Cryopreserved Peripheral Blood Mononuclear Cells Research Articles

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

Evaluating the Impact of Cryopreservation of PBMCs on CAR-T Therapy Efficacy and Safety in DLBCL Patients: An Informative Approach to Optimize Manufacturing Strategies

Background: Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a pivotal treatment modality for relapsed/refractory (R/R) B-cell malignancies. However, some patients are unable to receive CAR-T therapy due to challenges in obtaining sufficient lymphocytes through leukapheresis, often resulting from intensive prior treatments or impaired lymphocyte function after repeated tumor reductions under high tumor burden. This issue underscores the need to explore strategies such as advance collection and cryopreservation of peripheral blood mononuclear cells (PBMCs), which could provide enhanced flexibility in the pre-CAR-T treatment planning process and potentially widen the accessibility of CAR-T therapy. Aims: This retrospective study compares the safety, efficacy, and adverse reactions of anti-CD19 CAR-T cell therapy for Diffuse Large B-Cell Lymphoma (DLBCL) patients, using cryopreserved versus freshly harvested PBMCs. Methods: A total of 162 R/R DLBCL patients treated with an anti-CD19-CD3zeta-4-1BB CAR-T cell therapy from January 2019 to April 2023 were included in this study, excluding those who received concurrent autologous stem cell transplants. The median age was 52 (15-78) years old, and the majority (87.7%) patients had stage III-IV disease. With a median IPI score of 2 (range, 0-5), 71.0% (115/162) had extranodal lesions and 18.0% (29/162) had bulky disease > 7 cm. Among these, 136 patients received CAR-T cells derived from cryopreserved PBMCs, which were frozen with a controlled rate freezer and CryoSure-DEX40 cryoprotectant and stored in the liquid phase of liquid nitrogen. The remaining 26 patients received CAR-T cells that were directly cultured from freshly collected lymphocytes without any cryopreservation. For all patients, CAR-T cells were freshly infused post-manufacture. The study primarily explores the attainment of CAR-T cell infusion dose (achieving 2x10 6 cells/kg), in vivo expansion of CAR-T cells, and the persistence of CAR-T cells in the body. Secondary aims include assessing overall survival (OS), progression-free survival (PFS), 3-month complete response (CR) rate and objective response rate (ORR). The incidence of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and levels of cytokines were also recorded. In addition, baseline characteristics of both groups were taken into analysis, including factors such as Eastern Cooperative Oncology Group (ECOG) performance status, International Prognostic Index (IPI) score, age, gender, presence of bulky disease, and extranodal lesions. For statistical analysis, either chi-square test or Fisher's exact test was employed for categorical variables, whereas continuous variables were assessed using the rank-sum test. Survival curves between groups were compared using the log-rank test. All P-values were two-tailed. Results: Baseline characteristics between the cryopreserved and fresh groups showed no significant differences in age, gender, IPI score≥3, ECOG score≥3, and extranodal lesions (P all > .05), while bulky disease prevalence was higher in the cryopreserved group (20.6% vs. 3.85%, P = .049). Metrics of CAR-T cell expansion, including meeting the infusion dosage benchmark of 2x10 6 cells/kg (46.1% vs. 44.9%, P = .90), in vivo CAR-T cell persistence (median duration: 21 days for both groups, P= .48), and the peak proportion of CART19 cells in lymphocytes (median peak: 49.6% vs. 35.2%, P = .41), revealed no significant differences between the fresh and cryopreserved groups. Assessments of CAR-T therapy safety revealed no significant differences in the incidence of grade 3 or higher ICANS and CRS, or peak levels of cytokines including interferon gamma, IL-6, IL-10, soluble CD25, and TNFα (P all > .05) between the two groups. Clinical efficacy measurements (3-month CR and ORR, 1-year OS and PFS) were similar between the two groups, with no statistically significant differences in CR (46.2% vs. 45.5%), ORR (69.2% vs. 61.9%), 1-year OS (75.4% vs. 64.1%, Fig 1), and 1-year PFS (52.1% vs. 44.5%, Fig 2) (P all > .05). Conclusion: Our study affirms that cryopreservation of PBMCs has no significant influence on the safety and efficacy of anti-CD19 CAR-T therapy for patients with R/R DLBCL. These findings widen the therapeutic flexibility and accessibility of anti-CD19 CAR-T therapy for DLBCL patients. Key words: PBMC Cryopreservation, CAR-T, DLBCL

OMIP-099: 31-color spectral flow cytometry panel to investigate the steady-state phenotype of human T cells.

