Kammi J. Henriksen

In Vitro–expanded Antigen-specific Regulatory T Cells Suppress Autoimmune Diabetes

The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

Cutting Edge: CD28 Controls Peripheral Homeostasis of CD4+CD25+ Regulatory T Cells

CD28/B7 blockade leads to exacerbated autoimmune disease in the nonobese diabetic mouse strain as a result of a marked reduction in the number of CD4+CD25+ regulatory T cells (Tregs). Herein, we demonstrate that CD28 controls both thymic development and peripheral homeostasis of Tregs. CD28 maintains a stable pool of peripheral Tregs by both supporting their survival and promoting their self-renewal. CD28 engagement promotes survival by regulating IL-2 production by conventional T cells and CD25 expression on Tregs.

Distinct roles of CTLA-4 and TGF-β in CD4+CD25+regulatory T cell function

Both CTLA‐4 and TGF‐β have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA‐4 and TGF‐β in Treg function was examined. Blocking CTLA‐4 on wild‐type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF‐β had no effect, supporting a TGF‐β‐independent role for CTLA‐4 in Treg‐mediated suppression in vitro. In CTLA‐4‐deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA‐4‐deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA‐4‐deficient Treg expressed increased levels of the suppressive cytokines IL‐10 and TGF‐β, and in vitro suppression mediated by CTLA‐4–/– Treg was markedly reduced by neutralizing TGF‐β, suggesting that CTLA‐4‐deficient Treg develop a compensatory suppressive mechanism through CTLA‐4‐independent production of TGF‐β. Together, these data suggest that CTLA‐4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA‐4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.

Non-neoplastic renal diseases are often unrecognized in adult tumor nephrectomy specimens: a review of 246 cases.

The pathologic evaluation of tumor nephrectomy specimens focuses on the diagnosis, grading, and staging of the neoplasm. The presence of coincidental non-neoplastic diseases in these specimens may have significant implications for patient outcomes. The purpose of this study is to determine the spectrum of non-neoplastic disease processes that may be overlooked in tumor nephrectomies, and to ascertain the extent to which surgical pathologists are trained to recognize these lesions. We reviewed the hematoxylin and eosin-stained slides of 246 adult tumor nephrectomy specimens with an emphasis on the non-neoplastic renal parenchyma. Further analysis of cases with pathologic alterations included special stains and direct immunofluorescence microscopy. The surgical pathology reports were reviewed to determine whether the non-neoplastic lesions were originally identified. We also surveyed United States pathology residency programs to determine how many require training in medical renal pathology. Forty-one cases (16.7%) had alterations, such as diffuse and/or nodular mesangial sclerosis, mesangial hypercellularity, or glomerular basement membrane thickening that warranted further study. After further work-up and clinical correlation, the pathologic changes in 24 cases were categorized as follows: diabetic nephropathy (19 cases) of which one demonstrated atheroembolic disease, thrombotic microangiopathy (3 cases), sickle cell nephropathy (1 case), and focal segmental glomerulosclerosis (1 case). Twenty-one (88%) of these diagnoses were not identified at initial pathologic evaluation. Only 35 of 98 pathology residency programs that responded to our survey require any formal training in medical renal pathology. Although accurate pathologic evaluation of renal neoplasms remains essential, surgical pathologists should be aware that coincidental non-neoplastic renal diseases are common, often not recognized, and may have important implications for patient care. Further consideration should be given to the training requirements of pathology residents and the guidelines for evaluation of nephrectomy specimens to avoid missing non-neoplastic renal lesions.

CD28/B7 Regulation of Anti-CD3-Mediated Immunosuppression In Vivo

FcR-binding “classical” anti-CD3 mAb is a potent immunosuppressive drug that alters CD4+ and CD8+ T cell function in vivo via anergy induction and programmed cell death (PCD). Anti-CD3-mediated PCD was Fas independent but was mediated by the mitochondria-initiated apoptosis that was abrogated in Bcl-xL-transgenic T cells. The PCD was more pronounced in CD28-deficient mice consistent with defective Bcl-xL up-regulation. Residual T cells isolated from anti-CD3-treated wild-type, CD28−/−, and Bcl-xL-transgenic mice were hyporesponsive. The hyporesponsiveness was more pronounced in CD28−/− and wild-type mice treated with anti-B7-2, suggesting that CD28 interaction with B7-2 regulates T cell responsiveness in anti-CD3-treated animals. Finally, anti-CD3 treatment led to indefinite cardiac allograft survival in wild-type but not Bcl-xL animals. Together these results implicate CD28/B7 signaling in the regulation of both anti-CD3-induced T cell depletion and hyporesponsiveness in vivo, but T cell depletion, not hyporesponsiveness, appears to be critical for anti-CD3 mAb-mediated long-term immune regulation.

Nonneoplastic kidney diseases in adult tumor nephrectomy and nephroureterectomy specimens: common, harmful, yet underappreciated.

