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Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

Background: The HIV co-receptors CXCR4 and CCR5 play an important role in HIV infection and replication. Therefore we hypothesize that long-term non-progressors (LTNP) with viral control have lower expression of CCR5 and CXCR4 on CD4(+) cells, specifically on memory T-lymphocytes since they are the primary target cells of HIV., Methods: In this cross-sectional study, we included five HIV-infected LTNP with viral control (CD4 > 750 cell/μl & HIV < 50 copies for ≥2 years), thirteen HIV-infected and seven HIV-uninfected individuals at Radboud UMC Nijmegen, the Netherlands. We determined the CCR5 and CXCR4 expression among CD4(+) and CD8(+) lymphocyte subsets; memory (CD45RO(+)), naïve (CD45RA(+)) cells and regulatory T-cells (CD4(+)CD25(high)FoxP3(+)). In addition, CCR5∆32 polymorphism is related with disease progression and was therefore determined using polymerase chain reaction., Results: The percentage of CCR5-expressing CD4(+) cells of LTNP was comparable with healthy controls; whereas HIV-infected individuals showed more CCR5-expressing cells. This was observed in memory and naïve CD4(+) cells, but not in regulatory T-cells. The mean fluorescence intensity of CCR5-expressing CD4(+) cells was similar in all groups. All groups had comparable percentages of CXCR4-expressing cells. The mean fluorescence intensity of CXCR4-expressing cells was significantly higher in HIV-infected normally progressors in both memory and naïve CD4(+) cells, but not in CD8(+) cells. The CCR5∆32 polymorphism was not related to group., Conclusions: We show that HIV affects -directly or indirectly- the expression of CCR5 in CD4(+) T-lymphocytes; yet this effect is not seen in LTNP with viral control. Avoiding upregulation of CCR5 could be an important method via which LTNP counteracts the effects of HIV and suppresses viral replication. Exploring how LTNP suppress the upregulation of CCR5 could be an important step for discovering new therapeutics.

Vitamin D(3) affects both the innate as well as adaptive immune responses. Epidemiological studies have established that vitamin D(3) deficiency plays an important role in tuberculosis (TB) and viral influenza prevalence as well as susceptibility to active disease in TB. Vitamin D(3) status has been associated with the clinical course of HIV infection and drug interaction with anti-retroviral therapy. This article reviews the immunomodulatory capacity of vitamin D(3) and examines the impact of vitamin D(3) supplementation as a preventive or therapeutic intervention with the intent to uncover its potential therapeutic application in infectious diseases and to identify novel areas for future research. We present a review of randomized, controlled clinical studies conducted in humans which included assessment of the immune function or clinical outcome as study end points. Current data support vitamin D(3) supplementation as risk-modifying intervention in tuberculosis and viral respiratory tract infection, but the optimal dosage regimen remains to be determined. However, to date the knowledge on its role in fungal infection and sepsis is limited although a potential benefit could be harnessed from its ability to curtail the unrestrained pro-inflammatory response and therefore prevent excessive collateral tissue damage.

The generation of immunoregulatory T cells that block the B7(CD86/CD80)-CD28 and/or CD40-CD154 costimulatory pathways has great potential for the induction of long-term transplantation tolerance. In a human polyclonal in vitro model, combined monoclonal antibody (mAb) blocking of the costimulatory ligands CD40 and CD86 lead to allospecific T-cell anergy that cannot be reversed by antigenic rechallenge in the presence of IL-2. Although antigenic restimulation with IL-2 restored the proliferative response, subsequent antigenic restimulation of the restored anergic cells in a tertiary mixed lymphocyte culture still resulted in nonresponsiveness. Importantly, these anergic T cells suppress the response of naive alloreactive T cells in an antigen-specific way via linked recognition. Suppression may partially depend on local IL-10 production, while transforming growth factor-beta (TGF-beta) did not play a role. Irrespective of the monoclonal antibody combination used, blast formation occurred in a subset of CD4(+) cells. These cells were characterized by a sustained CD45RA expression, an increased T-cell receptor density, and a lower level of CD4 expression. A reduced number of CD45RO(+)/CD8(+) T cells was observed whenever anti-CD86 was combined with anti-CD40, which was reflected by an even more attenuated cytotoxic T-cell function. This indicates the importance of CD40-CD154 in the generation of cytotoxic T cells in this transplantation model. We hypothesize that in our model, anergy is induced in the CD4(+) T-cell subset, whereby CD8(+) cytotoxic effector function is impaired by the lack of both CD40-CD154 signaling and cytokine-mediated help. This costimulatory ligand-directed mAb approach might well be used for the ex vivo generation of antigen-specific immunoregulatory T cells applicable in adoptive immunotherapy.

