Standardized high-dimensional spectral cytometry protocol and panels for whole blood immune phenotyping in clinical and translational studies.

Flow cytometry is the method of choice for immunophenotyping in the context of clinical, translational, and systems immunology studies. Among the latter, the Milieu Intérieur (MI) project aims at defining the boundaries of a healthy immune response to identify determinants of immune response variation. MI used immunophenotyping of a 1000 healthy donor cohort by flow cytometry as a principal outcome for immune variance at steady state. New generation spectral cytometers now enable high-dimensional immune cell characterization from small sample volumes. Therefore, for the MI 10-year follow up study, we have developed two high-dimensional spectral flow cytometry panels for deep characterization of innate and adaptive whole blood immune cells (35 and 34 fluorescent markers, respectively). We have standardized the protocol for sample handling, staining, acquisition, and data analysis. This approach enables the reproducible quantification of over 182 immune cell phenotypes at a single site. We have applied the protocol to discern minor differences between healthy and patient samples and validated its value for application in immunomonitoring studies. Our protocol is currently used for characterization of the impact of age and environmental factors on peripheral blood immune phenotypes of >400 donors from the initial MI cohort.

Phase II trial of hypomethylating agent combined with nivolumab for acute myeloid leukaemia relapse after allogeneic haematopoietic cell transplantation-Immune signature correlates with response.

Acute myeloid leukaemia (AML) relapse after allogeneic haematopoietic cell transplantation (allo-HCT) is often driven by immune-related mechanisms and associated with poor prognosis. Immune checkpoint inhibitors combined with hypomethylating agents (HMA) may restore or enhance the graft-versus-leukaemia effect. Still, data about using this combination regimen after allo-HCT are limited. We conducted a prospective, phase II, open-label, single-arm study in which we treated patients with haematological AML relapse after allo-HCT with HMA plus the anti-PD-1 antibody nivolumab. The response was correlated with DNA-, RNA- and protein-based single-cell technology assessments to identify biomarkers associated with therapeutic efficacy. Sixteen patients received a median number of 2 (range 1-7) nivolumab applications. The overall response rate (CR/PR) at day 42 was 25%, and another 25% of the patients achieved stable disease. The median overall survival was 15.6 months. High-parametric cytometry documented a higher frequency of activated (ICOS+ , HLA-DR+ ), low senescence (KLRG1- , CD57- ) CD8+ effector T cells in responders. We confirmed these findings in a preclinical model. Single-cell transcriptomics revealed a pro-inflammatory rewiring of the expression profile of T and myeloid cells in responders. In summary, the study indicates that the post-allo-HCT HMA/nivolumab combination induces anti-AML immune responses in selected patients and could be considered as a bridging approach to a second allo-HCT. Trial-registration: EudraCT-No. 2017-002194-18.

Integrin activation enables rapid detection of functional Vδ1+ and Vδ2+ γδ T cells.

Conformational change of the ß2 integrin lymphocyte function-associated antigen 1 (LFA-1) is an early marker of T cell activation. A protocol using the mAb clone m24 recognizing the active, extended high-affinity conformation has been previously described for the assessment of functional CD4+ and CD8+ T cells in response to MHC-peptide stimulation. We investigated the applicability of the m24 mAb to detect the activation of γδ T cells in response to different soluble and immobilized stimuli. m24 mAb staining was associated with the expression of cytokines and was detectable as early as 10 min after stimulation, but with different kinetics depending on the nature of the stimulus. Hence, we conclude that this assay is suitable for the detection of functional γδ T cells and allows the assessment of activation more rapidly than alternative methods such as cytokine detection. Intracellular staining, protein trafficking inhibitors, or prior knowledge of the stimulating moiety recognized are no longer required for monitoring γδ T cell activation.

Evaluation of a fit-for-purpose assay to monitor antigen-specific functional CD4+ T-cell subpopulations in rheumatoid arthritis using flow cytometry-based peptide-MHC class-II tetramer staining.