We have developed a 31-color panel to define the steady-state phenotype of T cells in human peripheral blood (Table 1). The panel presented here was optimized using cryopreserved peripheral blood mononuclear cells (PBMC). The markers included in this panel were chosen in order to characterize the steady-state phenotype of T cells and includes markers (CD45RA, CD45RO, CCR7, CD95) to distinguish the main subsets (e.g., naïve, TEM , TCM , TEMRA , TSCM etc.) of CD4, CD8, and γδ T cells. This panel also includes markers for the identification of differentiation status (CD27, CD28), activation/antigen experience status (CD11a, CD49d, CD38, HLA-DR, CD56, and CD39), co-inhibitory marker expression (PD-1, TIM-3), and CD4 T helper subsets (CXCR3, CXCR5, CCR4, CCR6, Foxp3, CD25, and CD127). This optimized panel provides a broad assessment of the steady-state phenotype of human T cells.

OMIP-089: Cattle T-cell phenotyping by an 8-color panel.

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve ), central memory (TCM ), effector memory (TEM ) and terminal effector (TTE ) T cells. Activated cattle T cells (TAV ) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve , TAV , TCM , TEM , TTE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react.

SARS-CoV-2-specific CD4+ and CD8+ T cell responses can originate from cross-reactive CMV-specific T cells.

Detection of SARS-coronavirus-2 (SARS-CoV-2) specific CD4+ and CD8+ T cells in SARS-CoV-2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relative high frequency and prevalence of cross-reactive T cells, we hypothesized CMV may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved PBMCs with SARS-CoV-2 peptides revealed that frequencies of SARS-CoV-2-specific T cells were higher in CMV-seropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4+ and spike-specific CD8+ T cells originate from cross-reactive CMV-specific T cells. Spike-specific CD8+ T cells recognize SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA-B*35:01. These dual IPS/FVS-reactive CD8+ T cells were found in multiple donors as well as severe COVID-19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS-cross-reactivity is caused by a public TCR. In conclusion, CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV-2.

Abstract 511: Intrinsic Self-reactive T Cells To ApoB-100 Peptide P210 In Acute Coronary Syndrome Patients Are Associated With T Effector/Memory Response

Background: Antibodies to the apoB-100 peptide P210 have been reported in coronary artery disease (CAD) patients yet the nature of intrinsic self-reactive T cell response in these patients remains to be investigated. Given the reported presence of antibodies to P210 in CAD, we hypothesized that the P210 peptide would provoke T cell activation in peripheral blood mononuclear cells (PBMCs) of acute coronary syndrome (ACS) patients and would manifest as T Effector/Memory cells, previously demonstrated to correlate with atherosclerotic disease. Methods and Results: PBMCs from ACS patients were collected under an approved IRB and kept viable in cryo-preservation until assays. Cryo-preserved viable PBMCs from self-reported controls were purchased from a commercial vendor with their own IRB. T cell activation was assessed by stimulating PBMCs in vitro for 16 hours with P210 peptide. Negative control was no treatment and positive controls were stimulation with pooled CMV peptides or PMA/ionomycin cocktail. The Activation Induced Marker (AIM) flow cytometric assay was performed with the following antibodies: CD4, CD8, CD25, CD69, CD134, CD137, and CD154 using non-viable cells and CD14, CD16 and CD19 as dump gates. AIM+ cells were expressed as the ratio relative to no stimulation. T Effector/Memory responses were assessed after 72 hours of stimulation with P210. Negative control was no stimulation and positive control was stimulation with PMA/ionomycin. Viable T Effector/Memory cells were identified as positive for CD3, CD4, CD8, and CD45RO, but CD45RA(-), CD62L(-) and CCR7(-) by flow cytometry. T Effector/Memory cells were expressed as Response Index (negative control subtracted from P210 stimulation divided by the positive control for each sample). P210 stimulation significantly increased CD4+CD69+CD134+ (AIM+) cells in ACS (P<0.05; n=12) compared to controls (N=6). No other AIM+ cells were noted. P210 stimulation induced persistent CD8+ T Effector and T Effector Memory responses in ACS (N=13; P<0.05) compared to controls (N=14). Conclusion: P210 provokes self-reactive T cell activation and Effector/Memory responses in ACS patients consistent with the immunogenic role of P210 in coronary artery disease pathophysiology.

