Pre-Clinical Assessment of SAR442257, a CD38/CD3xCD28 Trispecific T Cell Engager in Treatment of Relapsed/Refractory Multiple Myeloma.

Current treatment strategies for multiple myeloma (MM) are highly effective, but most patients develop relapsed/refractory disease (RRMM). The anti-CD38/CD3xCD28 trispecific antibody SAR442257 targets CD38 and CD28 on MM cells and co-stimulates CD3 and CD28 on T cells (TCs). We evaluated different key aspects such as MM cells and T cells avidity interaction, tumor killing, and biomarkers for drug potency in three distinct cohorts of RRMM patients. We found that a significantly higher proportion of RRMM patients (86%) exhibited aberrant co-expression of CD28 compared to newly diagnosed MM (NDMM) patients (19%). Furthermore, SAR442257 mediated significantly higher TC activation, resulting in enhanced MM killing compared to bispecific functional knockout controls for all relapse cohorts (Pearson's r = 0.7). Finally, patients refractory to anti-CD38 therapy had higher levels of TGF-ß (up to 20-fold) compared to other cohorts. This can limit the activity of SAR442257. Vactoserib, a TGF-ß inhibitor, was able to mitigate this effect and restore sensitivity to SAR442257 in these experiments. In conclusion, SAR442257 has high potential for enhancing TC cytotoxicity by co-targeting CD38 and CD28 on MM and CD3/CD28 on T cells.

CD28null T cells in aging and diseases: From biology to assessment and intervention.

CD28null T cells, an atypical subset characterized by the loss of CD28 costimulatory molecule expression, exhibit functional variants and progressively expand with age. Moreover, T cells with these phenotypes are found in both typical and atypical humoral immune responses. Consequently, they accumulate during infectious diseases, autoimmune disorders, cardiovascular conditions, and neurodegenerative ailments. To provide an in-depth review of the current knowledge regarding CD28null T cells, we specifically focus on their phenotypic and functional characteristics as well as their physiological roles in aging and diseases. While uncertainties regarding the clinical utility remains, we will review the following two crucial research perspectives to explore clinical translational applications of the research on this specific T cell subset: 1) addressing the potential utility of CD28null T cells as immunological markers for prognosis and adverse outcomes in both aging and disease, and 2) speculating on the potential of targeting CD28null T cells as an interventional strategy for preventing or delaying immune aging processes and disease progression.

The Expression of LDL-R in CD8+ T Cells Serves as an Early Assessment Parameter for the Production of TCR-T Cells.

BACKGROUND/AIM: The quantity and the phenotypes of desired T cell receptor engineered T (TCR-T) cells in the final cell product determine their in vivo anti-tumor efficacy. Optimization of key steps in the TCR-T cell production process, such as T cell activation, has been shown to improve cell quality. MATERIALS AND METHODS: Using a modified TCR (mTCR) derived from mice transducing PBMCs, we assessed the proportions of low-density lipoprotein receptor (LDL-R) and mTCR expressing cells under the various activation conditions of CD3/CD28-Dynabeads or OKT3 via flow cytometry. RESULTS: We demonstrate that the proportion of T cells expressing LDL-R post activation is positively correlated with the percentage of mTCR+CD8+ T cells with their less differentiated subtypes in the final product. In addition, we show that shifting the CD3/CD28-Dynabeads activation duration from a typical 48 h to 24 h can significantly increase the production of the desired mTCR+CD8+ T cells. Importantly, the percentages of TCR-T cells with less-differentiated phenotypes, namely mTCR central memory T cells (TCM), were found to be preserved with markedly higher efficiency when T cell activation was optimized. CONCLUSION: Our findings suggest that the proportion of LDL-R+ T cells may serve as an early assessment parameter for evaluating TCR-T cell quality, possibly facilitating the functional and economical improvement of current adoptive therapy.

Assessment of tumor microenvironment expression and clinical significance of immune inhibitory molecule CTLA-4, ligand B7-1, and tumor-infiltrating regulatory cells in Hodgkin lymphoma.