Antigen-specific T cells can serve as a response biomarker in non-clinical or clinical immunotherapy studies in autoimmune disease. There are protocols with optimized multimer staining methods to detect peptide (p)MHCII+ CD4+ T cells, and some qualified and validated protocols for pMHCI+ CD8+ T cells. However, no protocol is fully or partially qualified to enumerate and characterize antigen-specific pMHCII+ CD4+ T cells from patient samples. Implementing such an assay requires a desired level of specificity and precision, in terms of assay repeatability and reproducibility. In transgenic type II collagen (CII)-immunized HLA-DR1/DR4 humanized mouse models of collagen-induced arthritis (CIA), CII259-273-specific T cells dominantly expand. Therefore antigen-specific T cells recognizing this epitope presented by rheumatoid arthritis (RA)-associated risk HLA-DR allomorphs are of interest to understand disease progression and responses to immunotherapy in RA patients. Using HLA-DRB1∗04:01 or ∗01:01-collagen type II (CII)259-273 tetramers, we evaluated parameters influencing precision and reproducibility of an optimized flow cytometry-based method for antigen-specific CD4+ T cells and eight specific subpopulations with and without tetramer positivity. We evaluated specificity, precision, and reproducibility for research environments and non-regulated laboratories. The assay has excellent overall precision with %CV<25% for intra-assay repeatability, inter-analyst precision, and inter-assay reproducibility. The precision of the assay correlated negatively with the cell viability after thawing, indicating that post-thaw viability is a critical parameter for reproducibility. This assay is suitable for longitudinal analysis of treatment response and disease activity outcome in RA patients, and adaptable for translational or immunotherapy clinical trial settings.

PR3-ANCAs predict relapses in ANCA-associated vasculitis patients after rituximab.

BACKGROUND: The primary challenge of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patient care is the early detection of relapses to prevent organ damage and increase survival. Potential biomarkers for relapses are ANCA and B cells, but their predictive value is a matter of debate. Therefore this study investigated how ANCA and B-cell status related to relapses in AAV patients treated with rituximab (RTX) as remission induction (RI). METHODS: This single-centre cohort study identified 110 ANCA-positive AAV patients treated with RTX between 2006 and 2018. Serial ANCA, CD19+ B-cell status and relapses were assessed >2 years. RESULTS: Patients (31/110) relapsed within 2 years after RTX RI treatment. Patients who achieved and maintained PR3-ANCA negativity (n = 29) had few relapses (3%), while persistent proteinase 3 (PR3)-ANCA positivity (n = 49) and reappearance of PR3-ANCAs (n = 10) associated significantly with more relapses (37%, P = 0.002 and 50%, P = 0.002). Patients with incomplete B-cell depletion (n = 11) had significantly more relapses (54%) as compared with patients with B-cell depletion [n = 76 (26%), P = 0.02]. Also, patients with repopulation of B cells (n = 58) had significantly more relapses (41%) as compared with patients without B-cell repopulation [n = 27 (15%), P = 0.03]. Overall, the absence of PR3- or myeloperoxidase (MPO)-ANCA positivity was highly predictive for remaining relapse-free. In PR3-ANCA-positive patients, 96% of the relapses occurred with persistent or reappearance of PR3-ANCAs and 81% with B-cell repopulation. In MPO-ANCA-positive patients, all relapses were restricted to patients with persistent MPO-ANCAs and B-cell repopulation. CONCLUSIONS: Upon RI treatment with RTX in AAV patients, ANCA and B-cell status were predictive of the majority of relapses and specifically their absence strongly predicted a relapse-free status. Therefore the implementation of ANCA and B-cell monitoring could guide therapeutic decision-making to prevent relapses in AAV patients treated with RTX.

Comparison of three immunoassays for infliximab trough level monitoring in paediatric inflammatory bowel diseases.

Many clinical studies in paediatric inflammatory bowel diseases (IBD) use infliximab trough level (IFX-TL) and detection of antibody against infliximab (ATI). Hence, comparison of commercially available assays is needed in paediatric samples to assess their reliability and their comparability. We measured IFX-TL and ATI-TL in sera samples of 53 IBD children using three ELISA kits: Lisa-Tracker® Duo Infliximab (Theradiag®), Ridascreen® IFX monitoring (R-Biopharm®) and Promonitor® IFX (Grifols®). Regarding IFX-TL, median values were comparable (p > 0.05), a good statistical correlation has been observed (0.73 ≤ R2 ≤ 0.85) between tested assays and the Bland-Altman analysis found an excellent agreement with a bias estimated between -0.56 and 0.12 and few values outside the 95% limits of agreement. However, qualitative comparison with therapeutic interval classifications showed some discrepancies (30.2%), mainly due to values near thresholds and more often than not with Theradiag® (22.6%). For ATI, because of non-standardized units, the qualitative comparison found a sensibly good agreement (98.1%). These data show a good agreement of IFX-TL and ATI measurement between three marketed ELISA assays with a small bias obtained. Variations in some results can lead to divergent therapeutic interval classifications and prompt us to be cautious in the interpretation of values near therapeutic thresholds.

