Ryoko Hamano

Co‐expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood

Previously, we found that co‐expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3+ cells present in CD25high, CD25low and even CD25– subsets of CD4+ cells expressed high levels of TNFR2. Consequently, TNFR2‐expressing CD4+CD25+ Treg included all of the FOXP3+ cells present in the CD4+CD25high subset as well as a substantial proportion of the FOXP3+ cells present in the CD4+CD25low subset. Flow cytometric analysis of PB identified five‐fold more Treg, determined by FOXP3 expression, in the CD4+CD25+TNFR2+ subset than in the CD4+CD25high subset. In addition, similar levels of FOXP3+ cells were identified in both the CD4+CD25+TNFR2+ and CD4+CD25+CD127low/− subsets. Furthermore, the CD4+CD25+TNFR2+ subset expressed high levels of CTLA‐4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4+CD25+TNFR2+ cells were anergic and markedly inhibited the proliferation and cytokine production of co‐cultured T‐responder cells. In contrast, CD4+CD25+TNFR2– and CD4+CD25–TNFR2+ T cells did not show inhibitory activity. As some non‐Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.

Expression of Costimulatory TNFR2 Induces Resistance of CD4+FoxP3− Conventional T Cells to Suppression by CD4+FoxP3+ Regulatory T Cells

Our previous study showed that TNFR2 is preferentially expressed by CD4+FoxP3+ regulatory T cells (Tregs), and expression of this receptor identified maximally suppressive Tregs. TNFR2 is also expressed by a small fraction of CD4+FoxP3− conventional T cells (Tconvs) in normal mice, and its expression is upregulated by T cell activation. This raises questions about the role of TNFR2 signaling in the function of Tconv cells. In this study, by using FoxP3/gfp knock-in mice, we showed that TNFR2 signaling did not induce FoxP3− CD4 cells to become suppressive. Ki-67, a marker of proliferation, was concomitantly expressed with TNFR2 by CD4 cells, independent of forkhead box P3 expression, in normal mice and Lewis lung carcinoma-bearing mice. TNFR2 is associated with greater suppressive functions when expressed by Tregs and is associated with greater resistance to suppression when expressed by Tconv cells. In mice bearing 4T1 breast tumor or Lewis lung carcinoma, intratumoral Tconv cells expressing elevated levels of TNFR2 acquired the capacity to resist suppression by lymph node-derived Tregs. However, they remained susceptible to inhibition by more suppressive tumor-infiltrating Tregs, which expressed higher levels of TNFR2. Our data indicate that TNFR2 also costimulates Tconv cells. However, intratumoral Tregs expressing more TNFR2 are able to overcome the greater resistance to suppression of intratumoral Tconv cells, resulting in a dominant immunosuppressive tumor environment.

TNF optimally activatives regulatory T cells by inducing TNF receptor superfamily members TNFR2, 4-1BB and OX40

TNF is a pleiotropic cytokine with intriguing biphasic pro‐inflammatory and anti‐inflammatory effects. Our previous studies demonstrated that TNF up‐regulated FoxP3 expression and activated and expanded CD4+FoxP3+ regulatory T cells (Tregs) via TNFR2. Furthermore, TNFR2‐expressing Tregs exhibited maximal suppressive activity. In this study, we show that TNF, in concert with IL‐2, preferentially up‐regulated mRNA and surface expression of TNFR2, 4‐1BB and OX40 on Tregs. Agonistic antibodies against 4‐1BB and OX40 also induced the proliferation of suppressive Tregs. Thus, TNF amplifies its stimulatory effect on Tregs by inducing TNF receptor superfamily (TNFRSF) members. In addition, administration of neutralizing anti‐TNF Ab blocked LPS‐induced expansion of splenic Tregs and up‐regulation of TNFR2, OX40 and 4‐1BB receptors on Tregs in vivo, indicating that the expansion of Tregs expressing these co‐stimulatory TNFRSF members in response to LPS is mediated by TNF. Altogether, our novel data indicate that TNF preferentially up‐regulates TNFR2 on Tregs, and this is amplified by the stimulation of 4‐1BB and OX40, resulting in the optimal activation of Tregs and augmented attenuation of excessive inflammatory responses.

