Safety, Immunogenicity, and Regimen Selection of Ad26.RSV.preF-Based Vaccine Combinations: A Randomized, Double-blind, Placebo-Controlled, Phase 1/2a Study.

BACKGROUND: Ad26.RSV.preF is an adenovirus serotype 26 vector-based respiratory syncytial virus (RSV) vaccine encoding a prefusion conformation-stabilized RSV fusion protein (preF) that demonstrated robust humoral and cellular immunogenicity and showed promising efficacy in a human challenge study in younger adults. Addition of recombinant RSV preF protein might enhance RSV-specific humoral immune responses, especially in older populations. METHODS: This randomized, double-blind, placebo-controlled, phase 1/2a study compared the safety and immunogenicity of Ad26.RSV.preF alone and varying doses of Ad26.RSV.preF-RSV preF protein combinations in adults aged ≥60 years. This report includes data from cohort 1 (initial safety, n = 64) and cohort 2 (regimen selection, n = 288). Primary immunogenicity and safety analyses were performed 28 days postvaccination (cohort 2) for regimen selection. RESULTS: All vaccine regimens were well tolerated, with similar reactogenicity profiles among them. Combination regimens induced greater humoral immune responses (virus-neutralizing and preF-specific binding antibodies) and similar cellular ones (RSV-F-specific T cells) as compared with Ad26.RSV.preF alone. Vaccine-induced immune responses remained above baseline up to 1.5 years postvaccination. CONCLUSIONS: All Ad26.RSV.preF-based regimens were well tolerated. A combination regimen comprising Ad26.RSV.preF, which elicits strong humoral and cellular responses, and RSV preF protein, which increases humoral responses, was selected for further development. Clinical Trials Registration. NCT03502707.

RSV A2-Based Prefusion F Vaccine Candidates Induce RSV A and RSV B Cross Binding and Neutralizing Antibodies and Provide Protection against RSV A and RSV B Challenge in Preclinical Models.

RSV is divided into two antigenic subtypes, RSV A and RSV B, which is largely based on the variation in the G protein, while the fusion protein F is more conserved and a target for antibody-mediated neutralization. Here we evaluate the breadth of the protective immune responses across RSV A and RSV B subtypes, induced by vaccines based on the RSV A-based fusion protein, stabilized in the prefusion conformation (preF) in preclinical models. Immunization of naïve cotton rats with preF subunit or preF encoded by a replication incompetent Adenoviral 26, induced antibodies capable of neutralizing recent RSV A and RSV B clinical isolates, as well as protective efficacy against a challenge with RSV A and RSV B strains. Similarly, induction of cross-neutralizing antibodies was observed after immunization with Ad26-encoded preF, preF protein or a mix of both (Ad26/preF protein) in RSV pre-exposed mice and African Green Monkeys. Transfer of serum of human subjects immunized with Ad26/preF protein into cotton rats provide protection against challenges with both RSV A and RSV B, with complete protection against both strains observed in the lower respiratory tract. In contrast, almost no protection against RSV A and B infection was observed after the transfer of a human serum pool isolated pre-vaccination. These results collectively show that the RSV A-based monovalent Ad26/preF protein vaccine induced neutralizing antibodies, as well as protection against both RSV A and RSV B subtypes in animals, including by passive transfer of human antibodies alone, suggesting that clinical efficacy against both subtypes can be achieved.

Combination Ad26.RSV.preF/preF protein vaccine induces superior protective immunity compared with individual vaccine components in preclinical models.

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously shown that a prefusion (preF) conformation-stabilized RSV F protein antigen and an adenoviral vector encoding RSV preF protein (Ad26.RSV.preF) are immunogenic and protective in animals when administered as single components. Here, we evaluated a combination of the 2 components, administered as a single injection. Strong induction of both humoral and cellular responses was shown in RSV-naïve and pre-exposed mice and pre-exposed African green monkeys (AGMs). Both components of the combination vaccine contributed to humoral immune responses, while the Ad26.RSV.preF component was the main contributor to cellular immune responses in both mice and AGMs. Immunization with the combination elicited superior protection against RSV A2 challenge in cotton rats. These results demonstrate the advantage of a combination vaccine and support further clinical development.