Immunomodulation by ionizing radiation-impact for design of radio-immunotherapies and for treatment of inflammatory diseases.

Ionizing radiation is often regarded as an element of danger. But, danger responses on the cellular and molecular level are often beneficial with regard to the induction of anti-tumor immunity and for amelioration of inflammation. We outline how in dependence of radiation dose and fraction, radiation itself-and especially in combination with immune modulators-impacts on the innate and adaptive immune system. Focus is set on radiation-induced changes of the tumor cell phenotype and the cellular microenvironment including immunogenic cancer cell death. Mechanisms how anti-tumor immune responses are triggered by radiotherapy in combination with hyperthermia, inhibition of apoptosis, the adjuvant AnnexinA5, or vaccination with high hydrostatic pressure-killed autologous tumor cells are discussed. Building on this, feasible multimodal radio-immunotherapy concepts are reviewed including overcoming immune suppression by immune checkpoint inhibitors and by targeting TGF-ß. Since radiation-induced tissue damage, inflammation, and anti-tumor immune responses are interconnected, the impact of lower doses of radiation on amelioration of inflammation is outlined. Closely meshed immune monitoring concepts based on the liquid biopsy blood are suggested for prognosis and prediction of cancer and non-cancer inflammatory diseases. Finally, challenges and visions for the design of cancer radio-immunotherapies and for treatment of benign inflammatory diseases are given.

"Tumor immunology meets oncology (TIMO) XV", April 25th-27th 2019, Halle/Saale, Germany.

Novel insights into basic and translational tumor immunology including immunotherapies were presented by national and international scientists and clinicians at the TIMO XV meeting in Halle.

Tracking immunodynamics by identification of S-G2/M-phase T cells in human peripheral blood.

The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G2/M phases of the cell-cycle (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "TDS" cells). Without protocol refinement, TDS could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of TDS cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV+ carriers; and recently-diagnosed T1D patients. TDS were reproducibly more abundant among CD8+ T cells and a defined subset of T-regulatory CD4+ T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive TDS cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking TDS cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful.

Monitoring of infliximab trough levels and anti-infliximab antibodies in inflammatory bowel diseases: A comparison of three commercially available ELISA kits.

BACKGROUND: There are many studies presenting data of biologics and several ELISA kits commercially available for monitoring infliximab serum trough levels (s-IFXt) and anti-drug antibodies (ADAb). We propose to compare technical characteristics and results of three different assays on a cohort of 35 patients under infliximab (IFX) and suffering from inflammatory bowel disease (IBD). PATIENTS AND METHODS: s-IFXt and ADAb were systematically measured with three ELISA kits: Lisa-Tracker® Duo infliximab (Theradiag®), Ridascreen® IFX Monitoring (R-Biopharm AG®) and Promonitor® IFX (Progenika Biopharma SA®). RESULTS: The main technical features that differed between kits for measuring s-IFXt were: (i) TNF coating, (ii) immune complexes revelation strategy and/or (iii) interference with other anti-TNFα agents. For kits measuring ADAb, they were revelation steps and unit of results. There was an excellent mathematical correlation of s-IFXt between assays however Bland-Altman analysis denoted (i) s-IFXt were on average 48 to 69% higher in Ridascreen® than in the other two assays, and (ii) elevated s-IFXt were higher with Promonitor® compared to Lisa-Tracker®. As a consequence, there were some substantial discrepancies between assays for classification of s-IFXt into concentration ranges. Despite unstandardized units, pairwise qualitative comparison showed a perfect agreement between the three pairs of ADAb assays. CONCLUSION: Our data show that the evaluated assays are not quantitatively interchangeable due to substantial variations in some results that could lead, for some patients, to divergent therapeutic decisions. We remind to be cautious when comparing study results issued from different kits and recommend using the same assay for the longitudinal follow-up of IBD patients.