Clonal relationship between infiltrating immunoglobulin G4 (IgG4)-positive plasma cells in lacrimal glands and circulating IgG4-positive lymphocytes in Mikulicz's disease

Mikuliczs disease (MD) is gaining acceptance as an immunoglobulin G4 (IgG4)‐related disease characterized by bilateral lacrimal and salivary gland swelling. The aetiology of MD and other IgG4‐related diseases is still unclear. The present work was performed to study the clonality of infiltrating IgG4‐positive plasma cells in lacrimal glands and circulating peripheral blood cells in patients with MD, and compare the clonal relationship between infiltrating and circulating IgG4 positive cells. Total cellular RNA was extracted from the lacrimal glands and peripheral blood in five MD patients. Reverse transcription polymerase chain reaction was performed with primers specific for activation‐induced cytidine deaminase (AID) and for Ig VH and IgG4. Sequences of Ig VH were compared with the structure of Ig VH of the lacrimal glands and the peripheral blood cells. AID was expressed to varying degrees in lacrimal glands of all MD patients. Most IgG4‐positive cells infiltrating lacrimal glands and in peripheral blood were polyclonal, although several clonally related pairs were detected. In one patient, two of the circulating IgG4 VH4‐59 clones shared identical CDR3 sequences with the clones within the lacrimal glands. In conclusion, while most tissue‐infiltrating and circulating IgG4‐positive cells in MD are polyclonal, some clonally related IgG4 positive cells exist between lacrimal gland and peripheral blood, accounting for the clinical features of MD as an IgG4‐related disease involving multiple organs.

Ag and IL‐2 immune complexes efficiently expand Ag‐specific Treg cells that migrate in response to chemokines and reduce localized immune responses

An intravenous administration of a high‐dose antigen (Ag) can induce immune tolerance and suppress the immune response, but the mechanism remains unclear. We recently proved that a combined i.v. administration of OVA and IL‐2‐anti‐IL‐2 Ab immune complexes (IL‐2 ICs) efficiently expands OVA‐specific Treg cells in the thymus and induces their migration into peripheral blood, by using OVA‐specific TCR Tg‐expressing DO11.10 mice. Here, we demonstrate that the expanded OVA‐specific Treg cells rapidly move into the air pouch after OVA injection in DO11.10 mice. The migration was inhibited by blocking the axis of a chemokine receptor, CCR2. Moreover, prior treatment with OVA and IL‐2 ICs enhanced OVA‐specific Treg‐cell migration and inhibited OVA‐induced delayed‐type hypersensitivity (DTH) reactions in the skin of BM chimeric mice with 15% of T cells expressing OVA‐specific TCR. Blocking the CCR2 axis reversed this suppression of DTH in these mice. Furthermore, prior treatment with OVA and IL‐2 ICs effectively reduced DTH reactions even in WT mice possessing only a very small population of OVA‐specific T cells. Thus, the treatment with Ag and IL‐2 ICs can efficiently expand Ag‐specific Treg cells with the capacity to migrate and reduce localized immune responses.

Characterization of MT-2 cells as a human regulatory T cell-like cell line

The paucity of naturally occurring CD4+FoxP3+ regulatory T cells (Tregs) and difficulties in the identification and isolation of such cells are major obstacles in the study of human Tregs. The availability of human Treg-like cell lines is likely to facilitate a better understanding of the molecular basis of Treg function and aid the discovery of pharmacological reagents to regulate Treg activity in a disease state. In this study, we examined the Treg phenotype and the immune suppressive effect of a human T-cell leukemia virus type 1 (HTLV-1) infected cell line, MT-2. The results clearly demonstrated that the MT-2 cell line has the phenotypic and functional characteristics of human Tregs and is a useful tool in the study of human Tregs. The MT-2 cell line was derived from normal human cord leukocytes of a healthy donor by co-cultivation with leukemic cells from an adult T-cell leukemia (ATL) patient.1 It has been shown that MT-2 cells express FoxP3, and when mixed at a ratio of 1∶2 (suppressors/responders), MT-2 cells inhibited the proliferative response of purified human peripheral blood (PB) CD4+CD25− cells stimulated with dendritic cells and anti-CD3 Ab.2 However, the Treg phenotypic markers and relative suppressive activity of MT-2 cells on both proliferation and cytokine production of cocultured responder cells need to be further evaluated.