Peripheral blood mononuclear cells cryopreservation and transportation

Chimeric antigen receptor-T (CAR-T) cells have emerged as a treatment option for patients with hematologic malignancies. Usually, their manufacture has been carried out by specialized and centralized laboratories. A useful strategy, which allows a better logistical organization, is the peripheral blood mononuclear cells (PBMC) cryopreservation and transportation. This article aims to discuss the most relevant points related to the PBMC cryopreservation and transportation process. In addition, it proposes a kind of standard operational protocol (SOP) that can be very helpful for the cell processing labs.

Single-Cell Phenotypic and Molecular Characterization of Circulating Tumor Cells Isolated from Cryopreserved Peripheral Blood Mononuclear Cells of Patients with Lung Cancer and Sarcoma.

The isolation of circulating tumor cells (CTCs) requires rapid processing of the collected blood due to their inherent fragility. The ability to recover CTCs from peripheral blood mononuclear cells (PBMCs) preserved from cancer patients could allow for retrospective analyses or multicenter CTC studies. We compared the efficacy of CTC recovery and characterization using cryopreserved PMBCs vs fresh whole blood from patients with non-small cell lung cancer (NSCLC; n = 8) and sarcoma (n = 6). Two epithelial cellular adhesion molecule (EpCAM)-independent strategies for CTC enrichment, based on Parsortix® technology or immunomagnetic depletion of blood cells (AutoMACS®) were tested, followed by DEPArray™ single-cell isolation. Phenotype and genotype, assessed by copy number alterations analysis, were evaluated at a single-cell level. Detection of target mutations in CTC-enriched samples from frozen NSCLC PBMCs was also evaluated by digital PCR (dPCR). The use of cryopreserved PBMCs from cancer patients allowed for the retrospective enumeration of CTCs and their molecular characterization, using both EpCAM-independent strategies that performed equally in capturing CTC. Cells isolated from frozen PBMCs were representative of whole blood-derived CTCs in terms of number, phenotype, and copy number aberration profile/target mutations. Long-term storage (≥3 years) did not affect the efficacy of CTC recovery. Detection of target mutations was also feasible by dPCR in CTC-enriched samples derived from stored PBMCs. Isolating CTCs from longitudinally collected PBMCs using an unbiased selection strategy can offer a wider range of retrospective genomic/phenotypic analyses to guide patients' personalized therapy, paving the way for sample sharing in multicenter studies.

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples

Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

Effects of long-term cryopreservation of PBMC on recovery of B cell subpopulations

Cryopreservation of human peripheral blood mononuclear cells (PBMC) is used in many clinical and research applications to avoid direct and on-site analysis of samples. Storage of PBMC further allows prequalification of donor cells for routine laboratory methods involving the evaluation of immune responses. Previous studies reported changes in cellular composition and phenotype of PBMC following the freezing procedure. In our 12-month follow-up study, we focused on B cells and proportional representation of B cell subpopulations during long-term storage at −80 °C. Over the 12-month period, we observed a gradual decline in B cell viability and recovery. Notably, no changes in the proportional representation of human B cell subpopulations occurred in this period and the functional response elicited by antigen and TLR9 ligand CpG remained comparable to that observed after short-term storage for one month.

Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies.

DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors' experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.

A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

We present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

The Cytokinesis-Block Micronucleus Assay on Human Isolated Fresh and Cryopreserved Peripheral Blood Mononuclear Cells.

The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G0 CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.

OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood.