Classical Hodgkin lymphoma represents a paradigm of tumor cell-microenvironment interactions as the neoplastic Hodgkin Reed-Sternberg (HRS) cells typically constitute less than 1% of the total tumor volume. CTLA-4, a member of the CD28/B7 immunoglobulin superfamily, and CD28 and their ligands B7-1 and B7-2 are critically important for the initial activation of naive T cells. Strategies aimed at interfering with the crosstalk between tumoral Reed-Sternberg cells and their cellular partners have been taken into account in the development of new immunotherapies that target different cell components of the HL microenvironment. The study included 50 histopathological confirmed cases of Hodgkin lymphoma. IHC staining for CTLA-4 and B7-1 was performed on archival paraffin-embedded biopsy. SPSS version 17 was used for statistical analysis. CTLA-4 IHC expression in HRS cells was negative in all cases, while in immune cells, CTLA-4 expression was observed in 45 (90%) cases. CD80 expression was present in all cases, both in HRS and immune cells. There was a significant association between HRS cell percentage and IPS score (p-value=0.001). Mean survival duration was longer in <50% immune cells compared to >50% groups, with an overall mean survival of 67.633 months. Considering the CTLA4 expression in immune cells within the microenvironment and the availability of targeted drugs like Iplimumab, which act through CTLA4 blockade, it may be appropriate to use this as targeted therapy in HL cases, particularly in those with refractory disease who are unable to achieve cure prior to ASCT.

CAR T-cells in acute lymphoblastic leukemia: Current results.

The marketing authorization of tisagenlecleucel, a 2nd generation of CD19-directed CAR T-cells, containing the 4-1 BB co-stimulatory domain, in 2017 in USA and in 2018 in EU, has revolutionized the therapeutic strategy in advanced B-cell acute lymphoblastic leukemia (B-ALL) in children, adolescents and young adults (AYAs) with relapsed or refractory disease. This innovative treatment, based on a "living drug", has shown very impressive short-term responses. However, safety profile and complex logistics require high expertise centers and tight collaborations between addressing and treating centers. Current research is exploring the possibility to move to first line ALL with high-risk features and/or first high-risk relapse. More efficient CAR T-cells products, are still lacking to counteract the escape mechanisms already described. Moreover, to define the bridge-to-CAR time for each patient remains a challenge to obtain optimal disease burden allowing expansion and persistence of CAR T-cells. Also difficult is to identify patients who will benefit from further therapy after infusion, such as allogeneic HSCT or may be immuno-modulatory treatment. Finally, CAR T-cells directed against T-ALL are only in their beginning but require more complex engineering process to avoid T- cell immune-deficiency or fratricide.

Design and Assessment of Novel Anti-CD30 Chimeric Antigen Receptors with Human Antigen-Recognition Domains.

Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T cell signaling domains. CD30 is a cell surface protein expressed on Hodgkin's lymphoma, some T cell lymphomas, and some B cell lymphomas. CD30 has a restricted expression pattern in normal cells, so CD30 has good potential as a clinical target for CAR T cells. We compared three different anti-CD30 CAR designs incorporating a single-chain variable fragment derived from the 5F11 fully human monoclonal antibody. 5F11-28Z has hinge, transmembrane, and costimulatory domains from CD28 and a CD3ζ T cell activation domain. 5F11-CD828Z has hinge and transmembrane domains from CD8α, a CD28 costimulatory domain, and a CD3ζ T cell activation domain. 5F11-CD8BBZ is identical to 5F11-CD828Z, except for the replacement of the CD28 moiety with a 4-1BB moiety. We found that T cells expressing 5F11-CD8BBZ had lower levels of CD30-specific degranulation and cytokine release compared with CD28-containing CARs. When compared to the CD28-containing CARs, T cells expressing 5F11-CD8BBZ had higher levels of nonspecific functional activity, including degranulation, cytokine release, and proliferation, when stimulated with CD30-negative target cells. We established tumors in nod-scid common gamma-chain deficient (NSG) mice and treated the tumors with T cells expressing different CARs. T cells expressing 5F11-28Z were most effective at eradicating tumors. T cells expressing 5F11-CD828Z had intermediate effectiveness, and T cells expressing 5F11-CD8BBZ were least effective. CD30+ T cells are lost from cultures of T cells containing 5F11-28Z-expressing T cells. This indicated the killing of CD30+ T cells by the 5F11-28Z-expressing T cells. Despite this, the number of T cells in the cultures consistently accumulated to numbers needed for use in a clinical trial. Based on all in vitro and murine experiments comparing the different CARs, we selected 5F11-28Z for further development, and we have initiated a clinical trial testing 5F11-28Z T cells.

