Antisense targeting of FOXP3+ Tregs to boost anti-tumor immunity.

Our goal is to improve the outcomes of cancer immunotherapy by targeting FOXP3+ T-regulatory (Treg) cells with a next generation of antisense oligonucleotides (ASO), termed FOXP3 AUMsilence ASO. We performed in vitro experiments with human healthy donor PBMC and clinical samples from patients with lung cancer, mesothelioma and melanoma, and tested our approach in vivo using ASO FOXP3 in syngeneic murine cancer models and in humanized mice. ASO FOXP3 had no effects on cell viability or cell division, did not affect expression of other FOXP members, but decreased expression of FOXP3 mRNA in PBMC by 54.9% and in cancer samples by 64.7%, with corresponding 41.0% (PBMC) and 60.0% (cancer) decreases of Treg numbers (all p<0.0001). Hence, intratumoral Treg were more sensitive to the effects of ASO FOXP3 than peripheral blood Tregs. Isolated human Treg, incubated with ASO FOXP3 for 3.5 hours, had significantly impaired suppressive function (66.4%) versus Scramble control. In murine studies, we observed a significant inhibition of tumor growth, while 13.6% (MC38) to 22% (TC1) of tumors were completely resorbed, in conjunction with ~50% decrease of Foxp3 mRNA by qPCR and decreased numbers of intratumoral Tregs. In addition, there were no changes in FOXP3 mRNA expression or in the numbers of Tregs in draining lymph nodes and in spleens of tumor bearing mice, confirming that intratumoral Treg had enhanced sensitivity to ASO FOXP3 in vivo compared to other Treg populations. ASO FOXP3 Treg targeting in vivo and in vitro was accompanied by significant downregulation of multiple exhaustion markers, and by increased expression of perforin and granzyme-B by intratumoral T cells. To conclude, we report that targeting the key Treg transcription factor FOXP3, with ASO FOXP3, has a powerful anti-tumoral effect and enhances T cell response in vitro and in vivo.

Methyl-CpG binding domain 2 (Mbd2) is an epigenetic regulator of autism-risk genes and cognition.

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2-/-) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2-/- mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2-/- mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

FOXP3+ regulatory T cells are associated with the severity and prognosis of sarcoidosis.

Rationale: Sarcoidosis is an inflammatory granulomatous disease of unknown etiology with predominant lung involvement. Organ involvement and disease severity, as well as the nature of immune alterations, vary among patients leading to a range of clinical phenotypes and outcomes. Our objective was to evaluate the association of disease course and immune responses in pulmonary sarcoidosis. Methods: In this prospective cohort study of 30 subjects, most of whom were followed for one year, we evaluated 14 inflammatory markers in plasma, 13 Treg/T cell flow cytometry markers and 8 parameters of FOXP3+ Treg biology, including suppressive function, epigenetic features and stability. Results: We identified a set of 13 immunological parameters that differ in sarcoidosis subjects in comparison with healthy donors. Five of those were inversely correlated with suppressive function of Tregs in sarcoidosis, and six (TNFα, TNFR I and II, sCD25, Ki-67 and number of Tregs) were particularly upregulated or increased in subjects with thoracic lymphadenopathy. Treg suppressive function was significantly lower in patients with thoracic lymphadenopathy, and in patients with higher burdens of pulmonary and systemic symptoms. A combination of five inflammatory markers, Ki-67 expression, Treg function, and lung diffusion capacity evaluated at study entry predicted need for therapy at one year follow-up in 90% of cases. Conclusion: Tregs may suppress ongoing inflammation at local and systemic levels, and TNFα, TNFR I and II, sCD25 and Ki-67 emerge as attractive biomarkers for in vivo sarcoid inflammatory activity.

Nuclear Coregulatory Complexes in Tregs as Targets to Promote Anticancer Immune Responses.

T-regulatory (Treg) cells display considerable heterogeneity in their responses to various cancers. The functional differences among this cell type are heavily influenced by multiprotein nuclear complexes that control their gene expression. Many such complexes act mechanistically by altering epigenetic profiles of genes important to Treg function, including the forkhead P3 (Foxp3) transcription factor. Complexes that form with certain members of the histone/protein deacetylase (HDAC) class of enzymes, like HDACs 1, 2, and 3, along with histone methyltransferase complexes, are important in the induction and stabilization of Foxp3 and Treg identity. The functional behavior of both circulating and intratumoral Tregs greatly impacts the antitumor immune response and can be predictive of patient outcome. Thus, targeting these regulatory complexes within Tregs may have therapeutic potential, especially in personalized immunotherapies.

