PRAME peptide-specific CD8+ T cells represent the predominant response against leukemia-associated antigens in healthy individuals.

Antigen-specific T cells isolated from healthy individuals (HIs) have shown great therapeutic potential upon adoptive transfer for the treatment of viremia in immunosuppressed patients. The lack of comprehensive data on the prevalence and characteristics of leukemia-associated antigen (LAA)-specific T cells in HIs still limits such an approach for tumor therapy. Therefore, we have investigated T-cell responses against prominent candidates comprising Wilms' tumor protein 1 (WT1), preferentially expressed antigen in melanoma (PRAME), Survivin, NY-ESO, and p53 by screening PBMCs from HIs using intracellular IFN-γ staining following provocation with LAA peptide mixes. Here, we found predominantly poly-functional effector/effector memory CCR7- /CD45RA+/- /CD8+ LAA peptide-specific T cells with varying CD95 expression in 34 of 100 tested HIs, whereas CD4+ T cells responses were restricted to 5. Most frequent LAA peptide-specific T cell responses were directed against WT1 and PRAME peptides with a prevalence of 20 and 17%, respectively, showing the highest magnitude (0.16% ± 0.22% (mean ± SD)) for PRAME peptides. Cytotoxicity of PRAME peptide-specific T cells was demonstrated by specific killing of PRAME peptide-pulsed T2 cells. Furthermore, the proliferative capacity of PRAME peptide-specific T cells was confined to HIs responsive toward PRAME peptide challenge corroborating the accuracy of the screening results. In conclusion, we identified PRAME as a promising target antigen for adoptive leukemia therapy.

Clinical grade manufacturing of genetically modified, CAR-expressing NK-92 cells for the treatment of ErbB2-positive malignancies.

BACKGROUND: The NK-92/5.28.z cell line (also referred to as HER2.taNK) represents a stable, lentiviral-transduced clone of ErbB2 (HER2)-specific, second-generation CAR-expressing derivative of clinically applicable NK-92 cells. This study addresses manufacturing-related issues and aimed to develop a GMP-compliant protocol for the generation of NK-92/5.28.z therapeutic doses starting from a well-characterized GMP-compliant master cell bank. MATERIALS AND METHODS: Commercially available GMP-grade culture media and supplements (fresh frozen plasma, platelet lysate) were evaluated for their ability to support expansion of NK-92/5.28.z. Irradiation sensitivity and cytokine release were also investigated. RESULTS: NK-92/5.28.z cells can be grown to clinically applicable cell doses of 5 × 108 cells/L in a 5-day batch culture without loss of viability and potency. X-Vivo 10 containing recombinant transferrin supplemented with 5% FFP and 500 IU/mL IL-2 in VueLife 750-C1 bags showed the best results. Platelet lysate was less suited to support NK-92/5.28.z proliferation. Irradiation with 10 Gy completely abrogated NK-92/5.28.z proliferation and preserved viability and potency for at least 24 h. NK-92/5.28.z showed higher baseline cytokine release compared to NK-92, which was significantly increased upon encountering ErbB2(+) targets [GZMB (twofold), IFN-γ (fourfold), IL-8 (24-fold) and IL-10 (fivefold)]. IL-6 was not released by NK cells, but was observed in some stimulated targets. Irradiation resulted in upregulation of IL-8 and downregulation of sFasL, while other cytokines were not impacted. CONCLUSION: Our concept suggests NK-92/5.28.z maintenance culture from which therapeutic doses up to 5 × 109 cells can be expanded in 10 L within 5 days. This established process is feasible to analyze NK-92/5.28.z in phase I/II trials.

Enumeration of WT1-specific CD8+ T cells for clinical application using an MHC Streptamer based no-wash single-platform flow-cytometric assay.

