Objective: In the sanroque mouse model of lupus, pathologic germinal centers (GCs) arise due to increased numbers of follicular helper T (Tfh) cells, resulting in high‐affinity anti–double‐stranded DNA antibodies that cause end‐organ inflammation, such as glomerulonephritis. The purpose of this study was to examine the hypothesis that this pathway could account for a subset of patients with systemic lupus erythematosus (SLE).Methods: An expansion of Tfh cells is a causal, and therefore consistent, component of the sanroque mouse phenotype. We validated the enumeration of circulating T cells resembling Tfh cells as a biomarker of this expansion in sanroque mice, and we performed a comprehensive comparison of the surface phenotype of circulating and tonsillar Tfh cells in humans. This circulating biomarker was enumerated in SLE patients (n = 46), Sjögren's syndrome patients (n = 17), and healthy controls (n = 48) and was correlated with disease activity and end‐organ involvement.Results: In sanroque mice, circulating Tfh cells increased in proportion to their GC counterparts, making circulating Tfh cells a feasible human biomarker of this novel mechanism of breakdown in GC tolerance. In a subset of SLE patients (14 of 46), but in none of the controls, the levels of circulating Tfh cells (defined as circulating CXCR5+CD4+ cells with high expression of Tfh‐associated molecules, such as inducible T cell costimulator or programmed death 1) were increased. This cellular phenotype did not vary with time, disease activity, or treatment, but it did correlate with the diversity and titers of autoantibodies and with the severity of end‐organ involvement.Conclusion: These findings in SLE patients are consistent with the autoimmune mechanism in sanroque mice and identify Tfh effector molecules as possible therapeutic targets in a recognizable subset of patients with SLE.

The transcription factor FOXP3 plays a key role in CD4⁺CD25⁺ regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4⁺CD25⁺ cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti‐FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow‐cytometric analysis showed that FOXP3 was expressed by the majority of CD4⁺CD25^high T cells in peripheral blood. By contrast, less than half of the CD4⁺CD25^int population were FOXP3⁺, providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4⁺CD25^high pool. Although FOXP3 expression was primarily restricted to CD4⁺CD25⁺ cells, it was induced following activation of both CD4⁺ and CD8⁺ T cell clones. These findings indicate that the frequency of FOXP3⁺ cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4⁺ and CD8⁺ T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use. See accompanying Commentary http://dx.doi.org/10.1002/eji.200526303

Previous studies showed that most cases of ALK⁺ anaplastic large-cell lymphoma (ALK⁺ALCL) do not express SHP1, a tyrosine phosphatase and an important negative regulator for cellular signaling pathways such as that of JAK/STAT. To fully assess the biologic significance of loss of SHP1 in ALK⁺ALCL, we transfected SHP1 plasmids into 2 SHP1^-, ALK⁺ALCL cell lines, Karpas 299 and SU-DHL-1. After 24 hours of transfection, pJAK3 and pSTAT3 were decreased, and these changes correlated with down-regulation of STAT3 downstream targets including cyclin D3, mcl-1, and bcl-2. Expression of SHP1 in these 2 cell lines also resulted in marked decreases in the protein levels of JAK3 and NPM-ALK, and these effects were reversible by proteosome inhibitor MG132. Conversely, when SHP1 expression in SUP-M2 (a SHP1⁺ ALK⁺ALCL cell line) was inhibited using siRNA, pSTAT3, pJAK3, JAK3, and NPM-ALK were all up-regulated. Coimmunoprecipitation studies showed that SHP1 was physically associated with JAK3 and NPM-ALK. SHP1 expression in Karpas 299 and SU-DHL-1 led to significant G₁ cell cycle arrest but not apoptosis. To conclude, loss of SHP1 contributes to the pathogenesis of ALK⁺ALCL by 2 mechanisms: (1) it leaves the tyrosine phosphorylation and activation of JAK3/STAT3 unchecked and (2) it decreases proteosome degradation of JAK3 and NPM-ALK.

Purpose: Recent studies of Hodgkin's lymphoma (HL) have suggested that the presence of regulatory T cells in the reactive background may explain the inhibition of the antitumoral host immune response observed in these patients. This study aimed to assess the relevance of regulatory T cells and CTLs present in the background of HL samples in the prognosis of a series of classic HL (cHL) patients. Experimental Design: Expression of granzyme B and TIA-1 (markers for CTL) and FOXP3 (a marker for regulatory T cells) were evaluated independently by immunohistochemistry in tissue microarrays of 257 cHL patients and correlated with patient outcome. Results: The combined influence of the presence of FOXP3⁺ and TIA-1⁺ cells distinguished three risk groups of patients with 5-year overall survival of 100%, 88%, and 73%. The presence of a small number of FOXP3⁺ cells and a high proportion of TIA-1⁺ cells in the infiltrate represent an independent prognostic factor that negatively influenced event-free survival and disease-free survival in cHL. Compared with the features at diagnosis, relapsed samples tended to have more TIA-1⁺ cells and a lower proportion of FOXP3⁺ cells in the reactive background. Conclusions: These data suggest that low infiltration of FOXP3⁺ cells in conjunction with high infiltration of TIA-1⁺ cells in cHL may represent biological markers predicting an unfavorable outcome. Moreover, the variation of these markers over the course of the disease implies a possible role for them in the progression of HL cases.

Purpose: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest–derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL. Design: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics. Results: Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine. Conclusions: Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients. Clin Cancer Res; 18(10); 2828–37. ©2012 AACR.