Antibody effector functions are associated with protection from respiratory syncytial virus.

Respiratory syncytial virus (RSV) infection is a major cause of severe lower respiratory tract infection and death in young infants and the elderly. With no effective prophylactic treatment available, current vaccine candidates aim to elicit neutralizing antibodies. However, binding and neutralization have poorly predicted protection in the past, and accumulating data across epidemiologic cohorts and animal models collectively point to a role for additional antibody Fc-effector functions. To begin to define the humoral correlates of immunity against RSV, here we profiled an adenovirus 26 RSV-preF vaccine-induced humoral immune response in a group of healthy adults that were ultimately challenged with RSV. Protection from infection was linked to opsonophagocytic functions, driven by IgA and differentially glycosylated RSV-specific IgG profiles, marking a functional humoral immune signature of protection against RSV. Furthermore, Fc-modified monoclonal antibodies able to selectively recruit effector functions demonstrated significant antiviral control in a murine model of RSV.

Immunogenicity and protective efficacy of adenoviral and subunit RSV vaccines based on stabilized prefusion F protein in pre-clinical models.

Respiratory Syncytial Virus (RSV) remains a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously described the derivation of an RSV Fusion protein (F) stabilized in its prefusion conformation (preF) as vaccine immunogen and demonstrated superior immunogenicity in naive mice of preF versus wild type RSV F protein, both as protein and when expressed from an Ad26 vaccine vector. Here we address the question if there are qualitative differences between the two vaccine platforms for induction of protective immunity. In naïve mice, both Ad26.RSV.preF and preF protein induced humoral responses, whereas cellular responses were only elicited by Ad26.RSV.preF. In RSV pre-exposed mice, a single dose of either vaccine induced cellular responses and strong humoral responses. Ad26-induced RSV-specific cellular immune responses were detected systemically and locally in the lungs. Both vaccines showed protective efficacy in the cotton rat model, but Ad26.RSV.preF conferred protection at lower virus neutralizing titers in comparison to RSV preF protein. Factors that may contribute to the protective capacity of Ad26.RSV.preF elicited immunity are the induced IgG2a antibodies that are able to engage Fcγ receptors mediating Antibody Dependent Cellular Cytotoxicity (ADCC), and the induction of systemic and lung resident RSV specific CD8 + T cells. These data demonstrate qualitative improvement of immune responses elicited by an adenoviral vector based vaccine encoding the RSV preF antigen compared to the subunit vaccine in small animal models which may inform RSV vaccine development.

Low-dose Ad26.COV2.S protection against SARS-CoV-2 challenge in rhesus macaques.

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26)-vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. To evaluate reduced doses of Ad26.COV2.S, 30 rhesus macaques were immunized once with 1 × 1011, 5 × 1010, 1.125 × 1010, or 2 × 109 viral particles (vp) Ad26.COV2.S or sham and were challenged with SARS-CoV-2. Vaccine doses as low as 2 × 109 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125 × 1010 vp were required for protection in nasal swabs. Activated memory B cells and binding or neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show enhancement of disease. These data demonstrate that a single immunization with relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques, although a higher vaccine dose may be required for protection in the upper respiratory tract.

Ad26.COV2.S protects Syrian hamsters against G614 spike variant SARS-CoV-2 and does not enhance respiratory disease.

Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy, and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1 × 109 and 1 × 1010 VP elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers that was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these preclinical data confirm efficacy of a one-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.

Low-Dose Ad26.COV2.S Protection Against SARS-CoV-2 Challenge in Rhesus Macaques.