The advent of novel strategies to generate leukemia-associated-antigen (LAA)-specific T cells for adoptive immunotherapies creates a demand for standardized good laboratory practice (GLP)-compliant enumeration assays to provide a secure clinical environment-whether it is to identify potential donors, define therapeutic doses for transplantation, or monitor clinical success. Here, we introduce a no-wash assay based on single-platform cell enumeration and Streptamer staining to determine the Wilms' tumor antigen 1 (WT1)-specific T cell immunity in clinical samples. We analyzed the performance of the WT1-specific MHC Streptamers in direct comparison to CMV- and EBV-specific MHC Streptamer staining by spiking antigen-specific T cells in PBMCs. The accuracy of the assay was high for all performed experiments with a mean recovery of 94% and a linear regression of 0.988. Differences were apparent regarding the limit of detection/quantification (LOD/LOQ). While results obtained for WT1 yielded an LOD/LOQ of 0.08 ± 0.04% and 0.11 ± 0.06% (1.33 ± 0.32 cells/µl and 1.9 ± 0.14 cells/µl), the overall LOD/LOQ was notably lower and accounted to 0.02 ± 0.02% and 0.05 ± 0.03% (0.60 ± 0.03 cells/µl and 1.27 ± 0.58 cells/µl). Subsequent screening of 22 healthy individuals revealed significantly higher values for WT1 (0.04 ± 0.02% and 1.5 ± 0.9 cells/µl) than for the irrelevant HIV pol (0.016 ± 0.01% and 0.5 ± 0.4 cells/µl). In contrast, no increased frequencies were observed for WT1-specific T cells compared to HIV-specific T cells using a classical wash-protocol. These findings strongly suggest the use of no-wash single-platform assays in combination with MHC Streptamer staining for the detection of low affinity LAA-specific T cells due to its high accuracy and sensitivity. © 2017 International Society for Advancement of Cytometry.

NK-92: an 'off-the-shelf therapeutic' for adoptive natural killer cell-based cancer immunotherapy.

Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-versus-host disease. Efficacy and clinical in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clinical NK cell lots to allow repeated clinical applications remains an additional challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufacturing practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purification methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a number of tumor types tested, which was confirmed in preclinical mouse studies. Subsequent clinical testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a number of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments.

Lowest numbers of primary CD8(+) T cells can reconstitute protective immunity upon adoptive immunotherapy.

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of today's virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentiated, and protected against L.m. infections after prophylactic application. Even progenies derived from a single CD62L(hi) L.m.-specific CD8(+) T cell could be protective against bacterial challenge. In analogy, low-dose transfers of Streptamer-enriched human CMV-specific CD8(+) T cells into allo-HSCT recipients led to strong pathogen-specific T-cell expansion in a compassionate-use setting. In summary, low-dose adoptive T-cell transfer (ACT) could be a promising strategy, particularly for prophylactic treatment of infectious complications after allo-HSCT.

Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.

Gamma irradiation preserves immunosuppressive potential and inhibits clonogenic capacity of human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are promising candidates for the treatment of graft-versus-host and autoimmune diseases. Here, by virtue of their immunosuppressive effects, they are discussed to exhibit inhibitory actions on various immune effector cells, including T lymphocytes that promote the underlying pathology. While it becomes apparent that MSCs exhibit their therapeutic effect in a transient manner, they are usually transplanted from third party donors into heavily immunocompromised patients. However, little is known about potential late complications of persisting third party MSCs in these patients. We therefore analysed the effect of gamma irradiation on the potency and proliferation of MSCs to elucidate an irradiation dose, which would allow inhibition of MSC proliferation while at the same time preserving their immunosuppressive function. Bone marrow-derived MSCs (BM-MSCs) were gamma-irradiated at increasing doses of 5, 10 and 30 Gy and subsequently assessed by colony formation unit (CFU)-assay, Annexin V-staining and in a mixed lymphocyte reaction, to assess colony growth, apoptosis and the immunosuppressive capacity, respectively. Complete loss of proliferative capacity measured by colony formation was observed after irradiation with a dose equal to or greater than 10 Gy. No significant decrease of viable cells was detected, as compared to non-irradiated BM-MSCs. Notably, irradiated BM-MSCs remained highly immunosuppressive in vitro for at least 5 days after irradiation. Gamma irradiation does not impair the immunosuppressive capacity of BM-MSCs in vitro and thus might increase the safety of MSC-based cell products in clinical applications.