The Selective Estrogen Receptor Modulator, Raloxifene, Is Protective Against Renal Ischemia-reperfusion Injury.

BACKGROUND: There is increasing evidence that estrogen is responsible for improved outcomes in female kidney transplant recipients. Although the exact mechanism is not yet known, estrogen appears to exert its protective effects by ameliorating ischemia-reperfusion injury (IRI). In this study, we have examined whether the beneficial effects of exogenous estrogen in renal IRI are replicated by therapy with any one of several selective estrogen receptor modulators. METHODS: C57BL/6 adult mice underwent standardized warm renal ischemia for 28 min after being injected with the selective estrogen receptor modulators, raloxifene, lasofoxifene, tamoxifen, bazedoxifene, or control vehicle (dimethyl sulfoxide), at 16 and 1 h before IRI. Plasma concentrations of blood urea nitrogen and creatinine were assessed 24, 48, 72, and 96 h post-IRI. Tissue was collected 30 d postischemia for fibrosis analysis using Sirius Red staining. RESULTS: Raloxifene treatment in female mice resulted in significantly lower blood urea nitrogen and creatinine after IRI and significantly lower fibrosis 30 d following IRI. CONCLUSIONS: Raloxifene is protective against both acute kidney injury and fibrosis resulting from renal IRI in a mouse model.

Kynurenine induces T cell fat catabolism and has limited suppressive effects in vivo.

BACKGROUND: L-kynurenine is a tryptophan-derived immunosuppressive metabolite and precursor to neurotoxic anthranilate and quinolinate. We evaluated the stereoisomer D-kynurenine as an immunosuppressive therapeutic which is hypothesized to produce less neurotoxic metabolites than L-kynurenine. METHODS: L-/D-kynurenine effects on human and murine T cell function were examined in vitro and in vivo (homeostatic proliferation, colitis, cardiac transplant). Kynurenine effects on T cell metabolism were interrogated using [13C] glucose, glutamine and palmitate tracing. Kynurenine was measured in tissues from human and murine tumours and kynurenine-fed mice. FINDINGS: We observed that 1 mM D-kynurenine inhibits T cell proliferation through apoptosis similar to L-kynurenine. Mechanistically, [13C]-tracing revealed that co-stimulated CD4+ T cells exposed to L-/D-kynurenine undergo increased ß-oxidation depleting fatty acids. Replenishing oleate/palmitate restored effector T cell viability. We administered dietary D-kynurenine reaching tissue kynurenine concentrations of 19 µM, which is close to human kidney (6 µM) and head and neck cancer (14 µM) but well below the 1 mM required for apoptosis. D-kynurenine protected Rag1-/- mice from autoimmune colitis in an aryl-hydrocarbon receptor dependent manner but did not attenuate more stringent immunological challenges such as antigen mismatched cardiac allograft rejection. INTERPRETATION: Our dietary kynurenine model achieved tissue concentrations at or above human cancer kynurenine and exhibited only limited immunosuppression. Sub-suppressive kynurenine concentrations in human cancers may limit the responsiveness to indoleamine 2,3-dioxygenase inhibition evaluated in clinical trials. FUNDING: The study was supported by the NIH, the Else Kröner-Fresenius-Foundation, Laffey McHugh foundation, and American Society of Nephrology.

A Biological Circuit Involving Mef2c, Mef2d, and Hdac9 Controls the Immunosuppressive Functions of CD4+Foxp3+ T-Regulatory Cells.