Immunosuppression, BK polyomavirus infections, and BK polyomavirus-specific T cells after pediatric kidney transplantation.

BACKGROUND: After kidney transplantation, immunosuppressive therapy increases risk of BK polyomavirus-associated nephropathy (BKPyVAN). Outcomes of BKPyV viremia are various and prognostic markers are missing. The impact of different immunosuppressive regimens on BKPyV infections is currently under discussion. METHODS: We analyzed immunosuppressive therapy and BKPyV-specific cellular immunity to distinguish patients at risk of BKPyVAN from those with self-limiting viremia for purposes of risk-stratified BKPyV management. In a retrospective analysis, 46 pediatric kidney recipients with BKPyV viremia were analyzed with regard to duration of BKPyV viremia and immunosuppressive therapy; in addition, in 37/46 patients, BKPyV-specific CD4 and CD8 T cells were measured. RESULTS: Nine patients showed persistent BKPyV viremia and BKPyVAN, and required therapeutic intervention, while 37 patients had asymptomatic, self-limiting viremia. At onset of viremia, 78% of patients with persistent viremia and BKPyVAN were treated with tacrolimus, whereas tacrolimus therapy was significantly less frequent in patients with self-limiting viremia (14%). The majority of patients with transient, self-limiting viremia received cyclosporine A (81%) and/or mTOR inhibitors (81%). Patients with persistent BKPyV viremia and BKPyVAN showed lack of BKPyV-specific CD4 and CD8 T cells (6/6), whereas the majority of patients with self-limiting viremia (27/31) had detectable BKPyV-specific CD4 and/or CD8 T cells ≥ 0.5 cells/µl (p < 0.001). CONCLUSIONS: These results indicate that tacrolimus enhances risk of BKPyVAN with need of therapeutic intervention, whereas under cyclosporine A and mTOR inhibitors, the majority of pediatric kidney recipients showed self-limiting viremia. In patients at risk of BKPyV infections, combination of cyclosporine A and mTOR inhibitor may be advantageous.

[Immunological monitoring of the efficacy of extracorporeal photopheresis for prevention of kidney transplant rejection].