CONTEXT Nonneoplastic kidney diseases, such as arterionephrosclerosis and/or diabetic nephropathy, are commonly encountered in tumor nephrectomy and nephroureterectomy specimens. Although any nonneoplastic kidney disease may be encountered in these resection specimens by chance, additional diseases that may be related to the underlying neoplasm or its treatment regimen include thrombotic microangiopathy, Amyloid A amyloidosis, membranous nephropathy, immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, pauci-immune crescentic glomerulonephritis, focal segmental glomerulosclerosis, minimal-change disease, acute interstitial nephritis, and xanthogranulomatous pyelonephritis. Given the morbidity of chronic kidney disease and the relatively favorable 5-year survival rates for urothelial and renal cell carcinomas, accurate evaluation of the nonneoplastic kidney parenchyma is important. OBJECTIVES We will discuss our approach for evaluating the nonneoplastic kidney parenchyma in tumor nephrectomy and nephroureterectomy specimens. The pathologic features of the aforementioned kidney diseases as well as pertinent references will be reviewed. The identification of glomerular abnormalities, including mesangial sclerosis or hypercellularity, segmental sclerosis, crescent formation, glomerulitis, or glomerular basement membrane alterations, should lead to additional immunofluorescence and electron microscopic studies. Safeguards to ensure that the nonneoplastic parenchyma is not overlooked include adding this important parameter to synoptic reports and obtaining periodic acid-Schiff and/or Jones methenamine silver stains prior to microscopic evaluation of the neoplasm. DATA SOURCES Relevant literature and University of Chicago Medical Center pathology archives. CONCLUSIONS The practicing surgical pathologist should be aware of the importance of both correctly classifying the resected renal or urothelial neoplasm and the concomitant nonneoplastic kidney disease that may be present in these specimens.

The Src Family Kinase Fyn Mediates Signals Induced by TCR Antagonists

FcR nonbinding anti-CD3ε mAbs elicit partial TCR signaling that leads to T cell unresponsiveness and tolerance in vivo. In this study, the membrane-proximal events that promote T cell inactivation by FcR nonbinding anti-CD3 mAbs were examined. In the context of FcR nonbinding anti-CD3, TCR complexes did not aggregate and failed to translocate into glycolipid-enriched membrane microdomains. Furthermore, FcR nonbinding anti-CD3 mAbs induced tyrosine phosphorylation of the Fyn substrate Cbl, but not the ZAP-70 substrate linker for activation of T cells. Overexpression of Fyn, but not Lck, restored the mitogenicity of FcR nonbinding anti-CD3 in primary T cells. Taken together, these results suggest that Fyn mediates the partial signaling induced by TCR antagonists.

Rare Association of Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, ANCAs, and Pauci-immune Crescentic Glomerulonephritis

We report a 69-year-old African American woman with hemoptysis and hematuria caused by a focally crescentic pauci-immune glomerular injury associated with the presence of antineutrophil cytoplasmic antibodies (ANCAs). An incidental diagnosis of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma also was established based on the kidney biopsy. Given that a subset of patients with CLL can develop autoantibodies to red blood cells, platelets, or, rarely, neutrophils, the simultaneous presence of CLL, ANCA, and a pauci-immune crescentic glomerulonephritis may not be a coincidence. Recent advances in the pathogenic role of ANCAs in pauci-immune crescentic glomerulonephritis may link the underlying CLL to this patients glomerular injury. Awareness of this possible association may be important for clinicians who manage patients with CLL, as well as for renal pathologists who diagnose pauci-immune crescentic glomerulonephritis.

Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation.

About 50% of all malignant peripheral nerve sheath tumors (MPNSTs) arise as neurofibromatosis type 1 associated lesions. In those patients malignant peripheral nerve sheath tumors are thought to arise through malignant transformation of a preexisting plexiform neurofibroma. The molecular changes associated with this transformation are still poorly understood. We sought to test the hypothesis that dysregulation of expression of kinases contributes to this malignant transformation. We analyzed expression of all 519 kinase genes in the human genome using the nanostring nCounter system. Twelve cases of malignant peripheral nerve sheath tumor arising in a background of preexisting plexiform neurofibroma were included. Both components were separately sampled. Statistical analysis compared global changes in expression levels as well as changes observed in the pairwise comparison of samples taken from the same surgical specimen. Immunohistochemical studies were performed on tissue array slides to confirm expression of selected proteins. The expression pattern of kinase genes can separate malignant peripheral nerve sheath tumors and preexisting plexiform neurofibromas. The majority of kinase genes is downregulated rather than overexpressed with malignant transformation. The patterns of expression changes are complex without simple recurring alteration. Pathway analysis demonstrates that differentially expressed kinases are enriched for kinases involved in the direct regulation of mitosis, and several of these show increased expression in malignant peripheral nerve sheath tumors. Immunohistochemical studies for the mitotic regulators BUB1B, PBK and NEK2 confirm higher expression levels at the protein level. These results suggest that the malignant transformation of plexiform neurofibroma is associated with distinct changes in the expression of kinase genes. The patterns of these changes are complex and heterogeneous. There is no single unifying alteration. Kinases involved in mitotic regulation are particularly enriched in the pool of differentially expressed kinases. Some of these are overexpressed and are therefore possible targets for kinase inhibitors.

CD43 Regulates Th2 Differentiation and Inflammation

CD43 is a highly glycosylated transmembrane protein that regulates T cell activation. CD43−/− T cells are hyperproliferative and the cytoplasmic tail of CD43 has been found to be sufficient to reconstitute wild-type proliferation levels, suggesting an intracellular mechanism. In this study, we report that upon TCR ligation CD43−/− T cells demonstrated no increase in tyrosine phosphorylation but a decreased calcium flux. Interestingly, CD43−/− T cells preferentially differentiated into Th2 cells in vitro, and CD43−/− T cells show increased GATA-3 translocation into the nucleus. In vivo, CD43−/− mice exhibited increased inflammation in two separate models of Th2-mediated allergic airway disease. In contrast, in Th1-mediated diabetes, nonobese diabetic CD43−/− mice did not significantly differ from wild-type mice in disease onset or progression. Th1-induced experimental autoimmune encephalomyelitis to MOG35–55 was also normal in the CD43−/− mice. Nonetheless, the CD43−/− mice produced more IL-5 when restimulated with MOG35–55 in vitro and demonstrated decreased delayed-type hypersensitivity responses. Together, these data demonstrate that although CD43−/− T cells preferentially differentiate into Th2 cells, this response is not sufficient to protect against Th1-mediated autoimmune responses.