The Mads/Mef2 (Mef2a/b/c/d) family of transcription factors (TFs) regulates differentiation of muscle cells, neurons and hematopoietic cells. By functioning in physiological feedback loops, Mef2 TFs promote the transcription of their repressor, Hdac9, thereby providing temporal control of Mef2-driven differentiation. Disruption of this feedback is associated with the development of various pathologic states, including cancer. Beside their direct involvement in oncogenesis, Mef2 TFs indirectly control tumor progression by regulating antitumor immunity. We recently reported that in CD4+CD25+Foxp3+ T-regulatory (Treg) cells, Mef2d is required for the acquisition of an effector Treg (eTreg) phenotype and for the activation of an epigenetic program that suppresses the anti-tumor immune responses of conventional T and B cells. We now report that as with Mef2d, the deletion of Mef2c in Tregs switches off the expression of Il10 and Icos and leads to enhanced antitumor immunity in syngeneic models of lung cancer. Mechanistically, Mef2c does not directly bind the regulatory elements of Icos and Il10, but its loss-of-function in Tregs induces the expression of the transcriptional repressor, Hdac9. As a consequence, Mef2d, the more abundant member of the Mef2 family, is converted by Hdac9 into a transcriptional repressor on these loci. This leads to the impairment of Treg suppressive properties in vivo and to enhanced anti-cancer immunity. These data further highlight the central role played by the Mef2/Hdac9 axis in the regulation of CD4+Foxp3+ Treg function and adds a new level of complexity to the analysis and study of Treg biology.

Lactate Limits T Cell Proliferation via the NAD(H) Redox State.

Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD+-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.

The CCR2/MCP-1 Chemokine Pathway and Lung Adenocarcinoma.

Host anti-tumor immunity can be hindered by various mechanisms present within the tumor microenvironment, including the actions of myeloid-derived suppressor cells (MDSCs). We investigated the role of the CCR2/MCP-1 pathway in MDSC-associated tumor progression in murine lung cancer models. Phenotypic profiling revealed maximal expression of CCR2 by tumor-resident MDSCs, and MCP-1 by transplanted TC1 tumor cells, respectively. Use of CCR2-knockout (CCR2-KO) mice showed dependence of tumor growth on CCR2 signaling. Tumors in CCR2-KO mice had fewer CCR2low MDSCs, CD4 T cells and Tregs than WT mice, and increased infiltration by CD8 T cells producing IFN-γ and granzyme-B. Effects were MDSC specific, since WT and CCR2-KO conventional T (Tcon) cells had comparable proliferation and production of inflammatory cytokines, and suppressive functions of WT and CCR2-KO Foxp3+ Treg cells were also similar. We used a thioglycolate-induced peritonitis model to demonstrate a role for CCR2/MCP-1 in trafficking of CCR2+ cells to an inflammatory site, and showed the ability of a CCR2 antagonist to inhibit such trafficking. Use of this CCR2 antagonist promoted anti-tumor immunity and limited tumor growth. In summary, tumor cells are the prime source of MCP-1 that promotes MDSC recruitment, and our genetic and pharmacologic data demonstrate that CCR2 targeting may be an important component of cancer immunotherapy.

MEF2D sustains activation of effector Foxp3+ Tregs during transplant survival and anticancer immunity.

The transcription factor MEF2D is important in the regulation of differentiation and adaptive responses in many cell types. We found that among T cells, MEF2D gained new functions in Foxp3+ T regulatory (Treg) cells due to its interactions with the transcription factor Foxp3 and its release from canonical partners, like histone/protein deacetylases. Though not necessary for the generation and maintenance of Tregs, MEF2D was required for the expression of IL-10, CTLA4, and Icos, and for the acquisition of an effector Treg phenotype. At these loci, MEF2D acted both synergistically and additively to Foxp3, and downstream of Blimp1. Mice with the conditional deletion in Tregs of the gene encoding MEF2D were unable to maintain long-term allograft survival despite costimulation blockade, had enhanced antitumor immunity in syngeneic models, but displayed only minor evidence of autoimmunity when maintained under normal conditions. The role played by MEF2D in sustaining effector Foxp3+ Treg functions without abrogating their basal actions suggests its suitability for drug discovery efforts in cancer therapy.

Donor bone-marrow CXCR4+ Foxp3+ T-regulatory cells are essential for costimulation blockade-induced long-term survival of murine limb transplants.

Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM. Surprisingly, success of either protocol required a bone-marrow-associated, radiation-sensitive cell population, since long-bone removal or pre-transplant donor irradiation prevented long-term engraftment. Rejection also occurred if Rag1-/- donors were used, or if donors were treated with a CXCR4 inhibitor to mobilize donor BM cells pre-transplant. Donor bone-marrow contained a large population of Foxp3+ T-regulatory (Treg) cells, and donor Foxp3+ Treg depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed diphtheria toxin receptor, restored rejection with either protocol. Rejection also occurred if CXCR4 was deleted from donor Tregs pre-transplant. Hence, long-term VCA survival is possible across a full MHC disparity using peritransplant costimulation blockade-based approaches, but unexpectedly, the efficacy of costimulation blockade requires the presence of a radiation-sensitive, CXCR4+ Foxp3+ Treg population resident within donor BM.

Inhibiting the coregulator CoREST impairs Foxp3+ Treg function and promotes antitumor immunity.

Foxp3+ Tregs are key to immune homeostasis, but the contributions of various large, multiprotein complexes that regulate gene expression remain unexplored. We analyzed the role in Tregs of the evolutionarily conserved CoREST complex, consisting of a scaffolding protein, Rcor1 or Rcor2, plus Hdac1 or Hdac2 and Lsd1 enzymes. Rcor1, Rcor2, and Lsd1 were physically associated with Foxp3, and mice with conditional deletion of Rcor1 in Foxp3+ Tregs had decreased proportions of Tregs in peripheral lymphoid tissues and increased Treg expression of IL-2 and IFN-γ compared with what was found in WT cells. Mice with conditional deletion of the gene encoding Rcor1 in their Tregs had reduced suppression of homeostatic proliferation, inability to maintain long-term allograft survival despite costimulation blockade, and enhanced antitumor immunity in syngeneic models. Comparable findings were seen in WT mice treated with CoREST complex bivalent inhibitors, which also altered the phenotype of human Tregs and impaired their suppressive function. Our data point to the potential for therapeutic modulation of Treg functions by pharmacologic targeting of enzymatic components of the CoREST complex and contribute to an understanding of the biochemical and molecular mechanisms by which Foxp3 represses large gene sets and maintains the unique properties of this key immune cell.

Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells.

Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransferases (HATs) in Foxp3+ T-regulatory (Treg) cells are unexplored, despite the general importance of these enzymes in cell biology. We now show that two prototypical GNAT family members, GCN5 (general control nonrepressed-protein 5, lysine acetyltransferase (KAT)2a) and p300/CBP-associated factor (p300/CBP-associated factor (PCAF), Kat2b) contribute to Treg functions through partially distinct and partially overlapping mechanisms. Deletion of Gcn5 or PCAF did not affect Treg development or suppressive function in vitro, but did affect inducible Treg (iTreg) development, and in vivo, abrogated Treg-dependent allograft survival. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acute allograft rejection. PCAF deletion also enhanced anti-tumor immunity in immunocompetent mice. Dual deletion of GCN5 and PCAF led to decreased Treg stability and numbers in peripheral lymphoid tissues, and mice succumbed to severe autoimmunity by 3-4 weeks of life. These data indicate that HATs of the GNAT family have contributions to Treg function that cannot be replaced by the functions of previously characterized Treg HATs (CBP, p300, and Tip60), and may be useful targets in immuno-oncology.

Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer.

Data from mouse tumor models suggest that tumor-associated monocyte/macrophage lineage cells (MMLCs) dampen antitumor immune responses. However, given the fundamental differences between mice and humans in tumor evolution, genetic heterogeneity, and immunity, the function of MMLCs might be different in human tumors, especially during early stages of disease. Here, we studied MMLCs in early-stage human lung tumors and found that they consist of a mixture of classical tissue monocytes and tumor-associated macrophages (TAMs). The TAMs coexpressed M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors. Functionally, TAMs did not primarily suppress tumor-specific effector T cell responses, whereas tumor monocytes tended to be more T cell inhibitory. TAMs expressing relevant MHC class I/tumor peptide complexes were able to activate cognate effector T cells. Mechanistically, programmed death-ligand 1 (PD-L1) expressed on bystander TAMs, as opposed to PD-L1 expressed on tumor cells, did not inhibit interactions between tumor-specific T cells and tumor targets. TAM-derived PD-L1 exerted a regulatory role only during the interaction of TAMs presenting relevant peptides with cognate effector T cells and thus may limit excessive activation of T cells and protect TAMs from killing by these T cells. These results suggest that the function of TAMs as primarily immunosuppressive cells might not fully apply to early-stage human lung cancer and might explain why some patients with strong PD-L1 positivity fail to respond to PD-L1 therapy.