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26) vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. In this study, we evaluated the immunogenicity and protective efficacy of reduced doses of Ad26.COV2.S. 30 rhesus macaques were immunized once with 1×10 11 , 5×10 10 , 1.125×10 10 , or 2×10 9 vp Ad26.COV2.S or sham and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes. Vaccine doses as low as 2×10 9 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125×10 10 vp were required for protection in nasal swabs. Activated memory B cells as well as binding and neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show evidence of virologic, immunologic, histopathologic, or clinical enhancement of disease compared with sham controls. These data demonstrate that a single immunization with a relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques. Moreover, our findings show that a higher vaccine dose may be required for protection in the upper respiratory tract compared with the lower respiratory tract.

A Double-Blind, Randomized, Placebo-Controlled Phase 1 Study of Ad26.ZIKV.001, an Ad26-Vectored Anti-Zika Virus Vaccine.

BACKGROUND: Zika virus (ZIKV) may cause severe congenital disease after maternal-fetal transmission. No vaccine is currently available. OBJECTIVE: To assess the safety and immunogenicity of Ad26.ZIKV.001, a prophylactic ZIKV vaccine candidate. DESIGN: Phase 1 randomized, double-blind, placebo-controlled clinical study. (ClinicalTrials.gov: NCT03356561). SETTING: United States. PARTICIPANTS: 100 healthy adult volunteers. INTERVENTION: Ad26.ZIKV.001, an adenovirus serotype 26 vector encoding ZIKV M-Env, administered in 1- or 2-dose regimens of 5 × 1010 or 1 × 1011 viral particles (vp), or placebo. MEASUREMENTS: Local and systemic adverse events; neutralization titers by microneutralization assay (MN50) and T-cell responses by interferon-γ enzyme-linked immunospot and intracellular cytokine staining; and protectivity of vaccine-induced antibodies in a subset of participants through transfer in an exploratory mouse ZIKV challenge model. RESULTS: All regimens were well tolerated, with no safety concerns identified. In both 2-dose regimens, ZIKV neutralizing titers peaked 14 days after the second vaccination, with geometric mean MN50 titers (GMTs) of 1065.6 (95% CI, 494.9 to 2294.5) for 5 × 1010 vp and 956.6 (595.8 to 1535.8) for 1 × 1011 vp. Titers persisted for at least 1 year at a GMT of 68.7 (CI, 26.4-178.9) for 5 × 1010 vp and 87.0 (CI, 29.3 to 258.6) for 1 × 1011 vp. A 1-dose regimen of 1 × 1011 vp Ad26.ZIKV.001 induced seroconversion in all participants 56 days after the first vaccination (GMT, 103.4 [CI, 52.7 to 202.9]), with titers persisting for at least 1 year (GMT, 90.2 [CI, 38.4 to 212.2]). Env-specific cellular responses were induced. Protection against ZIKV challenge was observed after antibody transfer from participants into mice, and MN50 titers correlated with protection in this model. LIMITATION: The study was conducted in a nonendemic area, so it did not assess safety and immunogenicity in a flavivirus-exposed population. CONCLUSION: The safety and immunogenicity profile makes Ad26.ZIKV.001 a promising candidate for further development if the need reemerges. PRIMARY FUNDING SOURCE: Janssen Vaccines and Infectious Diseases.

Adenovector 26 encoded prefusion conformation stabilized RSV-F protein induces long-lasting Th1-biased immunity in neonatal mice.

While RSV is a major cause of respiratory morbidity in infants, vaccine development is hindered by the immaturity and Th2-bias of the infant immune system and the legacy of enhanced respiratory disease (ERD) after RSV infection following immunization with formalin inactivated (FI)-RSV vaccine in earlier clinical trials. Preclinical studies have demonstrated that an adenoviral vector-based RSV F vaccine candidate (Ad26.RSV.FA2) induces Th1-biased protective immune responses, without signs of ERD upon subsequent RSV challenge. We here developed an Ad26 vector encoding the RSV F protein stabilized in its prefusion conformation (Ad26.RSV.preF). In adult mice, Ad26.RSV.preF induced superior, Th1-biased IgG2a-dominated humoral responses as compared to Ad26.RSV.FA2, while maintaining the strong Th1-biased cellular responses. Similar to adult mice, Ad26.RSV.preF induced robust and durable humoral immunity in neonatal mice, again characterized by IgG2a-dominated RSV F-binding antibodies, and high and stable virus-neutralizing titers. In addition, vaccine-elicited cellular immune responses were durable and characterized by IFN-γ-producing CD4+ and CD8+ T cells, with a profound Th1 bias. In contrast, immunization of neonatal mice with FI-RSV resulted in IgG1 RSV F-binding antibodies associated with a Th2 phenotype, no detectable virus-neutralizing antibodies, and a Th2-biased cellular response. These results are supportive for the clinical development of Ad26.RSV.preF for use in infants.