Clinical-scale isolation of 'minimally manipulated' cytomegalovirus-specific donor lymphocytes for the treatment of refractory cytomegalovirus disease.

BACKGROUND AIMS: Reactivation of cytomegalovirus (CMV) after hematopoietic stem cell transplantation remains a major cause of morbidity despite improved antiviral drug therapies. Selective restoration of CMV immunity by adoptive transfer of CMV-specific T cells is the only alternative approach that has been shown to be effective and non-toxic. We describe the results of clinical-scale isolations of CMV-specific donor lymphocytes with the use of a major histocompatibility (MHC) class I peptide streptamer-based isolation method that yields minimally manipulated cytotoxic T cells of high purity. METHODS: Enrichment of CMV-specific cytotoxic T lymphocytes (CTLs) was performed by labeling 1 × 10(10) leukocytes from a non-mobilized mononuclear cell (MNC) apheresis with MHC class I streptamers and magnetic beads. Thereafter, positively labeled CMV-specific CTLs were isolated through the use of CliniMACS (magnetic-activated cell sorting), and MHC streptamers were released through the use of d-biotin. The purity of enriched CMV-specific CTLs was determined on the basis of MHC streptamer staining and fluorescence-activated cell sorting. RESULTS: A total of 22 processes were performed with the use of five different MHC class I streptamers. The median frequency of CMV-specific CTLs in the starting apheresis product was 0.41% among CD3+ T cells. The isolation process yielded a total of 7.77 × 10(6) CMV-specific CTLs, with a median purity of 90.2%. Selection reagents were effectively removed from the final cell product; the CMV-specific CTLs displayed excellent viability and cytotoxicity and were stable for at least 72 h at 4°C after MNC collection. CONCLUSIONS: Clinical-scale isolation of "minimally manipulated" CMV-specific donor CTLs through the use of MHC class I streptamers is feasible and yields functional CTLs at clinically relevant dosages.

PD-1 expression on Melan-A-reactive T cells increases during progression to metastatic disease.

Programmed death 1 (PD-1) is known as an important factor for the development of tolerogenicity. This has been proven in chronic viral infections and different tumor models. To address the role of PD-1 and its ligand programmed death ligand 1 (PD-L1) in different stages of malignant melanoma, we investigated peripheral blood and tumor tissues in regard to overall survival (OS) and prognostic relevance. One hundred samples of peripheral blood mononuclear cells from HLA-A2(+) patients with malignant melanoma (Stages I-IV) were analyzed in seven color FACS combined with multimer analyses for the immunodominant epitope of Melan-A (peptide A2/Melan-A(p26-35mod) ). Corresponding formalin-fixed paraffin-embedded tissues of primary tumor and distant organ metastases from 37 cases were analyzed by immunohistochemistry for Melan-A, PD-L1 and PD-1 expression. Compared to the total CD8(+) T cell population, PD-1 expression by A2/Melan-A(+) CD8(+) T cells was over-represented in melanoma stages III and IV (p < 0.001). Although elevation of PD-1(+) Melan-A(+) CD8(+) T cells had no significant influence on OS, a positive correlation was observed between PD-L1 expression on melanoma cells and OS (p = 0.05). Correlation of advanced tumor stage with increased A2/Melan-A-multimer(+) PD-1(+) T cells in the peripheral blood suggest that blocking of PD-1 could have therapeutic potential in advanced stage melanoma.

SARS-CoV-2-specicific humoral immunity in convalescent patients with mild COVID-19 is supported by CD4+ T-cell help and negatively correlated with Alphacoronavirus-specific antibody titer.