PURPOSE AND APPROPRIATE SAMPLE TYPESThis 40‐color flow cytometry‐based panel was developed for in‐depth immunophenotyping of the major cell subsets present in human peripheral blood. Sample availability can often be limited, especially in cases of clinical trial material, when multiple types of testing are required from a single sample or timepoint. Maximizing the amount of information that can be obtained from a single sample not only provides more in‐depth characterization of the immune system but also serves to address the issue of limited sample availability. The panel presented here identifies CD4 T cells, CD8 T cells, regulatory T cells, γδ T cells, NKT‐like cells, B cells, NK cells, monocytes and dendritic cells. For each specific cell type, the panel includes markers for further characterization by including a selection of activation and differentiation markers, as well as chemokine receptors. Moreover, the combination of multiple markers in one tube might lead to the discovery of new immune phenotypes and their relevance in certain diseases. Of note, this panel was designed to include only surface markers to avoid the need for fixation and permeabilization steps. The panel can be used for studies aimed at characterizing the immune response in the context of infectious or autoimmune diseases, monitoring cancer patients on immuno‐ or chemotherapy, and discovery of unique and targetable biomarkers. Different from all previously published OMIPs, this panel was developed using a full spectrum flow cytometer, a technology that has allowed the effective use of 40 fluorescent markers in a single panel. The panel was developed using cryopreserved human peripheral blood mononuclear cells (PBMC) from healthy adults (Table 1). Although we have not tested the panel on fresh PBMCs or whole blood, it is anticipated that the panel could be used in those sample preparations without further optimization. @ 2020 Cytek Biosciences, Inc. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Cryopreservation of peripheral blood mononuclear cells using uncontrolled rate freezing.

Peripheral blood mononuclear cells are widely used as source material for anticancer immunotherapies. The conventional cryopreservation method for peripheral blood mononuclear cells is time-consuming and expansive, which involves controlled rate freezing followed by storage in liquid nitrogen. Instead, the convenient uncontrolled rate freezing cryopreservation method had been reported successfully in peripheral blood hematopoietic stem cells and peripheral blood progenitor cells. Therefore, we hypothesized that uncontrolled rate freezing cooling method maybe also applied to peripheral blood mononuclear cells cryopreservation. In this study, we evaluated the performance of uncontrolled rate freezing and controlled rate freezing cooling methods through cell recovery rate, viability, differentiation potential into cytokine-induced killer cells and the cellular properties of the cultured cytokine-induced killer cells. The results showed similar post-thaw viability and recovery rate in both controlled rate freezing and uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells. Importantly, the uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells exhibited higher growth ratio and earlier cell clustering during ex-vivo cytokine-induced killer cell culture than the controlled rate freezing ones. These two groups of expanded cytokine-induced killer cells also exhibited similar effector cell subset ratio and tumoricidal activity. In general, the performance of cryopreserved peripheral blood mononuclear cells using uncontrolled rate freezing cooling method, with the commercial cryoprotective agent CellBanker 2, was equal or better than the controlled rate freezing method. Our study implied that the combined use of cryoprotective agent CellBanker 2 and uncontrolled rate freezing could be a convenient cryopreservation method for peripheral blood mononuclear cells.

Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73.

CD39 and CD73 are ecto-nucleotidases present on human peripheral blood mononuclear cells (PBMCs) and are emerging biomarkers on these cells in various disorders including cancer. Many factors influence PBMC quality, so it is essential to validate sample processing methods prior to incorporation in clinical studies. This study examined the impact of both PBMC cryopreservation and PBMC isolation using SepMate density gradient centrifugation on CD39 and CD73 expressing subsets. First, PBMCs were isolated from the peripheral blood of 11 healthy donors by routine Ficoll-Paque density gradient centrifugation, cryopreserved and compared with freshly isolated PBMCs by flow cytometry. The proportions of T and B cells expressing combinations of CD39 and CD73 were relatively stable over 6-month cryopreservation, although some T cell combinations revealed small but significant changes. Second, peripheral blood was collected from six healthy donors to compare PBMCs isolated by SepMate or Ficoll-Paque density gradient centrifugation. Compared with Ficoll-Paque, the more rapid SepMate method yielded 9.1% less PBMCs but did not alter cell viability or proportions of T and B cells expressing combinations of CD39 and CD73. The present study reveals that cryopreservation is suitable for studying T and B cells expressing combinations of CD39 and CD73. However, caution should be exercised when observing small differences in these cryopreserved subsets between different cohorts. Further, SepMate and Ficoll-Paque methods of PBMC isolation show similar results for T and B cell subset analysis; however, SepMate is a faster and easier approach.

A Method of Assessment of Human Natural Killer Cell Phenotype and Function in Whole Blood.

