD19+ B cell leukaemia,
however some of the patient treatments with CAR T cells
failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily
pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes
for CD20 CAR T cells from healthy donor leukapheresis using
the automated CliniMACS ProdigyÂź platform. Starting with
a CD4/CD8-positive selection, a high purity of a median
of 97% T cells with a median 65-fold cell expansion was
achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached
in a median of 23%. CD20 CAR T cells showed a good specific IFN-Îł secretion after cocultivation with CD20+ target
cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase
in telomere length during the manufacturing process, while
telomere length remained consistent in one and decreased
in another process. In conclusion, this shows for the first time
that beside heterogeneity among healthy donors, CAR T cell
products also differ regarding cell senescence, even for cells
manufactured in a standardised automated process
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