Soluble CD163: a novel independent prognostic biomarker in patients with metastatic renal cell carcinoma.

The hemoglobin-haptoglobin scavenger receptor CD163 is present in both a membrane-bound form on monocytes and macrophages (mCD163) and a shed soluble circulating form (sCD163). CD163 is a well-described marker of M2-like tumor-associated macrophages, but in patients with metastatic renal cell carcinoma (mRCC), monocyte mCD163 and serum sCD163 levels have not previously been investigated and associated with patient overall survival (OS). Here, we report mCD163 expression on peripheral blood monocytes, as well as sCD163 serum levels, in samples from 89 patients newly diagnosed with mRCC and 20 healthy controls. We found that in mRCC patients, compared to healthy controls, monocyte mCD163 levels were reduced (P < 0.001) whereas serum sCD163 levels were increased (P = 0.004). Moreover, an inverse correlation between mCD163 and sCD163 levels (P = 0.04) was shown. In survival analyses, intermediary levels of monocyte mCD163 were associated with longest OS, compared to both lower and higher mCD163 levels, which were both associated with worse outcomes (P < 0.01). Further, higher levels of sCD163 at diagnosis were associated with poor OS in both univariate (P < 0.001) and multivariate analysis (HR = 1.28; 95%CI 1.09-1.50, P = 0.002). Importantly, stratification by low vs. high sCD163 was able to separate patients with International Metastatic RCC Database Consortium (IMDC) intermediate risk (IMDCINT) into two subgroups with different OS (P = 0.03): IMDCINT-sCD163LOW showed survival similar to IMDCFAV patients, and IMDCINT-sCD163HIGH showed survival similar to IMDCPOOR patients. Thus, baseline sCD163 is a novel independent biomarker of OS in mRCC, and using sCD163 as an add-on biomarker may improve prognostic value for patients in the heterogenous IMDC intermediate group.

STAT3 is over-activated within CD163pos bone marrow macrophages in both Multiple Myeloma and the benign pre-condition MGUS.

Tumour-associated macrophages (TAMs) support cancer cell survival and suppress anti-tumour immunity. Tumour infiltration by CD163pos TAMs is associated with poor outcome in several human malignancies, including multiple myeloma (MM). Signal transducer and activator of transcription 3 (STAT3) is over-activated in human cancers, and specifically within TAMs activation of STAT3 may induce an immunosuppressive (M2-like) phenotype. Therefore, STAT3-inhibition in TAMs may be a future therapeutic strategy.We investigated TAM markers CD163, CD206, and activated STAT3 (pSTAT3) in patients with MGUS (n = 32) and MM (n = 45), as well as healthy controls (HCs, n = 13).Blood levels of the macrophage biomarkers sCD163 and sCD206, and circulating cytokines, as well as bone marrow mRNA expression of CD163 and CD206, were generally increased in MGUS and MM patients, compared to HCs, but to highly similar levels. By immunohistochemistry, bone marrow levels of pSTAT3 were increased specifically within CD163pos cells in both MGUS and MM patients.In conclusion, macrophage-related inflammatory changes, including activation of STAT3, were present already at the MGUS stage, at similar levels as in MM. Specific increase in pSTAT3 levels within CD163pos cells supports that the CD163 scavenger receptor may be a useful target for future delivery of STAT3-inhibitory drugs to TAMs in MM patients.

The effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma.

OBJECTIVE: The subset distribution and immunophenotype of circulating immune cells ("peripheral blood immune cell profile") may reflect tumor development and response to cancer treatment. In order to use the peripheral blood immune cell profile as biomarker to monitor patients over time, it is crucial to know how immune cell subsets respond to therapeutic interventions. In this study, we investigated the effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma (CC). METHODS: The subset distribution and immunophenotype of T cells (CD3+CD56-), CD56dim NK cells (CD3-CD56dim), CD56bright NK cells (CD3-CD56bright) and NKT-like cells (CD3+CD56+) were studied in preoperative and postoperative peripheral blood mononuclear cell (PBMC) samples of 24 patients with CC by multiparameter flow cytometry. Changes in immunophenotype of circulating immune cells after tumor resection were studied in patients treated with and without (capecitabine-based) adjuvant therapy. RESULTS: The NKT-like cell (% of total PBMCs) and CD8+ T cell (% of total T cells) populations expanded in the peripheral blood of non-adjuvant-treated CC patients after surgery. NK- and NKT-like cells showed upregulation of activating receptors and downregulation of inhibitory receptors in non-adjuvant-treated CC patients after surgery. These changes were not observed in the peripheral blood of adjuvant-treated CC patients. CONCLUSIONS: Our results suggest tumor-induced suppression of NK- and NKT-like cells in CC patients, an effect that could not be detected after tumor resection. In contrast, adjuvant therapy maintained tumor-induced immunosuppression of NK- and NKT-like cells in CC patients.