Background: Milky spots in the human greater omentum are preformed specific accumulations of primarily macrophages within the stroma of the greater omentum. To obtain a better understanding of milky spots in the human greater omentum, the development and the earliest forms of milky spots in the human greater omentum were studied, with special attention to the macrophage population., Methods: Specimens of human greater omentum were obtained from fetuses of 20 to 40 weeks gestation and one newborn three days old (n = 6). Using mature macrophages (RFD 7), activated macrophages (RFD 1), B-lymphocytes (CD 22), and T-lymphocytes (CD 2), and immunoperoxydase labeling, the percentage of these cells in developing milky spots and the development of milky spots were studied by light microscopy. A time-dependent increase in the percentage of positive staining cells and the size of clusters was analyzed using the non-parametric Spearman rank correlation test., Results: Small accumulations of cells with about 50% monocytes/macrophages were present at 20 weeks of gestation. With increasing gestational age the number of clusters of cells increased significantly (P < 0.01) as well as their size (P < 0.01). Starting at 29 weeks, vascularized clusters of cells were seen; true milky spots were present at 35 weeks. A significant (P < 0.05) increase in the percentage of mature macrophages was found in developing milky spots, whereas no activated macrophages were seen. The percentage of B-lymphocytes and T-lymphocytes found in the clusters of cells and milky spots increased significantly (P < 0.05) but did not exceed 10% of the total number of cells., Conclusions: From our data it can be concluded that milky spots are specific structures in the greater omentum formed between the 20th and 35th week of gestation. Further, we concluded that immature cells (promonocytes) mature locally in developing milky spots.

Male Wag rats were pretreated for 7 days with 1000 U/ml recombinant murine granulocyte macrophage colony stimulating factor (rmGM-CSF). Rat Kupffer cells (KC) were isolated by a enzymatic method. We injected decreasing numbers of CC531 tumor cells in the portal system. Mean KC yield increased from 1.5 +/- 0.2 to 2.2 +/- 0.2 (p < 0.05). Mean percentage of KC-mediated cytotoxicity against CC531 increased from 20.0 +/- 0.5 to 42 +/- 1.0 after rmGM-CSF (p < 0.05). At 1 x 10(5) CC531 tumor cells we demonstrated prevention of formation of small foci of CC52+ tumor cells. We demonstrate increased isolated KC with enhanced cytotoxic capacity after rmGM-CSF. rmGM-CSF induced prevention of minimal residual disease in the rat liver.

Milky spots in the greater omentum are well organized perivascular infiltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity. In milky spots, macrophages are the predominant cell type forming a distinct population of cells. To investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cytotoxicity of macrophages from milky spots in the greater omentum against tumor cells in vitro. In order to obtain a cell suspension, greater omenta of unstimulated healthy male WAG/RIJ rats were incubated in collagenase/DNase suspension and filtered. Subsequently, macrophages were isolated and purified using flow cytometry by sorting unstained cells on the basis of size and internal complexity. Macrophages and other cells were identified by routine May-Grünwald-Giemsa staining and by immunophenotyping with the specific macrophage monoclonal antibody ED 1. Furthermore, macrophage subtypes were characterized by ultrastructural analysis. Functional cytotoxicity of the isolated macrophages was assayed against the syngeneic CC 531 tumor cell line in a colorimetric MTT assay. From three greater omenta of healthy rats 1.16 +/- 0.16 x 10(6) macrophages were isolated with a purity of 83 +/- 2% and a viability of > or = 96%. The macrophages were of the exudate (monocytic), exudate-resident and resident cell type and were in equal proportions. The contaminating cells were mainly mesothelial. A maximum cytotoxicity of approximately 30% was reached with the macrophage fraction at an effector-to-target ratio of 10. Furthermore, it was established that the mesothelial cells did not exhibit cytotoxicity.

DNase/collagenase treatments are widely used to obtain single-cell suspensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) from solid tumours. Since the functional integrity of such cells has been questioned, we have studied whether treatments with commonly used preparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With peripheral-blood-derived T cells as a model, flow-cytometric analysis revealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to protease contaminations present in all but the purest enzyme preparations tested. Addition of serum or trypsin inhibitor abolished the effects. Since serum-free media are widely used to expand tumour-infiltrating T cells for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following the enzyme treatments, re-expression of the affected membrane markers was still far from complete. On the other hand, despite strongly reduced expression of CD2 molecules on the lymphocyte membrane, anti-CD2-induced proliferation was not affected, showing the redundancy of this signal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expensive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.

Patients: We studied the course and particularly the social consequences of Crohn's disease (n = 107) and ulcerative colitis extending beyond the sigmoid colon (n = 22) during the first 6.1 respectively 6.3 years after onset., Results: Under common treatment, the disease activity decreased and the body mass index increased significantly in the majority of patients, but about 30% of them reached no continuous remission. The professional training was delayed in 23% of patients with Crohn's disease and 9% of those with ulcerative colitis. However, by objective datas and in the patients' own opinion the effect of both diseases on the professional qualification and occupational status which were reached in the end was small. The percentage of partly occupied patients and those without work compared to that of healthy ones. 11% of patients with Crohn's disease and 14% with ulcerative colitis had to retire prematurely. The patients' family status and the number of their children matched largely that of healthy men and women of same age.