Adenoviral vector type 26 encoding Zika virus (ZIKV) M-Env antigen induces humoral and cellular immune responses and protects mice and nonhuman primates against ZIKV challenge.

In 2015, there was a large outbreak of Zika virus (ZIKV) in Brazil. Despite its relatively mild impact on healthy adults, ZIKV infection during pregnancy has been associated with severe birth defects. Currently, there is no ZIKV vaccine available, but several vaccine candidates based on the ZIKV membrane (M) and envelope (Env) structural proteins showed promising results in preclinical and clinical studies. Here, the immunogenicity and protective efficacy of a non-replicating adenoviral vector type 26 (Ad26) that encodes the ZIKV M-Env antigens (Ad26.ZIKV.M-Env) was evaluated in mice and non-human primates (NHP). Ad26.ZIKV.M-Env induced strong and durable cellular and humoral immune responses in preclinical models. Humoral responses were characterized by Env-binding and ZIKV neutralizing antibody responses while cellular responses were characterized by ZIKV reactive CD4+ and CD8+ T cells. Importantly, a single immunization with a very low dose of 4x107 vp of Ad26.ZIKV.M-Env protected mice from ZIKV challenge. In NHP, a single immunization with a typical human dose of 1x1011 vp of Ad26.ZIKV.M-Env also induced Env-binding and ZIKV neutralizing antibodies and Env and M specific cellular immune responses that associated with complete protection against viremia from ZIKV challenge as measured in plasma and other body fluids. Together these data provide the rationale to progress the Ad26.ZIKV.M-Env candidate vaccine to clinical testing.

Evaluation of Immunophenotypic and Molecular Biomarkers for Sézary Syndrome Using Standard Operating Procedures: A Multicenter Study of 59 Patients.

Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses.

EPHA4 is overexpressed but not functionally active in Sézary syndrome.

EPHA4 belongs to the largest subfamily of receptor tyrosine kinases. In addition to its function during development, overexpression of EPHA4 in tumors has been correlated with increased proliferation, migration and poor survival. Several genome-wide transcription profiling studies have demonstrated high EPHA4 expression in Sézary syndrome (SS), a leukemic variant of cutaneous CD4+ T-cell lymphoma (CTCL) with an aggressive clinical course and poor prognosis. In this study we set out to explore the functional role of EPHA4 in SS. Both high EPHA4 mRNA and protein expression was found in circulating SS-cells of patients compared to healthy CD4+ T-cells. However, using a phosphospecific EPHA4 antibody, phosphorylation of the EPHA4 kinase domain was not detected in either circulating or skin residing SS cells. Moreover, treatment with the phosphatase inhibitor pervanadate did not result in detectable phosphorylation of the EPHA4 kinase domain, in either SS cells or in healthy CD4+ T-cells. Thus, the results from our study confirm high EPHA4 expression in SS cells both on the mRNA and protein levels, making EPHA4 a good diagnostic marker. However, the overexpressed EPHA4 does not appear to be functionally active and its overexpression might be secondary to other oncogenic drivers in SS, like STAT3 and TWIST1.

Exploring the IL-21-STAT3 axis as therapeutic target for Sézary syndrome.

Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease.

Caspase-14-deficient mice are more prone to the development of parakeratosis.