BACKGROUND: The concept of the formation of immunological tolerance is a promising direction for correcting the renal transplant rejection. One of these methods is extracorporeal photochemotherapy (ECP), however, according to the literature, there is no single concept of its mechanisms of action in the formation of immunological tolerance in transplantology. AIM: To assess the effect of the preventive use of extracorporeal photochemotherapy on the factors of cellular immunity that contribute to the development of long-term tolerance in patients after kidney transplantation. MATERIALS AND METHODS: A total of 24 patients after a cadaveric kidney transplantation with group matching were included in the study. During the first six months after transplantation, 15 patients of the main group (MG) underwent 10 sessions of ECP in combination with the standard immunosuppression protocol, and 9 patients of the control group (CG) received only standard immunosuppressive therapy. Immunological studies were carried out by the 3rd year after transplantation. The number of cells expressing the antigens CD3, CD4, CD8, CD19, CD16 and CD56, the expression of co-stimulating molecules CD25, CD28 on T-lymphocytes, the number of T-regulatory cells with the CD3+ CD4+ CD25+ (hi) CD127- phenotype was evaluated. RESULTS: Compared with early post-transplant period, the number of naive CD3+CD4+CD45RO-CD28+ T-cells and CD28+ antigen expression was not different between two groups by 3 years after transplantation and with a group of otherwise healthy individuals (p=0.47 and p=0.26, respectively). Three years after transplantation, the T-helper lymphocyte count (CD3+CD4+) in MG were significantly higher than in CG (48.5+/-7.3% vs. 43.0+/-4.6%, respectively; p=0,04), cytotoxic T-lymphocytes count (CD3+CD8+) was 29.5+/-8.9% in MG, compared to 36.1+/-8.6% in CG (p=0.09), the ratio of CD4+/CD8+ in MG was significantly higher (1.83+/-0.72) than in CG (1.29+/-0.49) (p=0.04). CD19+ lymphocytes count was significantly below normal values in both groups, but in the CG it was more pronounced than in the MG (5.06+/-2.1% and 7.73+/-3%, respectively, (p=0.02) In the long-term period, CD3+CD4+CD25+(hi)CD127- T-regulatory cells count in MG was significantly higher than in CG (20.6+/-10.76*106/L and 12.9+/-4.97*106/l, respectively) (p=0.04). CONCLUSION: ECP initiates immunological tolerance through the activation of a second co-activation pathway between B-7 and CTLA-4 molecules in the early period after kidney transplantation. As a result, a clone of tolerogenic CD3+CD4+ T-lymphocytes is formed, which differentiates into T-regulatory cells and maintains immunological tolerance in the long-term period. Using ECP as a part of combination therapy allows to normalize the indicators of cellular immunity in the long-term period. BACKGROUND: The concept of the formation of immunological tolerance is a promising direction for correcting the renal transplant rejection. One of these methods is extracorporeal photochemotherapy (ECP), however, according to the literature, there is no single concept of its mechanisms of action in the formation of immunological tolerance in transplantology. AIM: To assess the effect of the preventive use of extracorporeal photochemotherapy on the factors of cellular immunity that contribute to the development of long-term tolerance in patients after kidney transplantation. MATERIALS AND METHODS: A total of 24 patients after a cadaveric kidney transplantation with group matching were included in the study. During the first six months after transplantation, 15 patients of the main group (MG) underwent 10 sessions of ECP in combination with the standard immunosuppression protocol, and 9 patients of the control group (CG) received only standard immunosuppressive therapy. Immunological studies were carried out by the 3rd year after transplantation. The number of cells expressing the antigens CD3, CD4, CD8, CD19, CD16 and CD56, the expression of co-stimulating molecules CD25, CD28 on T-lymphocytes, the number of T-regulatory cells with the CD3+ CD4+ CD25+ (hi) CD127- phenotype was evaluated. RESULTS: Compared with early post-transplant period, the number of naive CD3+CD4+CD45RO-CD28+ T-cells and CD28+ antigen expression was not different between two groups by 3 years after transplantation and with a group of otherwise healthy individuals (p=0.47 and p=0.26, respectively). Three years after transplantation, the T-helper lymphocyte count (CD3+CD4+) in MG were significantly higher than in CG (48.5+/-7.3% vs. 43.0+/-4.6%, respectively; p=0,04), cytotoxic T-lymphocytes count (CD3+CD8+) was 29.5+/-8.9% in MG, compared to 36.1+/-8.6% in CG (p=0.09), the ratio of CD4+/CD8+ in MG was significantly higher (1.83+/-0.72) than in CG (1.29+/-0.49) (p=0.04). CD19+ lymphocytes count was significantly below normal values in both groups, but in the CG it was more pronounced than in the MG (5.06+/-2.1% and 7.73+/-3%, respectively, (p=0.02) In the long-term period, CD3+CD4+CD25+(hi)CD127- T-regulatory cells count in MG was significantly higher than in CG (20.6+/-10.76*106/L and 12.9+/-4.97*106/l, respectively) (p=0.04). CONCLUSION: ECP initiates immunological tolerance through the activation of a second co-activation pathway between B-7 and CTLA-4 molecules in the early period after kidney transplantation. As a result, a clone of tolerogenic CD3+CD4+ T-lymphocytes is formed, which differentiates into T-regulatory cells and maintains immunological tolerance in the long-term period. Using ECP as a part of combination therapy allows to normalize the indicators of cellular immunity in the long-term period.

Adhering to adhesion: assessing integrin conformation to monitor T cells.

Monitoring T cells is of major importance for the development of immunotherapies. Recent sophisticated assays can address particular aspects of the anti-tumor T-cell repertoire or support very large-scale immune screening for biomarker discovery. Robust methods for the routine assessment of the quantity and quality of antigen-specific T cells remain, however, essential. This review discusses selected methods that are commonly used for T-cell monitoring and summarizes the advantages and limitations of these assays. We also present a new functional assay, which specifically detects activated ß2 integrins within a very short time following CD8+ T-cell stimulation. Because of its unique and favorable characteristics, this assay could be useful for implementation into our T-cell monitoring toolbox.