MEF2 and the tumorigenic process, hic sunt leones.

While MEF2 transcription factors are well known to cooperate in orchestrating cell fate and adaptive responses during development and adult life, additional studies over the last decade have identified a wide spectrum of genetic alterations of MEF2 in different cancers. The consequences of these alterations, including triggering and maintaining the tumorigenic process, are not entirely clear. A deeper knowledge of the molecular pathways that regulate MEF2 expression and function, as well as the nature and consequences of MEF2 mutations are necessary to fully understand the many roles of MEF2 in malignant cells. This review discusses the current knowledge of MEF2 transcription factors in cancer.

Human neutrophils can mimic myeloid-derived suppressor cells (PMN-MDSC) and suppress microbead or lectin-induced T cell proliferation through artefactual mechanisms.

We report that human conventional CD15+ neutrophils can be isolated in the peripheral blood mononuclear cell (PBMC) layer during Ficoll gradient separation, and that they can impair T cell proliferation in vitro without concomitant neutrophil activation and killing. This effect was observed in a total of 92 patients with organ transplants, lung cancer or anxiety/depression, and in 18 healthy donors. Although such features are typically associated in the literature with the presence of certain myeloid-derived suppressor cell (PMN-MDSC) populations, we found that commercial centrifuge tubes that contained membranes or gels for PBMC isolation led to up to 70% PBMC contamination by CD15+ neutrophils, with subsequent suppressive effects in certain cellular assays. In particular, the suppressive activity of human MDSC should not be evaluated using lectin or microbead stimulation, whereas assays involving soluble or plate-bound antibodies or MLR are unaffected. We conclude that CD15+ neutrophil contamination, and associated effects on suppressor assays, can lead to significant artefacts in studies of human PMN-MDSC.

Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors.

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin's favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.

Active site-targeted covalent irreversible inhibitors of USP7 impair the functions of Foxp3+ T-regulatory cells by promoting ubiquitination of Tip60.

Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7) is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-generation USP7 inhibitor with improved potency and selectivity. P217564 selectively targets the catalytic cleft of USP7 and modifies its active site cysteine (C223) by forming a covalent adduct. Irreversible inhibition of USP7 results in durable downstream biological responses in cells, including down-regulation of Tip60 and consequent impairment of Treg suppressive function. In addition, we demonstrate that both USP7 and various USP7 substrates are subjected to Lys48-mediated ubiquitin modification, consistent with increased proteasomal degradation of these proteins because of USP7 inhibition.

The Effects of Tacrolimus on T-Cell Proliferation Are Short-Lived: A Pilot Analysis of Immune Function Testing.

BACKGROUND: Optimal immunosuppression after organ transplant should balance the risks of rejection, infection, and malignancy while minimizing barriers to adherence including frequent or time-sensitive dosing. There is currently no reliable immune function assay to directly measure the degree of immunosuppression after transplantation. METHODS: We developed an immune function assay to mea//sure T-cell proliferation after exposure to immunosuppression in vivo. We tested the assay in mice, and then piloted the approach using single time point samples, 11 pediatric kidney transplant recipients prescribed tacrolimus, mycophenolate, and prednisone 6 months to 5 years posttransplant, with no history of rejection, opportunistic infection, or cancer. Twelve healthy adults were controls. RESULTS: We demonstrated that our assay can quantify suppression of murine T-cell proliferation after tacrolimus treatment in vivo. In humans, we found a mean 25% reduction in CD4 and CD8 T-cell proliferation in pediatric renal transplant recipients on triple immunosuppression compared with adult healthy controls, but the pilot results were not statistically significant nor correlated with serum tacrolimus levels. We observed that cell processing and washing reduced the effects of tacrolimus on T-cell proliferation, as did discontinuation of tacrolimus treatment shortly before sampling. CONCLUSIONS: T-cell proliferation is currently not suitable to measure immunosuppression because sample processing diminishes observable effects. Future immune function testing should focus on fresh samples with minimal washing steps. Our results also emphasize the importance of adherence to immunosuppressive treatment, because T-cell proliferation recovered substantially after even brief discontinuation of tacrolimus.