Caspase-14 is an important protease in the proper formation of a fully functional skin barrier. Newborn mice that are deficient in caspase-14 exhibit increased transepidermal water loss and are highly sensitive to UVB-induced photodamage. Decreased caspase-14 expression and incomplete caspase-14 processing in lesional psoriatic parakeratotic stratum corneum has been reported previously. In this study, we show that caspase-14-deficient skin frequently displays incompletely cornified cells in the transitional zone between the granular and the cornified layers, pointing to a delay in cornification. We also demonstrate that after challenge of epidermal permeability barrier function by repetitive acetone treatment, a higher incidence of large parakeratotic plaques was observed in caspase-14-deficient skin. Furthermore, caspase-14-deficient mice are more prone than control mice to the development of parakeratosis upon induction of psoriasis-like dermatitis by imiquimod treatment. These results show that lack of caspase-14 expression predisposes to the development of parakeratosis and that caspase-14 has an important role in keratinocyte terminal differentiation and the maintenance of normal stratum corneum, especially in conditions causing epidermal hyperproliferation.

A novel mouse model for Sézary syndrome using xenotransplantation of Sézary cells into immunodeficient RAG2(-/-) γc(-/-) mice.

Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma with CD4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG2(-/-) γc(-/-) mice that are completely devoid of T-, B- and NK-cell activity. Injection of the SS cell line SeAx led to long-term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT-78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG2(-/-) γc(-/-) mice results in long-term systemic repopulation of the mice, thereby providing a novel mouse model for Sézary syndrome.

NOTCH1 signaling as a therapeutic target in Sézary syndrome.

NOTCH signaling is important for development and tissue homeostasis and is activated in many human cancers. We investigated a role for NOTCH1 signaling in Sézary syndrome (SS), a cutaneous T-cell lymphoma in which CD4+ tumor cells (Sézary cells) are present in the skin, lymph nodes, and peripheral blood. We show consistent expression of activated NOTCH1 by Sézary cells isolated from peripheral blood of SS patients, as well as the SS-derived cell lines SeAx and HuT78. In addition, immunohistochemical stainings of skin biopsies from SS patients showed consistent expression of nuclear NOTCH1 and its downstream target hairy/enhancer of split-1 (HES1) by Sézary cells. We demonstrate that this persistent NOTCH1 activation is not caused by mutations in the coding regions of NOTCH1 and F-box and WD40 domain protein 7 (FBWX7) genes. Inhibition of NOTCH1 signaling by gamma secretase inhibitors decreased cellular viability and induced apoptosis of Sézary cells. These observations argue that NOTCH1 signaling is functionally involved in the pathogenesis of SS, and inhibition of NOTCH1 signaling represents a new therapeutic target for the treatment of SS.

Autocrine IL-21 stimulation is involved in the maintenance of constitutive STAT3 activation in Sézary syndrome.

Sézary syndrome (SS) is a cutaneous T-cell lymphoma (CTCL) with malignant CD4+ T cells (SS cells) in skin, lymph nodes, and blood. Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in SS cells, whereas this activation is lost upon in vitro culturing, indicating that STAT3 activation observed in vivo is the result of activating factors in the micromilieu of the malignant cells. We investigated which factors are involved in STAT3 activation in SS, focusing on cytokines of the common γ-chain family because of their crucial role in T-cell activation. Furthermore, downstream effects of STAT3 signaling in SS cells were assayed. In SS cells, STAT3 was strongly activated by IL-21, and increased expression of IL-21 and its receptor chains was observed in peripheral blood SS cells. IL-21 and IL-21R protein expression was detectable on neoplastic cells in SS skin biopsies. Using short-term culturing experiments, we demonstrate that IL-21 itself and the α-chain of the IL-2 receptor are STAT3 target genes in SS cells, thereby rendering cells more sensitive to stimulation with the T-cell proliferation and activating cytokine IL-2. Combined, our data point toward a pivotal role for an autocrine positive feedback loop involving IL-21 and consequent persistent STAT3 activation in the pathogenesis of SS, thereby indicating IL-21 and IL-21R as new therapeutical targets.