Monte Carlo cross-validation analysis screens pathway cross-talk associated with Parkinson's disease.

We purposed to identify underlying functional pathway cross-talk in Parkinson's disease (PD) through Monte Carlo cross-validation analysis. Microarray data set of E-GEOD-6613 was downloaded from ArrayExpress database. First, the identification of differentially expressed genes (DEGs) was implemented, following by extracting the potential disrupted pathway enriched by DEGs. In addition, a discriminating score (DS) was computed based on the distribution of gene expression levels by quantifying their pathway cross-talk for each pair of pathways. Furthermore, random forest (RF) classification model was utilized to identify the top ten paired pathways with high AUC between PD and healthy control samples using the tenfold cross-validation method. Finally, Monte Carlo cross-validation was repeated 50 times to explore the best pairs of pathways. After quantile normalization, a total of 9331 genes with higher than 0.25-fold quantile average across all samples were obtained. Totally, 42 DEGs and 19 differential pathways enriched from DEGs were identified. We then ranked each pathway according to their AUC values, the pair of pathways, phosphatidylcholine biosynthesis I, and PPAR signaling obtained the best AUC value of 0.942. Moreover, the paired pathways of mTOR signaling and CD28 signaling in T helper cells had higher AUC value of 0.837 in five bootstraps. Two paired pathways, including phosphatidylcholine biosynthesis I and PPAR signaling, as well as mTOR signaling and CD28 signaling in T helper cells were able to accurately classify PD and healthy control samples. Significantly, these paired pathways might be underlying biomarkers for early diagnosis and therapy of PD.

Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-κB, NFAT and AP-1.

Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.

Mechanistic Assessment of PD-1H Coinhibitory Receptor-Induced T Cell Tolerance to Allogeneic Antigens.

PD-1H is a recently identified cell surface coinhibitory molecule of the B7/CD28 immune modulatory gene family. We showed previously that single injection of a PD-1H agonistic mAb protected mice from graft-versus-host disease (GVHD). In this study, we report two distinct mechanisms operate in PD-1H-induced T cell tolerance. First, signaling via PD-1H coinhibitory receptor potently arrests alloreactive donor T cells from activation and expansion in the initiation phase. Second, donor regulatory T cells are subsequently expanded to maintain long-term tolerance and GVHD suppression. Our study reveals the crucial function of PD-1H as a coinhibitory receptor on alloreactive T cells and its function in the regulation of T cell tolerance. Therefore, PD-1H may be a target for the modulation of alloreactive T cells in GVHD and transplantation.

Quantitative assessment of the associations between CD28 T > C polymorphism (rs3116496) and cancer risk.

Many studies have examined the association between CD28 T > C polymorphism (rs3116496) and cancer risk in various populations. However, results remained controversial. To assess this relationship more precisely, a meta-analysis was performed. A comprehensive literature search was performed using the PubMed database for relevant articles published (updated to January 1, 2014). Odds ratios (ORs) and 95 % confidence intervals (CIs) were used to assess the strength of the association. A total of nine studies were selected for this meta-analysis, including 3,878 cases and 4,424 controls. The results indicated that CD28 T > C polymorphism (rs3116496) was not associated with the risk of cancer in overall population (CC + CT vs. TT, OR = 1.17, 95 %CI = 0.94-1.47, P H = 0.00; CC vs. CT + TT, OR = 1.26, 95 %CI = 0.92-1.73, P H = 0.86; CC vs. TT, OR = 1.27, 95 %CI = 0.92-1.74, P H = 0.85; CT vs. TT, OR = 1.15, 95 %CI = 0.91-1.46, P H = 0.00; and C vs. T, OR = 1.17, 95 %CI = 0.97-1.41, P H = 0.00). In subgroup analysis according to cancer type, no significant association was found in cervical cancer or other cancer. However, in the subgroup analysis by ethnicity, the significant risk was found among Asians (CC + CT vs. TT, OR = 1.51, 95 %CI = 1.24-1.83, P H = 0.05; C vs. T, OR = 1.46, 95 %CI = 1.22-1.74, P H = 0.11), but not among Caucasians. The result of this meta-analysis suggested that CD28 T > C polymorphism (rs3116496) may have an increased risk of cancer in Asians.