Application Settings versus Fixed Photomultiplier Tube Voltages for Optimal Cytometer Stability over Time, and Effect on Reusing a Compensation Matrix.

In flow cytometry, a compensation matrix is commonly reused over time, especially in clinical laboratories, to save time and reagents. However, generating a same-day compensation matrix is considered the best practice by many experts. BD Biosciences developed Cytometer Setup and Tracking software to deliver proper instrument characterization, performance tracking, and stability. BD's "Application Settings" enable daily cytometer adjustments of photomultiplier tube (PMT) settings to correct for day-to-day variations in instrument performance. Here, we investigated if using Application Settings would improve data stability over time, including the impact on data stability when reusing a compensation matrix. We consecutively analyzed peripheral blood mononuclear cell (PBMC) aliquots from a single healthy donor together with 8-peak Rainbow beads and daily compensation controls (22 runs in total over 6.5 months). We found larger variation within both PBMC subset quantifications and median fluorescence intensity (MFI) levels when using Application Settings (i.e., daily adjusted PMTs) compared to fixed PMT voltages (both with the same Day 0 compensation matrix applied). This larger variation was partly due to errors in compensation, but was also seen for Rainbow beads MFI data (not impacted by compensation), and thus likely produced by imprecise adjustments of PMTs by Applications Settings. Notably, the larger variation observed with Application Settings was most pronounced on a few days of the experiment with very large deviations, whereas on most days Application Settings and Fixed PMTs performed similar. The present results call for caution in using Application Settings in longitudinal studies, especially if also reusing a compensation matrix. In contrast, reusing a compensation matrix over time with fixed PMT voltages yielded stable results comparable with running same-day compensation controls. © 2020 International Society for Advancement of Cytometry.

Circulating extracellular vesicle-associated CD163 and CD206 in multiple myeloma.

OBJECTIVES: Extracellular vesicles (EVs) are important for intercellular signalling in cancer. Tumour-associated macrophages, expressing the haemoglobin-haptoglobin and mannose receptors CD163 and CD206, are crucial for cancer progression. We recently identified CD163 on EVs in the circulation as a fraction of total soluble CD163 (sCD163). Here, we investigated the presence of CD163 and CD206-positive EVs (EV-CD163, EV-CD206) in patients with multiple myeloma (MM). METHODS: We enrolled patients with MM (n = 32), monoclonal gammopathy of undetermined significance (MGUS) (n = 8) and healthy donors (n = 16). Plasma protein levels were determined by ELISA before and after vesicle precipitation. Monocytes were examined by flow cytometry, and leucocyte CD163 mRNA by qPCR. RESULTS: Fractions of EV-CD163 and EV-CD206 were significantly elevated in patients with newly diagnosed MM (median = 39.8%, 76.5%, respectively) compared to patients with relapse (15.6%, P = .02, 42.5%, P = .003), remission (16.9%, P < .0001, 25.2%, P < .0001), MGUS (17.8%, P < .01, 33.1%, P = .0005) and healthy donors (14.8%, P < .0001, 35.5%, P < .0001). Whole blood CD163 mRNA did not vary between the groups. The intermediate monocyte subset showed a higher CD163 expression in newly diagnosed patients. CONCLUSIONS: Our results indicate that macrophage-derived EVs may play a role in the late phase of malignant progression of MM, and encourage further EV investigations in functional experiments.

CD163 as a Biomarker in Colorectal Cancer: The Expression on Circulating Monocytes and Tumor-Associated Macrophages, and the Soluble Form in the Blood.

The macrophage-associated molecule CD163 has been reported as a prognostic biomarker in different cancer types, but its role in colorectal cancer (CRC) is unclear. We studied CD163 in the tumor microenvironment and circulation of patients with CRC in relation to clinicopathological parameters. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum sCD163 levels and multiparameter flow cytometry was used to study the peripheral blood monocytes and their CD163 expression in CRC patients (N = 78) and healthy donors (N = 50). The distribution of tumor-associated macrophages (TAMs) was studied in primary colorectal tumors with multiplex immunofluorescence. We showed that CRC patients with above-median sCD163 level had a shorter overall survival (OS, p = 0.035) as well as disease-free survival (DFS, p = 0.005). The above-median sCD163 remained significantly associated with a shorter DFS in the multivariate analysis (p = 0.049). Moreover, a shorter OS was observed in CRC patients with an above-median total monocyte percentage (p = 0.007). The number and phenotype of the stromal and intraepithelial TAMs in colorectal tumors were not associated with clinical outcome. In conclusion, sCD163 and monocytes in the circulation may be potential prognostic biomarkers in CRC patients, whereas TAMs in the tumor showed no association with clinical outcome. Thus, our results emphasize the importance of the innate systemic immune response in CRC disease progression.