This study aimed at investigating the nature of SARS-CoV-2-specific immunity in patients with mild COVID-19 and sought to identify parameters most relevant for the generation of neutralizing antibody responses in convalescent COVID-19 patients. In the majority of the examined patients a cellular as well as humoral immune response directed to SARS-CoV-2 was detected. The finding of an anti-SARS-CoV-2-reactive cellular immune response in healthy individuals suggests a pre-existing immunity to various common cold HCoVs which share close homology with SARS-CoV-2. The humoral immunity to the S protein of SARS-CoV-2 detected in convalescent COVID-19 patients correlates with the presence of SARS-CoV-2-reactive CD4+ T cells expressing Th1 cytokines. Remarkably, an inverse correlation of SARS-CoV-2 S protein-specific IgGs with HCoV-NL63 and HCoV-229E S1 protein-specific IgGs suggests that pre-existing immunity to Alphacoronaviruses might have had an inhibitory imprint on the immune response to SARS-CoV-2-infection in the examined patients with mild COVID-19.

Circulating Dickkopf1 Parallels Metabolic Adaptations and Predicts Disease Trajectories in Patients With COVID-19.

CONTEXT AND AIMS: Coronavirus disease 19 (COVID-19) trajectories show high interindividual variability, ranging from asymptomatic manifestations to fatal outcomes, the latter of which may be fueled by immunometabolic maladaptation of the host. Reliable identification of patients who are at risk of severe disease remains challenging. We hypothesized that serum concentrations of Dickkopf1 (DKK1) indicate disease outcomes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals. METHODS: We recruited hospitalized patients with PCR-confirmed SARS-CoV-2 infection and included 80 individuals for whom blood samples from 2 independent time points were available. DKK1 serum concentrations were measured by ELISA in paired samples. Clinical data were extracted from patient charts and correlated with DKK1 levels. Publicly available datasets were screened for changes in cellular DKK1 expression on SARS-CoV-2 infection. Plasma metabolites were profiled by nuclear magnetic resonance spectroscopy in an unbiased fashion and correlated with DKK1 data. Kaplan-Meier and Cox regression analysis were used to investigate the prognostic value of DKK1 levels in the context of COVID-19. RESULTS: We report that serum levels of DKK1 predict disease outcomes in patients with COVID-19. Circulating DKK1 concentrations are characterized by high interindividual variability and change as a function of time during SARS-CoV-2 infection, which is linked to platelet counts. We further find that the metabolic signature associated with SARS-CoV-2 infection resembles fasting metabolism and is mirrored by circulating DKK1 abundance. Patients with low DKK1 levels are twice as likely to die from COVID-19 than those with high levels, and DKK1 predicts mortality independent of markers of inflammation, renal function, and platelet numbers. CONCLUSION: Our study suggests a potential clinical use of circulating DKK1 as a predictor of disease outcomes in patients with COVID-19. These results require validation in additional cohorts.

Recruitment of highly cytotoxic CD8+ T cell receptors in mild SARS-CoV-2 infection.

T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8+ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8+ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8+ TCRs-classic features of protective immunity-are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8+ T cell immunity.

Enumeration of viable CD34(+) cells by flow cytometry in blood, bone marrow and cord blood: results of a study of the novel BD™ stem cell enumeration kit.