A quantitative assessment of costimulation and phosphatase activity on microclusters in early T cell signaling.

T cell signaling is triggered through stimulation of the T cell receptor and costimulatory receptors. Receptor activation leads to the formation of membrane-proximal protein microclusters. These clusters undergo tyrosine phosphorylation and organize multiprotein complexes thereby acting as molecular signaling platforms. Little is known about how the quantity and phosphorylation levels of microclusters are affected by costimulatory signals and the activity of specific signaling proteins. We combined micrometer-sized, microcontact printed, striped patterns of different stimuli and simultaneous analysis of different cell strains with image processing protocols to address this problem. First, we validated the stimulation protocol by showing that high expression levels CD28 result in increased cell spreading. Subsequently, we addressed the role of costimulation and a specific phosphotyrosine phosphatase in cluster formation by including a SHP2 knock-down strain in our system. Distinguishing cell strains using carboxyfluorescein succinimidyl ester enabled a comparison within single samples. SHP2 exerted its effect by lowering phosphorylation levels of individual clusters while CD28 costimulation mainly increased the number of signaling clusters and cell spreading. These effects were observed for general tyrosine phosphorylation of clusters and for phosphorylated PLCγ1. Our analysis enables a clear distinction between factors determining the number of microclusters and those that act on these signaling platforms.

PD-1 analysis on CD28(-) CD27(-) CD4 T cells allows stimulation-independent assessment of CMV viremic episodes in transplant recipients.

Expression of the inhibitory receptor programmed death 1 (PD-1) on cytomegalovirus (CMV)-specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28(-) CD27(-) double negativity is known as a typical phenotype of CMV-specific CD4 T cells. Therefore, the co-expression of inhibitory receptors on CD28(-) CD27(-) CD4 T cells was assessed as a rapid, stimulation-independent parameter for monitoring CMV complications after transplantation. Ninety-three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross-sectional and longitudinal manner. CMV-specific CD4 T cell levels quantified after stimulation were compared to levels of CD28(-) CD27(-) CD4 T cells. PD-1 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expression on CD28(-) CD27(-) CD4 T cells were related to viremia. A percentage of ≥0.44% CD28(-) CD27(-) CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV-specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD-1 expression levels on CD28(-) CD27(-) CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA-4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD-1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD-1 expression on CD28(-) CD27(-) CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation-independent parameter to guide duration of antiviral therapy.

Assessment of oxidative stress in lymphocytes with exercise.

This study investigated whether changes in the cellular composition of blood during exercise could partly account for observations of exercise-induced changes in lymphocyte oxidative stress markers. Markers of oxidative stress were assessed before and after 60 min of intense treadmill running. Samples were collected from 16 men (means ± SD: age 33 ± 13 yr; body mass index 23.8 ± 2.5 kg/m(2); maximal oxygen uptake 59.7 ± 5.2 ml·kg(-1)·min(-1)). Peripheral blood lymphocytes were assayed for protein carbonyl concentration, and plasma was assessed for lipid peroxides and antioxidant capacity. In a separate study, intracellular thiol concentration was determined in lymphocyte subsets from eight characteristically similar men by flow cytometry, of which T-cell memory populations were further identified on the basis of CD27, CD28, and CD45RA expression. Total lymphocyte protein carbonyls were transiently increased with exercise and returned to baseline within 15 min (P < 0.001). This change was accompanied by an increase in plasma lipid peroxides (P < 0.05) and total antioxidant capacity (P < 0.001). Correlation analyses showed that lymphocyte protein carbonyl content was not related to changes in the cellular composition of peripheral blood during exercise. Natural killer cells (CD3(-)CD56(+)) and late-differentiated/effector memory cells (CD4(+)/CD8(+)CD27(-)CD28(-)/CD45RA(+)), which mobilized most with exercise, showed high intracellular thiol content (P < 0.001). High thiol content suggests a lower oxidative load carried by these lymphocytes. Thus vigorous exercise resulted in a transient increase in lymphocyte oxidative stress. Results suggest this was unrelated to the alterations in the cellular composition of peripheral blood.