STAT3 inhibition specifically in human monocytes and macrophages by CD163-targeted corosolic acid-containing liposomes.

Tumor-associated macrophages (TAMs) are of major importance in cancer-related immune suppression, and tumor infiltration by CD163pos TAMs is associated with poor outcome in most human cancers. Therefore, therapeutic strategies for reprogramming TAMs from a tumor-supporting (M2-like) phenotype towards a tumoricidal (M1-like) phenotype are of great interest. Activation of the transcription factor STAT3 within the tumor microenvironment is associated with worse prognosis, and STAT3 activation promotes the immunosuppressive phenotype of TAMs. Therefore, we aimed to develop a drug for inhibition of STAT3 specifically within human TAMs by targeting the endocytic CD163 scavenger receptor, which is highly expressed on TAMs. Here, we report the first data on a CD163-targeted STAT3-inhibitory drug consisting of corosolic acid (CA) packaged within long-circulating liposomes (LCLs), which are CD163-targeted by modification with monoclonal anti-CD163 antibodies (αCD163)-CA-LCL-αCD163. We show, that activation of STAT3 (by phosphorylation) was inhibited by CA-LCL-αCD163 specifically within CD163pos cells, with minor effect on CD163neg cells. Furthermore, CA-LCL-αCD163 inhibited STAT3-regulated gene expression of IL-10, and increased expression of TNFα, thus indicating a pro-inflammatory effect of the drug on human macrophages. This M1-like reprogramming at the mRNA level was confirmed by significantly elevated levels of pro-inflammatory cytokines (IFNγ, IL-12, TNFα, IL-2) in the culture medium. Since liposomes are attractive vehicles for novel anti-cancer drugs, and since direct TAM-targeting may decrease adverse effects of systemic inhibition of STAT3, the present results encourage future investigation of CA-LCL-αCD163 in the in vivo setting.

Characterization of circulating T-, NK-, and NKT cell subsets in patients with colorectal cancer: the peripheral blood immune cell profile.

OBJECTIVE: As the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data. METHODS: Using multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56-), CD56dim NK cells (CD3-CD56dim), CD56bright NK cells (CD3-CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors. RESULTS: CRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients. CONCLUSION: The altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.

Probiotic treatment protects against the pro-depressant-like effect of high-fat diet in Flinders Sensitive Line rats.

Major depressive disorder (MDD) is highly associated with dysmetabolic conditions, such as obesity and diabetes mellitus type 2, and the gut microbiota may interact with both disease entities. We have previously shown that a high-fat diet (HFD) exacerbated depressive-like behaviour uniquely in Flinders Sensitive Line (FSL) rats that inherently present with an increased level of depressive-like behaviour compared with Flinders Resistant Line (FRL) rats. We therefore investigated whether multispecies probiotics possessed anti-depressant-like effect in FSL rats or protected against the pro-depressant-like effect of HFD. We also examined blood and cerebral T cell subsets as well as plasma cytokines. Lastly, we investigated the effect of HFD in outbred Sprague-Dawley (SD) rats to substantiate the association between depressive-like behaviour and any immunological measures affected by HFD. HFD exacerbated the depressive-like behaviour in FSL rats in the forced swim test, whereas SD rats remained unaffected. Probiotic treatment completely precluded the pro-depressant-like effect of HFD, but it did not affect FSL rats on control diet. Cerebral T lymphocyte CD4/8 ratios closely mirrored the behavioural changes, whereas the proportions of Treg and Th17 subsets were unaltered. No association between blood and brain CD4/8 ratios were evident; nor did plasma cytokine levels change as a consequence of HFD of probiotic treatment. Our findings suggest that MDD may hold a dysmetabolic component that responds to probiotic treatment. This finding has wide implications owing to the high metabolic comorbidity in MDD. Furthermore, the close association between depressive-like behaviour and cerebral T cell populations demonstrate lymphocyte-brain interactions as a promising future research area in the field of psychoneuroimmunology.

Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line.