BACKGROUND AIMS: Enumeration of CD34(+) cells in leukocyte-rich cell suspensions is important for clinical decision-making in stem cell transplantation. Single-platform flow cytometry assays offer the significant advantages of speed and reproducibility, and have therefore become the gold standard in stem cell enumeration. The clinical community has recently defined the need for stem cell enumeration kits that incorporate viability dyes. The purpose of this study was to evaluate a novel assay, BD Biosciences' (BD) stem cell enumeration kit (SCE kit(‡)), in relation to Beckman Coulter's (BC) commercially available BC Stem-Kit™. METHODS: Fresh/freeze-thawed samples from leukapheresis, bone marrow and cord blood, and fresh normal/mobilized blood, were analyzed with both assays (simultaneous detection of side/forward scatter and three fluorescence signals) on two flow cytometry platforms, BD FACSCanto II and BD FACSCalibur. Results. Results from both assays were highly congruent, with an overall r(2) ≥ 0.99 (all specimen types included), a linear correlation across all CD34(+) cell frequencies and concentrations, and an almost ideal steepness of the trend line. CONCLUSIONS: Both assays functioned reliably. Being based on single-platform International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines and similar staining methods, both assays essentially come to identical results. For most specimen types, the viability of CD34(+) cells was equal to overall leukocyte viability. In summary, in the hands of an experienced technician, the BD™ SCE kit and the BC Stem-Kit are equivalent. The infrequent user might derive benefit from the fact that counting spheres are pre-pipetted into the Trucount tube for the SCE kit, making this assay less susceptible to pipetting inaccuracy.

Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic stem cell transplantation. For the clearance of CMV, CD8+ T cells are pivotal. STUDY DESIGN AND METHODS: Here, the novel streptamer technology was used at good manufacturing practice (GMP) level for adoptive transfer of CMV-specific T cells into acute leukemia patients with recurrent high CMV antigenemia after allogeneic stem cell transplantation. RESULTS: After a single transfusion, the frequency of CMV-specific CD8+CD45RA+CCR7- effector T cells increased dramatically from 0.0% to a maximum of 27.1% of all T cells. These T cells were clearly donor derived and did not stem from intrinsic reconstitution, as demonstrated by analysis of 1) donor chimerism through single-tandem repeats, 2) T-cell receptor excision circles, and 3) Vß-chain typing by polymerase chain reaction. Clinically, the specific T-cell transfer resulted in a persistent clearance of the CMV antigenemia, which allowed the patients to discontinue toxic antiviral drug therapy without further high-level reactivation of CMV, demonstrating the power of the streptamer technology. CONCLUSION: Taken together, the streptamer technology offers the advantage of selecting virus-specific CD8+ T cells at GMP level for adoptive T-cell transfer, thus inducing long-lasting specific CD8+ T-cell responses without increasing the risk for graft-versus-host disease.

Antigen-shift in varicella-zoster virus-specific T-cell immunity over the course of Fingolimod-treatment in relapse-remitting multiple sclerosis patients.

BACKGROUND: Fingolimod (FTY) applied as treatment regimen of relapsing-remitting multiple sclerosis (RRMS) induces downregulation of sphingosine-1-phosphate receptors on the lymphocytes. As a result CC chemokine receptor type 7 (CCR7) expressing lymphocytes are retained within the peripheral lymph nodes thus suppressing their accumulation into the cerebrospinal fluid of multiple sclerosis (MS) patients and hampering disease progress. Unfortunately, MS patients treated with FTY suffer from an increased incidence of varicella-zoster virus (VZV) infections which has been associated with a decrease of VZV immediate early 63 (IE63)-specific T-cell immunity. To elucidate VZV-specific T-cell immunity over the course of FTY-treatment, we analyzed T-cell immunity for immediate early, early and late VZV-antigens. METHODS: T-cell immune responses were detected via intracellular IFN-γ staining after stimulation with VZV-specific peptide mixes for IE62 and IE63 and recombinant proteins for open reading frame 26 (ORF26), ORF9 and glycoprotein E (gE) using flow cytometry. Analyzed samples comprised of different groups including 18 patients with RRMS at baseline (BL), 6 and 12 months after FTY-treatment start, 12 patients with long-term (LT) FTY-treatment, one FTY-treated patient, before and after VZV-reactivation. In addition, VZV-specific IgG and IgM titers were assessed by ELISA. RESULTS: After FTY-treatment start, absolute numbers of CCR7 expressing CD4+ T cells and CD8+T cells dropped rapidly. However, VZV-specific immunity could be detected in the majority of RRMS patients throughout FTY-treatment with increasing prevalence after 6 months of treatment. We found an increase in the prevalence of VZV-specific IFN-γ+CD8+ T-cell immunity in FTY-treated patients after six months of therapy, while in parallel VZV-specific IFN-γ+CD4+ T cells declined dramatically. Additionally, a strong correlation between VZV-specific IgG serum titers and the percentage of RRMS patients with detectable VZV-specific T cells was observed (r = 0.985). Most remarkably, FTY-treated RRMS patients presented a shift in the predominant CD8+ T cell-mediated antigen-response from immediate early (IE62) to early virus antigens (ORF26) six months after treatment in parallel to a decrease of VZV-specific CD4+ T-cell immunity. ORF26-specific CD8+ T cells still dominated the VZV-specific cellular immunity at month 12 after FTY-treatment start and in LT FTY-treated MS patients. In a RRMS patient an increase of VZV-specific CD4+ T cells at VZV-reactivation accompanied with a four-fold increase of a VZV-specific IgG titer was detected which might indicate an important role in cellular immune control of VZV-infections. CONCLUSION: Monitoring VZV-specific T-cell immunity might provide a valuable tool to RRMS patient risk management during FTY-treatment.

CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations.

Despite the important role immunoglobulin G (IgG)-secreting plasma cells play in memory immune responses, the differentiation and homeostasis of these cells are not completely understood. Here, we studied the differentiation of human IgG-secreting cells ex vivo and in vitro, identifying these cells by the cellular affinity matrix technology. Several subpopulations of IgG-secreting cells were identified among the cells isolated from tonsils and bone marrow, particularly differing in the expression levels of CD9, CD19, and CD38. CD38 low IgG-secreting cells were present exclusively in the tonsils. A major fraction of these cells appeared to be early plasma cell precursors, as upon activation of B cells in vitro, IgG secretion preceded up-regulation of CD38, and on tonsillar sections, IgG-containing, CD38 low cells with a plasmacytoid phenotype were found in follicles, where plasma cell differentiation starts. A unitary phenotype of migratory peripheral blood IgG-secreting cells suggests that all bone marrow plasma cell populations share a common precursor cell. These data are compatible with a multistep model for plasma cell differentiation and imply that a common CD38 low IgG-secreting precursor gives rise to a diverse plasma cell compartment.

Extracellular vesicles secreted by bone marrow- and adipose tissue-derived mesenchymal stromal cells fail to suppress lymphocyte proliferation.

Recently, mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have been suggested as an alternative to MSCs for the treatment of various inflammatory disorders. However, while a first case report observed beneficial therapeutic effects of repeated MSC-EV infusions in a patient with therapy-refractory graft-versus-host disease, in vitro findings revealed that MSC-EVs were significantly less immunosuppressive than their parental cells. In this study, we compared the immunosuppressive potency of MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs), with their secreted EVs in a standardized lymphocyte proliferation assay (LPA). Both BM-MSCs and AT-MSCs exhibited a remarkable inhibition of lymphocyte proliferation (LP) (88.1%±1.5% and 75.5%±1.5%, respectively), while isolated EVs derived from them failed to suppress LP at dose levels up to 100 µg/mL. Thus, our data further substantiate previous reports suggesting that cell-cell contact plays an important role on the immunosuppressive potential mediated by MSCs. Hence, MSC-EVs are still a matter of debate and might not be a reasonable substitute for MSCs with regard to the immunosuppressive function. Collectively, these contrasting findings may also reflect the importance of relevant translational aspects when designing new studies. Standardization of MSC culture conditions before EV collection as well as isolation and characterization methods with regard to EV purity are urged. Moreover, before clinical use, dose-finding studies evaluating MSC-EV preparations in suitable preclinical models are warranted.

The role of long-lived plasma cells in autoimmunity.

Recent results on the biology of plasma cells have shown that these cells can survive as long as memory B cells. Possibly, such long-lived plasma cells are also involved in the production of autoantibodies. Here, we discuss the potential involvement of long-lived plasma cells in the pathogenesis of autoimmune disease and the consequences it has for the development of effective therapeutic strategies.