Toward experimental assessment of receptor occupancy: TGN1412 revisited.

In March 2006, 6 healthy volunteers experienced serious adverse reactions during a first-in-human clinical trial of the superagonistic anti-CD28 mAb TGN1412. A first investigation excluded contaminations of the drug product or protocol irregularities as the root cause. Later, an expert scientific group convened in the United Kingdom to develop recommendations pertinent to minimizing risks of first-in-human clinical trials. The expert scientific group concluded from in silico calculations that at the initial dose of 0.1 mg/kg, which was adjusted on the basis of the no observed adverse effect level, approximately 86.2% to 90.9% CD28 receptor occupancy was obtained. Here we developed a flow cytometric method that revealed receptor occupancy of approximately 45% to 80% under the above conditions. Thus we present a method to experimentally determine receptor occupancy that can be taken as one parameter to define the minimal anticipated biological effect level as the basis for calculating safer starting doses for first-in-human clinical trials for products in which a potential risk has been identified. Additional measures are being discussed that will help to significantly improve safety of first-in-human clinical trials.

A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells.

Many cell types release ATP in response to mechanical or biochemical stimulation. The mechanisms responsible for this release, however, are not well understood and may differ among different cell types. In addition, there are numerous difficulties associated with studying the dynamics of ATP release immediately outside the cell membrane. Here, we report a new method that allows the visualization and quantification of ATP release by fluorescence microscopy. Our method utilizes a two-enzyme system that generates NADPH when ATP is present. NADPH is a fluorescent molecule that can be visualized by fluorescence microscopy using an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The method is capable of detecting ATP concentrations <1 microM and has a dynamic range of up to 100 microM. Using this method, we visualized and quantified ATP release from human polymorphonuclear leukocytes and Jurkat T cells. We show that upon cell stimulation, the concentrations of ATP can reach levels of up to 80 microM immediately outside of the cell membrane. This new method should prove useful for the study of the mechanisms of release and functional role of ATP in various cell systems, including individual cells.

Global assessment of cross-hybridization for oligonucleotide arrays.

Cross-hybridization is the tendency for chains of nucleic acids to bind to other chains of nucleic acids that have similar but not identical sequences. This has the potential to make the interpretation of microarray experiments difficult since intensity at a spot on the array does not simply depend on the quantity of target in the sample. We propose a method for evaluating the extent of cross-hybridization in oligonucleotide arrays for data arising from a typical microarray experiment. We show that the level of cross-hybridization can be quite substantial. We argue that this makes the interpretation of the difference calls provided by MAS 5.0 difficult.

Assessment of selected co-stimulatory, adhesion and activatory molecules and cytokines of Th(1)/Th(2) balance in acute lymphoblastic leukemia in children.