A phase I/IIa study of the mRNA-based cancer immunotherapy CV9201 in patients with stage IIIB/IV non-small cell lung cancer.

CV9201 is an RNActive®-based cancer immunotherapy encoding five non-small cell lung cancer-antigens: New York esophageal squamous cell carcinoma-1, melanoma antigen family C1/C2, survivin, and trophoblast glycoprotein. In a phase I/IIa dose-escalation trial, 46 patients with locally advanced (n = 7) or metastatic (n = 39) NSCLC and at least stable disease after first-line treatment received five intradermal CV9201 injections (400-1600 µg of mRNA). The primary objective of the trial was to assess safety. Secondary objectives included assessment of antibody and ex vivo T cell responses against the five antigens, and changes in immune cell populations. All CV9201 dose levels were well-tolerated and the recommended dose for phase IIa was 1600 µg. Most AEs were mild-to-moderate injection site reactions and flu-like symptoms. Three (7%) patients had grade 3 related AEs. No related grade 4/5 or related serious AEs occurred. In phase IIa, antigen-specific immune responses against ≥ 1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD+CD38hi B cells increased > twofold in 18/30 (60%) evaluable patients. 9/29 (31%) evaluable patients in phase IIa had stable disease and 20/29 (69%) had progressive disease. Median progression-free and overall survival were 5.0 months (95% CI 1.8-6.3) and 10.8 months (8.1-16.7) from first administration, respectively. Two- and 3-year survival rates were 26.7% and 20.7%, respectively. CV9201 was well-tolerated and immune responses could be detected after treatment supporting further clinical investigation.

Immunomonitoring of Tacrolimus in Healthy Volunteers: The First Step from PK- to PD-Based Therapeutic Drug Monitoring?

Therapeutic drug monitoring is routinely performed to maintain optimal tacrolimus concentrations in kidney transplant recipients. Nonetheless, toxicity and rejection still occur within an acceptable concentration-range. To have a better understanding of the relationship between tacrolimus dose, tacrolimus concentration, and its effect on the target cell, we developed functional immune tests for the quantification of the tacrolimus effect. Twelve healthy volunteers received a single dose of tacrolimus, after which intracellular and whole blood tacrolimus concentrations were measured and were related to T cell functionality. A significant correlation was found between tacrolimus concentrations in T cells and whole blood concentrations (r = 0.71, p = 0.009), while no correlation was found between tacrolimus concentrations in peripheral blood mononuclear cells (PBMCs) and whole blood (r = 0.35, p = 0.27). Phytohemagglutinin (PHA) induced the production of IL-2 and IFNγ, as well as the inhibition of CD71 and CD154 expression on T cells at 1.5 h post-dose, when maximum tacrolimus levels were observed. Moreover, the in vitro tacrolimus effect of the mentioned markers corresponded with the ex vivo effect after dosing. In conclusion, our results showed that intracellular tacrolimus concentrations mimic whole blood concentrations, and that PHA-induced cytokine production (IL-2 and IFNγ) and activation marker expression (CD71 and CD154) are suitable readout measures to measure the immunosuppressive effect of tacrolimus on the T cell.

Accurate quantification of T-cells expressing PD-1 in patients on anti-PD-1 immunotherapy.

Increasing numbers of trials employing anti-PD-1 immunotherapy emphasize the requirement for predictive biomarkers of clinical response. Many studies examine the cell surface expression of PD-1 and other key regulators of T-cell activation and inhibition. Here, we compared common commercially available anti-PD-1 diagnostic antibodies and tested whether they can bind the PD-1 receptor in the presence of the therapeutic antagonists pembrolizumab and nivolumab. We observed that currently no antibodies are available that can reliably stain all PD-1 receptors on T-cells from patients treated with anti-PD-1 antibodies. Furthermore, none of the diagnostic antibodies detected the entire population of PD-1+ T-cells relative to indirect staining using the therapeutic antibodies themselves. To overcome this problem, here we present a reliable method for quantifying PD-1 expression on immune cells from treated patients which can be included in any conventional flow or mass cytometry antibody panel used for patient monitoring.

Deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses.