Situs inversus and cystic kidney disease: Two adult patients with this Heterogeneous syndrome

Summary Background: Situs inversus is a rare complication of cystic kidney diseases. Only three genes, INVS (NPHP2), NPHP3 and PKD2 have been proved to be responsible for some cases, while the responsible genes in many others are still unknown. Case Reports: Here we report two male patients with situs inversus combined with cystic kidney disease without any family history of polycystic kidney disease. Their renal function was normal in childhood but culminated in end stage renal disease in middle age. No pathogenic mutations were found in mutation analysis of INVS, IFT88, PKD2, UMOD or NPHP3 in them. Conclusions: Past reported cases of situs inversus and cystic kidney diseases were divided into three groups, i.e., gestational lethal renal dysplasia group, infantile or juvenile nephronophthisis group and polycystic kidney disease group. The present patients are different from each of these groups. Moreover, the renal lesions of the present two cases are quite different from each other, with one showing mildly atrophic kidneys with small numbers of cysts and the other an enlarged polycystic kidney disease, suggesting very heterogeneous entities.

TNF by inducing co-stimulatory TNF receptor superfamily members TNFR2, 4-1BB and OX40 augments the number and function of mouse CD4+FoxP3+ regulatory T cells

TNF is a pleiotropic cytokine with intriguing biphasic pro‐inflammatory and anti‐inflammatory effects. Our previous studies demonstrated that TNF up‐regulated FoxP3 expression and activated and expanded CD4+FoxP3+ regulatory T cells (Tregs) via TNFR2. Furthermore, TNFR2‐expressing Tregs exhibited maximal suppressive activity. In this study, we show that TNF, in concert with IL‐2, preferentially up‐regulated mRNA and surface expression of TNFR2, 4‐1BB and OX40 on Tregs. Agonistic antibodies against 4‐1BB and OX40 also induced the proliferation of suppressive Tregs. Thus, TNF amplifies its stimulatory effect on Tregs by inducing TNF receptor superfamily (TNFRSF) members. In addition, administration of neutralizing anti‐TNF Ab blocked LPS‐induced expansion of splenic Tregs and up‐regulation of TNFR2, OX40 and 4‐1BB receptors on Tregs in vivo, indicating that the expansion of Tregs expressing these co‐stimulatory TNFRSF members in response to LPS is mediated by TNF. Altogether, our novel data indicate that TNF preferentially up‐regulates TNFR2 on Tregs, and this is amplified by the stimulation of 4‐1BB and OX40, resulting in the optimal activation of Tregs and augmented attenuation of excessive inflammatory responses.

A case developing minimal change disease during the course of IgG4-related disease

We describe a 66-year-old male with immunoglobulin G4-related disease (IgG4-RD) presenting with minimal change disease (MCD). Three years prior to this admission, the patient had been diagnosed with IgG4-RD. The development of sudden massive proteinuria (4+; 16.7 g/gCr) with a weight gain of 8 kg within a two-week period was noted, and nephrotic syndrome was suspected. The patients serum IgG4 level did not increase and hypocomplementemia was not found. A renal biopsy showed no cellular infiltration in the renal interstitium, and no spiking or bubbling was found on periodic acid methenamine silver staining. On electron microscopy, foot process effacement was seen, but no subepithelial electron-dense deposits were found. The patient was diagnosed with MCD. Ten days after starting prednisolone (60 mg/day), proteinuria was negative. Since IgG4-RD and MCD share a T-helper 2-dominant immunoreaction, the development of MCD in IgG4-RD patients may reflect more than a mere coincidence.

Efficacy of Coronary Artery Screening Tests and Intervention in Hemodialysis Patients

Although cardiovascular disease (CVD) is an important cause of death in patients on hemodialysis, evidence of a beneficial effect of percutaneous intervention (PCI) on stable heart disease is scarce. We investigated the cardiovascular outcomes of hemodialysis patients under our policy of encouraging coronary artery screening tests to the extent possible. A total of 147 hemodialysis patients have been treated in our clinic so far. In 98 of them, coronary artery screening tests were performed, three in unstable and 95 in asymptomatic patients. Significant coronary artery stenosis was detected in 29 at the first tests and in 11 during subsequent tests (40/98, 40.8%), and PCI or coronary artery bypass grafting (CABG) was performed. Multiple PCIs were needed in 21 patients. In the other 49 patients, coronary artery screening tests were not undertaken based on the nephrologists decision or patient refusal. At the end of the study, 73 (74.5%) patients with tests, and 14 (28.6%) without tests were still outpatients (P < 0.01). Of 40 patients transferred to other hospitals for medical reasons or who died before transfer, there was cerebrovascular accident in eight, malignancy in six, congestive heart failure without CVD in four, infection in three, sudden cardiac death in one, and others 18. No patient with tests died of CVD and the only patient who died of sudden cardiac death probably due to myocardial infarction was a patient who had declined the screening tests. Coronary artery screening tests, intervention and subsequent periodic tests for asymptomatic hemodialysis patients can reduce the occurrence of cardiovascular events in this population.