The majority of data on human Natural Killer (NK) cell phenotype and function has been generated using cryopreserved peripheral blood mononuclear cells (PBMCs). However, cryopreservation can have adverse effects on PBMCs. In contrast, investigating immune cells in whole blood can reduce the time, volume of blood required, and potential artefacts associated with manipulation of the cells. Whole blood collected from healthy donors and cancer patients was processed by three separate protocols that can be used independently or in parallel to assess extracellular receptors, intracellular signaling protein phosphorylation, and intracellular and extracellular cytokine production in human NK cells. To assess extracellular receptor expression, 200 μL of whole blood was incubated with an extracellular staining (ECS) mix and cells were subsequently fixed and RBCs lysed prior to analysis. The phosphorylation status of signaling proteins was assessed in 500 μL of whole blood following co-incubation with interleukin (IL)-2/12 and an ECS mix for 20 min prior to cell fixation, RBC lysis, and subsequent permeabilization for staining with an intracellular staining (ICS) mix. Cytokine production (IFNγ) was similarly assessed by incubating 1 mL of whole blood with PMA-ionomycin or IL-2/12 prior to incubation with ECS and subsequent ICS antibodies. In addition, plasma was collected from stimulated samples prior to ECS for quantification of secreted IFNγ by ELISA. Results were consistent, despite inherent inter-patient variability. Although we did not investigate an exhaustive list of targets, this approach enabled quantification of representative ECS surface markers including activating (NKG2D and DNAM-1) and inhibitory (NKG2A, PD-1, TIGIT, and TIM-3) receptors, cytokine receptors (CD25, CD122, CD132, and CD212) and ICS markers associated with NK cell activation following stimulation, including signaling protein phosphorylation (p-STAT4, p-STAT5, p-p38 MAPK, p-S6) and IFNγ in both healthy donors and cancer patients. In addition, we compared extracellular receptor expression using whole blood vs. cryopreserved PBMCs and observed a significant difference in the expression of almost all receptors. The methods presented permit a relatively rapid parallel assessment of immune cell receptor expression, signaling protein activity, and cytokine production in a minimal volume of whole blood from both healthy donors and cancer patients.

Optimization of the monocyte activation test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine

Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: the rabbit pyrogen test (RPT), the bacterial endotoxin test, the recombinant factor C test, and the monocyte activation test (MAT). Here, we explored the possibility to replace the RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV). The testing was carried out using cryopreserved peripheral blood mononuclear cells as cell source. IL-6 release was selected as readout for the detection of both endotoxin and non-endotoxin contaminants. MAT applicability for pyrogen testing of the TBEV vaccine was assessed through preparatory tests and resulted in the establishment of a very sensitive assay (limit of detection (LOD) = 0.04 EU/mL; sensitivity = 0.1 EU/mL). Both quantitative Method A and semiquantitative Method B were used for data analysis. Our studies revealed that for a vaccine without intrinsic pyrogenicity, such as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD to define a stable maximum valid dilution of the product. In conclusion, we describe the challenges of MAT implementation for anti-TBEV vaccine following the current Ph. Eur. chapter 2.6.30 and propose a re-evaluation of the validity criteria of Methods A and B in order to set a semi-quantitative or limit test suitable for those products for which a reference lot comparison analysis is not applicable or favorable.

Optimization of the monocyte-activation-test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine_suppl

Pyrogen content is a key quality feature that must be checked in all injectable products, including vaccines. Four tests are currently available in the European Pharmacopoeia to monitor pyrogen/endotoxin presence: the rabbit pyrogen test (RPT), the bacterial endotoxin test, the recombinant factor C test, and the monocyte activation test (MAT). Here, we explored the possibility to replace the RPT with the MAT in the quality control of a vaccine against tick-borne encephalitis virus (TBEV). The testing was carried out using cryopreserved peripheral blood mononuclear cells as cell source. IL-6 release was selected as readout for the detection of both endotoxin and non-endotoxin contaminants. MAT applicability for pyrogen testing of the TBEV vaccine was assessed through preparatory tests and resulted in the establishment of a very sensitive assay (limit of detection (LOD) = 0.04 EU/mL; sensitivity = 0.1 EU/mL). Both quantitative Method A and semiquantitative Method B were used for data analysis. Our studies revealed that for a vaccine without intrinsic pyrogenicity, such as that against TBEV, sensitivity (the lowest endotoxin value of the standard curve) should be used instead of LOD to define a stable maximum valid dilution of the product. In conclusion, we describe the challenges of MAT implementation for anti-TBEV vaccine following the current Ph. Eur. chapter 2.6.30 and propose a re-evaluation of the validity criteria of Methods A and B in order to set a semi-quantitative or limit test suitable for those products for which a reference lot comparison analysis is not applicable or favorable.