Multicolor flow cytometry is a technology of choice for phenotyping of immune cells, and it can be used routinely for the follow up of patients in clinical trials. But it is challenging to define combinations of conjugated antibodies that efficiently allow the detailed analysis of major immune cell subsets and the identification of rare cell populations. In a collaborative work among the Immunology, Immunopathology, Immunotherapy (I3 ) laboratory, and the laboratory of immunomonitoring in oncology (L.I.O), we developed and validated 12 different 10-color flow cytometry panels that allow the deep immunophenotyping of cells from whole blood for the follow up of autoimmune and cancer patients. Here, we describe these optimized flow cytometry panels, showing that they provide the advanced analysis of T cells (including regulatory T cells), B cells, NK cells, MAIT cells, myeloid cells, monocytes, and dendritic cells. Most of the panels have been dried to improve standardization of the labeling and the entire procedure can be performed on less than 2 ml of whole blood. These deep immunophenotyping flow cytometry panels constitute a powerful tool for the monitoring of immune blood cells and will hopefully lead to the discovery of new biomarkers and potential therapeutic targets in autoimmune and cancer clinical trials. © 2018 International Society for Advancement of Cytometry.

Clinical validation of a novel enzyme-linked immunosorbent spot assay-based in vitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study.

Impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of CMV reactivation and associated complications in solid-organ transplantation. Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T-Track® CMV, a novel IFN-γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE-I CMV proteins, to monitor CMV-CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate-risk renal transplant recipients. CMV-CMI, CMV viral load, and clinical complications were monitored over 6 months post-transplantation. Ninety-five percent and 88-92% ELISpot assays were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65-specific response was ninefold higher in patients with self-clearing viral load compared to antivirally treated patients prior to first viral load detection (P < 0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T-Track® CMV is a highly sensitive IFN-γ ELISpot assay, suitable for the immunomonitoring of CMV-seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV-related clinical complications (ClinicalTrials.gov Identifier: NCT02083042).

Standardized whole blood stimulation improves immunomonitoring of induced immune responses in multi-center study.

Functional immune responses are increasingly important for clinical studies, providing in depth biomarker information to assess immunotherapy or vaccination. Incorporating functional immune assays into routine clinical practice has remained limited due to challenges in standardizing sample preparation. We recently described the use of a whole blood syringe-based system, TruCulture®, which permits point-of-care standardized immune stimulation. Here, we report on a multi-center clinical study in seven FOCIS Centers of Excellence to directly compare TruCulture to conventional PBMC methods. Whole blood and PBMCs from healthy donors were exposed to LPS, anti-CD3 anti-CD28 antibodies, or media alone. 55 protein analytes were analyzed centrally by Luminex multi-analyte profiling in a CLIA-certified laboratory. TruCulture responses showed greater reproducibility and improved the statistical power for monitoring differential immune response activation. The use of TruCulture addresses a major unmet need through a robust and flexible method for immunomonitoring that can be reproducibly applied in multi-center clinical studies. ONE SENTENCE SUMMARY: A multi-center study revealed greater reproducibility from whole blood stimulation systems as compared to PBMC stimulation for studying induced immune responses.

Identification of new MUC1 epitopes using HLA-transgenic animals: implication for immunomonitoring.

BACKGROUND: The success of immunotherapeutics in oncology and the search for further improvements has prompted revisiting the use of cancer vaccines. In this context, knowledge of the immunogenic epitopes and the monitoring of the immune response cancer vaccines generate are essential. MUC1 has been considered one of the most important tumor associated antigen for decades. METHODS: To identify HLA-restricted MUC1 peptides we used eight human MHC class I transgenic mouse lines, together covering more than 80% of the human population. MUC1 peptides were identified by vaccinating each line with full length MUC1 coding sequences and using an IFNγ ELIspot restimulation assay. Relevant peptides were tested in a flow cytometry-based tetramer assay and for their capacity to serve as target in an in vivo killing assay. RESULTS: Four previously identified MUC1 peptides were confirmed and five are described here for the first time. These nine peptide-MHC combinations were further characterized. Six gave above-background tetramer staining of splenocytes from immunized animals and three peptides were induced more than 5% specific in vivo killing. CONCLUSIONS: These data describe for the first time five new HLA class I-restricted peptides and revisit some that were previously described. They also emphasize the importance of using in vivo/ex vivo models to screen for immunogenic peptides and define the functions for individual peptide-HLA combinations.