INTRODUCTION: Recent years have seen a rise in the importance of cytokine production and co-stimulatory/activatory molecule expression in the immune response in leukemia. The aim of our study was to assess the function of T lymphocytes in children with acute lymphoblastic leukemia (ALL) during remission induction based on selected cytokine and co-stimulatory/activatory molecule expression. MATERIAL/METHODS: The study group consisted of 50 children with ALL (B cell precursor). Peripheral blood samples were taken before treatment (day 0), after the prednisone prophase (day 8), and during (day 15) and after (day 33) remission induction. The percentages of T cells with interferon (IFN)-gamma (Th(1)), interleukin (IL)-4 (Th(2)) and IL-2 receptor (IL-2R), CD28, CTLA-4, CD38, ICAM-1, and HLA-DR expression were assessed by tricolor flow cytometry. RESULTS: At the time of diagnosis we noted higher percentages of T cells with adhesion molecule ICAM-1, activation molecule CD38 expression, and an increased population of Th(2 )cells (IL-4) compared with the control group. During and after remission induction we observed a decreased population of CD38(+) T cells, elevated percentages of helper T lymphocytes with IL-2R expression, and a rise in helper T lymphocytes producing IFN-gamma (Th(1)). During fever/infection, higher levels of activated T lymphocytes (CD4(+)HLA-DR(+), CD8(+)HLA-DR(+)), a rise in Th1, and no change in Th(2 )populations were observed. CONCLUSIONS: The results suggest T cell activation and Th(2 )predominance at the time of diagnosis and during remission induction in ALL in children. These results confirm the involvement of cellular immunity in the leukemic process and can be used in immune therapy in leukemia.

Kinetic assessment of general gene expression changes during human naive CD4+ T cell activation.

The consequence of naive CD4+ T cell activation is the differentiation and generation of effector cells. How the engagement of T cell receptors and co-stimulatory receptors leads to profound differential changes is not fully understood. To assess the transcription changes during T cell activation, we developed human T cell specific cDNA microarray gene filters and examined the gene expression profiles in human naive CD4+ T cells for 10 continuous time points during the first 24 h after anti-CD3 plus anti-CD28 (anti-CD3/CD28) stimulation. We report here a global and kinetic analysis of gene expression changes during naive CD4+ T cell activation and identify 196 genes having expression levels that significantly changed after activation. Based on the temporal change, there are 15 genes that changed between 0-1 h (early), 25 genes between 2-8 h (middle) and 156 genes between 16-24 h (late) after stimulation. Further analyses of the functions of those genes indicate their roles in maintenance of resting status, activation, adhesion/migration, cell cycle progression and cytokine production. However, a significant majority of these genes are novel to T cells and their functions in T cell activation require further study. Together, these results present a kinetic view of the gene expression changes of naive CD4+ T cells in response to T cell receptor-mediated activation for the first time, and provide a basis in understanding how the complex network of gene expression regulation is programmed during CD4+ T cell activation.

Individual variability in cyclosporin A sensitivity: the assessment of functional measures on CD28-mediated costimulation of human whole blood T lymphocytes.

The quantitative analysis of cyclosporin A (CsA) effects might be helpful for optimizing immunosuppressive treatment after allogeneic organ transplantation in individual patients, as rejection can occur despite the existence of CsA blood levels within therapeutic ranges. Previous investigations found that costimulation of the CD28 pathway generally mediates CsA-resistant proliferation of T cell receptor (TCR)-activated T lymphocytes. However, here we describe considerable interindividual variation regarding the immunosuppressive effects of CsA (1000 microg/L) on anti-CD3/CD28 T cell costimulation in a human whole blood assay. In the in vitro study, we found a significant reduction of T cell proliferation, activation marker expression (CD25, CD69) on the T cell surface, and interleukin-2 (IL-2) protein expression in whole blood samples of all healthy subjects (n = 11). However, the investigation of cytokine mRNA profiles revealed variable results of in vitro CsA sensitivity. Whole blood samples of 3 of 11 healthy individuals demonstrated a marked suppression of IL-2 mRNA expression (>50%) and a partial inhibition of IL-4, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) mRNA expression on addition of CsA. In contrast, the remaining 8 healthy individuals had cytokine mRNA expression levels that were unaffected or even increased when CsA was administered in vitro. In patients undergoing CsA monotherapy (ex vivo study, n = 9), we found a significant suppression of IL-2 mRNA levels in 4 of 9 patients ex vivo. Thus, we cannot confirm a universal CsA resistance of T cells on anti-CD3/CD28 costimulation. Instead, our results suggest an individual degree of CsA sensitivity that might be more consistent with clinical experience. Prospective studies are necessary to determine if individual degrees of CsA sensitivity correlate with clinical events and are associated with a low or high risk of transplant rejection.