The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differentiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting and in normal leukocyte subsets by flow cytometry and found a heterogeneous expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor (M3-mAChR) and α7 nicotinic acetylcholine receptor (α7-nAChR). We then evaluated NNCS role in differentiation of human NB-4 acute promyelocytic leukemia cell line and discovered a dramatic induction of M3-mAChR after all-trans retinoic acid (ATRA) treatment (p<0.0001). Adding carbachol which is a cholinergic agonist to the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with ATRA treatment alone (p<0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 maturation. The combination of carbachol and ATRA treatment for 72h also resulted in decreased viability and increased cleaved caspase-3 expression when compared with ATRA treatment alone (p<0.05). However, this combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4 cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced maturation and death of ATRA-induced differentiated NB-4 cells.

Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages.

Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. In contrast, B-cells, T-cells, and NK-cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc-blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. Previously, isotype controls have been widely used in flow cytometry assays. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 µg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs. © 2016 International Society for Advancement of Cytometry.

The Immunogenicity of Colorectal Cancer in Relation to Tumor Development and Treatment.

Although most cancer types have been viewed as immunologically silent until recently, it has become increasingly clear that the immune system plays key roles in the course of tumor development. Remarkable progress towards understanding cancer immunogenicity and tumor-immune system interactions has revealed important implications for the design of novel immune-based therapies. Natural immune responses, but also therapeutic interventions, can modulate the tumor phenotype due to selective outgrowth of resistant subtypes. This is the result of heterogeneity of tumors, with genetic instability as a driving force, and obviously changes the immunogenicity of tumors. In this review, we discuss the immunogenicity of colorectal cancer (CRC) in relation to tumor development and treatment. As most tumors, CRC activates the immune system in various ways, and is also capable of escaping recognition and elimination by the immune system. Tumor-immune system interactions underlie the balance between immune control and immune escape, and may differ in primary tumors, in the circulation, and in liver metastases of CRC. Since CRC immunogenicity varies between tumors and individuals, novel immune-based therapeutic strategies should not only anticipate the molecular profile, but also the immunological profile of a specific tumor.

Cytotoxic T lymphocytes and natural killer cells display impaired cytotoxic functions and reduced activation in patients with alcoholic hepatitis.

The dynamics and role of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and NKT cells in the life-threatening inflammatory disease alcoholic hepatitis is largely unknown. These cells directly kill infected and damaged cells through, e.g., degranulation and interferon-γ (IFNγ) production, but cause tissue damage if overactivated. They also assist tissue repair via IL-22 production. We, therefore, aimed to investigate the frequency, functionality, and activation state of such cells in alcoholic hepatitis. We analyzed blood samples from 24 severe alcoholic hepatitis patients followed for 30 days after diagnosis. Ten healthy abstinent volunteers and 10 stable abstinent alcoholic cirrhosis patients were controls. Using flow cytometry we assessed cell frequencies, NK cell degranulation capacity following K562 cell stimulation, activation by natural killer group 2 D (NKG2D) expression, and IL-22 and IFNγ production. In alcoholic hepatitis we found a decreased frequency of CTLs compared with healthy controls (P < 0.001) and a similar trend for NK cells (P = 0.089). The NK cell degranulation capacity was reduced by 25% compared with healthy controls (P = 0.02) and by 50% compared with cirrhosis patients (P = 0.04). Accordingly, the NKG2D receptor expression was markedly decreased on NK cells, CTLs, and NKT cells (P < 0.05, all). The frequencies of IL-22-producing CTLs and NK cells were doubled compared with healthy controls (P < 0.05, all) but not different from cirrhosis patients. This exploratory study for the first time showed impaired cellular cytotoxicity and activation in alcoholic hepatitis. This is unlikely to cause hepatocyte death but may contribute toward the severe immune incompetence. The results warrant detailed and mechanistic studies.

Tubulointerstitial nephritis in a patient with probable autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is caused by a nonmalignant defective Fas-mediated apoptosis. The main clinical manifestations are chronic lymphadenopathy, splenomegaly, and autoimmune cytopenia. Most patients with ALPS have a FAS germline mutation. ALPS has occasionally been associated with glomerulonephritis and we present the first report of tubulointerstitial nephritis associated with probable ALPS. A 5-year-old girl presented with fever, vomiting, hypertension, and azotemia. No autoantibodies, viral, or streptococcal antibodies were detected. A renal biopsy showed small-vessel vasculitis with normal glomeruli and inflammation in the interstitium. The patient responded to prednisolone treatment and obtained a full renal recovery. Symptoms of connective tissue disorder supervened and after the development of more pronounced splenomegaly, a diagnosis of ALPS was confirmed.