Deficit of Circulating CD19+CD24hiCD38hi Regulatory B Cells in Severe Aplastic Anemia

Background: Immune aplastic anemia (AA) is caused by cytotoxic T cells (CTLs) that destroy hematopoietic stem and progenitor cells. Regulatory T cells (Tregs) are reduced in AA and increase in response to immunosuppressive therapy (IST; Solomou E et al, Blood 2007). Recent studies suggested an immune regulatory role of regulatory B cells (Bregs). Human CD19+CD24hiCD38hi Bregs suppress Th1 response of CD4+ T cells as well as IFN-γ production by CD8+ CTLs (Mauri C, Menon M, J Clin Invest 2017). The quantity and/or function of Bregs are impaired in autoimmune diseases, malignancies, chronic graft-versus-host disease, and during rejection of transplanted organs. Methods: We investigated B cell phenotypes including CD24hiCD38hi Bregs in previously untreated severe AA (SAA) and very severe AA (VSAA) patients, and healthy individuals aged 18 years and older, and tested their correlation with severity and response to IST. Absolute numbers of lymphocyte subsets, including CD19+ B cells, CD8+ T cells, CD4+ T cells, and NK cell (TBNK), were quantified in fresh blood. Percentages of B cell subsets among total CD19+ B cells, including CD24hiCD38hi Bregs, CD24loCD38lo mature naïve B cells, CD24hiCD38lo memory B cells and CD24loCD38hi plasma cells/plasmablasts, were analyzed using cryopreserved peripheral blood mononuclear cells (PBMCs). Blood samples were obtained from patients close to time of diagnosis and before institution of definitive therapy. All patients were treated with horse anti-thymocyte globulin, cyclosporine, and eltrombopag between 2012 and 2018 at the Hematology Branch, NHLBI (clinicaltrials.gov NCT01623167). Results: TBNK analysis revealed no significant difference in total B cell counts in 104 AA patients compared to 40 healthy individuals (median, 137/μl [IQR, 73-212] vs 163/μl [106-242], P=.11); NK cells were significantly decreased in patients with AA, as previously reported (Gascon P et al, Blood 1986). Total B cell count did not correlate with severity of AA (P=.89) nor with overall response at six months (P=.93). CD8+ T cells and NK cells were lower in VSAA patients compared to SAA patients. None of the TBNK subsets was predictive of overall response in six months after IST. When we assessed the phenotype of B cells among 60 AA patients whose cryopreserved PBMCs were available, CD24hiCD38hi Bregs were markedly decreased as compared to 29 healthy individuals (0.31% [0.14-0.85%] vs 1.9% [1.3-3.6%], P=3×10-7; Figure, Table), while there was no significant difference in other B cell phenotypes. Among these 60 patients, the percentage of CD24hiCD38hi Bregs was especially decreased in VSAA patients compared to SAA (0.18% [0.11-0.34%] vs 0.50% [0.17-1.4%], P=.017). In contrast, CD24loCD38lo mature naïve B cells were higher in VSAA than in SAA (69% [58-86%] vs 60% [42-70%], P=.024). CD24hiCD38hi Breg frequency was positively associated with neutrophil and reticulocyte counts (correlation coefficients [r], 0.34 and 0.26, respectively), while the frequency of CD24loCD38lo mature naïve B cells was negatively correlated (r, -0.34 and -0.40). CD24loCD38lo mature naïve B cells before IST were significantly lower in 47 patients who achieved overall responses at six months compared to 13 non-responders (64% [42-71%), vs 73% [58-88%], P=.014), but CD24hiCD38hi Breg frequency was not correlated with IST responses. At six months after IST, CD24hiCD38hi Bregs in AA patients had recovered to levels present in healthy individuals (2.3% [0.98-4.8%]), in both 34 responders and five non-responders; non-responders showed non-significant increased CD24loCD38lo mature naïve B cells at six months (P=.068). Discussion: A deficit of circulating CD24hiCD38hi Bregs in immune AA with recovery after IST, as occurs with Tregs, suggests Bregs may contribute to the immune pathophysiology in AA. We unexpectedly observed a higher percentage of CD24loCD38lo mature naïve B cells to be associated with more severe disease and a lower probability of responses to IST. B cell phenotype analysis may be beneficial for monitoring of AA and predicting outcomes of therapy. Disclosures No relevant conflicts of interest to declare.