Increased plasma levels of IL-21 and IL-23 in spondyloarthritis are not associated with clinical and MRI findings.

We have investigated the role of the Th17-related cytokines interleukin-17A (IL-17A), IL-21, and IL-23 in spondyloarthritis (SpA) by examining their association with disease activity and magnetic resonance imaging (MRI) findings in patients with SpA (n = 80). Furthermore, to investigate the cellular origins of the cytokines, paired mononuclear cells from blood and synovial fluid were examined for the expression of IL-17A, IL-21, and IL-23R using multicolor flow cytometry. Both IL-21 and IL-23 levels were increased in plasma from SpA patients compared with healthy volunteers (P < 0.05), whereas IL-17A was not. A significant correlation was observed between individual levels of IL-21 and IL-23 (r = 0.7, P < 0.001). No association between individual levels of IL-17A, IL-21, and IL-23 with C-reactive protein (CRP), MRI changes, and clinical scoring (BASMI, BASFI, and BASDAI) were observed. The frequency of CD4+CD45RO+ T cells expressing IL-21 and IL-23R was increased in the inflamed SpA joint compared to peripheral blood (P < 0.05). This study demonstrate that the plasma levels of the Th17-related cytokines IL-21 and IL-23, but not IL-17A, are increased in SpA patients, but we did not find evidence that the level of these cytokines reflect disease activity in SpA.

Differential associations between white blood cell counts and fatigue in young and older adults.

BACKGROUND AND AIMS: The aims of this exploratory study were to study whether fatigue might be related to the cellular immune system by 1) analysing if the number of white blood cell subsets are related to fatigue and 2) if possible relationships vary in younger and older community-dwelling individuals. METHODS: The participants were recruited from nine general practitioners in Aarhus County, Denmark and included 196 individuals aged 20-35 years and 314 individuals aged 70-85 years. The white blood cell counts included number of total leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. General fatigue was measured by a question from the SF-12 Vitality-Scale and mobility-related fatigue by the Avlund Mob-T Scale. RESULTS: Total number of lymphocytes was associated with fatigue in the old sample, both in the crude and adjusted analyses. Total number of leukocytes and netrophils were associated with fatigue in both age groups in the crude analyses. In the old sample the estimates were attenuated to non-significance when adjusting for physical activity and disability. In the young sample the estimates stayed statistically significant in the fully adjusted analyses regarding number of neutrophils, while the associations between fatigue and number of leukocytes were attenuated to non-significance when adjusting for depressive mood. CONCLUSION: Total number of leukocytes, lymphocytes and neutrophils were associated with fatigue in both age groups, while the explanatory factors for the associations differed by age group, in that the associations were partly explained by physical activity and disability in the old sample and partly by depressive mood in the young sample. The findings provide initial insight into the potential role of leukocyte, neutrophil and lymphocyte counts in the development of fatigue.

TLR3 ligand polyinosinic:polycytidylic acid induces IL-17A and IL-21 synthesis in human Th cells.

TLR3 and TLR9 recognize the pathogen-associated microbial patterns dsRNA and unmethylated DNA, respectively. The recent discovery that these receptors also recognize endogenous ligands from necrotic material has drawn increased attention to their involvement in autoimmunity. Th cell cytokines IL-17A and IL-21 have been assigned with pivotal roles in the regulation of such autoimmune diseases. IL-17A is the hallmark cytokine of the recently discovered proinflammatory Th cell subset T(H)17. By contrast, the expression of IL-21 does not seem to be limited to a single distinct Th cell subset. We investigated the expression of IL-17A and IL-21 in human CD4+ T cells in response to stimulation with the TLR3 ligand polyinosinic:polycytidylic acid (poly(I:C)) and the TLR9 ligand CpG. We discovered that poly(I:C) induced synthesis of both IL-17A and IL-21. Moreover, we found that poly(I:C) was able to drive the differentiation of naive Th cells into an IL-21 but not into an IL-17A-producing phenotype and did this without affecting the levels of transcription factors T-bet, GATA-3, or retinoic acid receptor-related orphan receptor C. Finally, we found that the IL-21-producing cells that were differentiated in response to poly(I:C) expressed the chemokine receptor CXCR3, which is important in the recruitment of T cells into inflamed joints in rheumatoid arthritis. This is the first report to show that the TLR3 ligand poly(I:C) can directly induce the synthesis of IL-17A and IL-21 and drive differentiation of human naive CD4+ T cells.