Carla C. Baan

Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is characterized by a weak immune response to HBV. Regulatory T cells (Treg) can suppress the function of effector T cells and may thus be key players in this impaired immune response. Changes in the functionality or number of Treg could explain the decreased antiviral response in chronic HBV patients. To investigate the role of Treg in chronic HBV infection, we compared the proportional frequency and functionality of Treg in peripheral blood of 50 chronic HBV patients, 23 healthy controls, and 9 individuals with a resolved HBV infection. A higher percentage of Treg, defined as CD4, CD25, CD45RO, and cytotoxic T‐lymphocyte–associated antigen 4–positive cells, was detected within the population of CD4+ cells in peripheral blood of chronic HBV patients compared with healthy controls and individuals with a resolved HBV infection. Accordingly, chronic HBV patients displayed a higher FoxP3 messenger RNA level than healthy controls. Depletion of CD25+ cells from peripheral blood mononuclear cells (PBMC) of chronic HBV patients resulted in an enhanced proliferation after stimulation with HBV core antigen. Reconstitution of these depleted PBMC with CD4+CD25+ Treg resulted in a dose‐dependent reduction of both HBV‐specific proliferation and interferon γ production. In conclusion, chronic HBV patients harbor an increased percentage of Treg in peripheral blood compared with controls. Treg have an immunosuppressive effect on HBV‐specific T helper cells. The presence of HBV‐specific Treg could contribute to an inadequate immune response against the virus, leading to chronic infection. (HEPATOLOGY 2005;41:771–778.)

Mesenchymal stem cells are short-lived and do not migrate beyond the lungs after intravenous infusion

Mesenchymal stem cells (MSC) are under investigation as a therapy for a variety of disorders. Although animal models show long term regenerative and immunomodulatory effects of MSC, the fate of MSC after infusion remains to be elucidated. In the present study the localization and viability of MSC was examined by isolation and re-culture of intravenously infused MSC. C57BL/6 MSC (500,000) constitutively expressing DsRed-fluorescent protein and radioactively labeled with Cr-51 were infused via the tail vein in wild-type C57BL/6 mice. After 5 min, 1, 24, or 72 h, mice were sacrificed and blood, lungs, liver, spleen, kidneys, and bone marrow removed. One hour after MSC infusion the majority of Cr-51 was found in the lungs, whereas after 24 h Cr-51 was mainly found in the liver. Tissue cultures demonstrated that viable donor MSC were present in the lungs up to 24 h after infusion, after which they disappeared. No viable MSC were found in the other organs examined at any time. The induction of ischemia-reperfusion injury in the liver did not trigger the migration of viable MSC to the liver. These results demonstrate that MSC are short-lived after i.v. infusion and that viable MSC do not pass the lungs. Cell debris may be transported to the liver. Long term immunomodulatory and regenerative effects of infused MSC must therefore be mediated via other cell types.

Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the induction of FOXP3 in human T cells.

Background. The transcription factor FOXP3 has been identified as the molecule associated with the regulatory function of CD25+ T cells. Methods. To understand the biology of FOXP3+ T cells in allogeneic reactions, we measured FOXP3 mRNA expression levels in allostimulated CD25 bright+ cells and CD25 intermediate( int)/- cells and in peripheral blood mononuclear cells (PBMC). The effect of immunosuppressive drugs on FOXP3 expression was studied in mixed lymphocyte reactions (MLR) in the presence and absence of calcineurin inhibitors (CNI), &agr;CD25 mAb, and Rapamycin (Rapa), and analyzed in biopsies from cardiac allograft recipients during acute rejection by quantitative (Q)-PCR. Results. FOXP3 mRNA expression was restricted to the CD25 bright+ population that inhibited the proliferation of allostimulated CD25 int/- cells. In the MLR FOXP3 was readily induced after allostimulation. Kinetic examination of the MLR showed a 10–20-fold higher FOXP3 mRNA expression level after 5 days of culture. The CNI Cyclosporin and Tacrolimus, and &agr;CD25 mAb inhibited in vitro induced FOXP3 gene transcription (range 70%–90%), whereas Rapa did not inhibit the induction. After clinical heart transplantation the highest FOXP3 mRNA expression levels were measured in biopsies during acute rejection (P=0.03). Conclusions. The high FOXP3 mRNA levels during allogeneic responses in vivo and in vitro suggests that regulatory activities of CD25 bright+ T cells or the generation of these cells is an intrinsic part of activation. CNI and &agr;CD25 mAb in contrast to Rapa, did interfere with this immunosuppressive counter-mechanism and as a result might have an inhibitory effect to tolerance induction after transplantation.

The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy.

There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohns disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

Genetic complementation analysis of ataxia telangiectasia and Nijmegen breakage syndrome: a survey of 50 patients

Cultured cells from patients with ataxia telangiectasia (AT) or Nijmegen breakage syndrome (NBS) are hypersensitive to ionizing radiation. After radiation exposure, the rate of DNA replication is inhibited to a lesser extent than in normal cells, whereas the frequency of chromosomal aberrations is enhanced. Both of these features have been used in genetic complementation studies on a limited series of patients. Here we report the results of extended complementation studies on fibroblast strains from 50 patients from widely different origins, using the radioresistant DNA replication characteristic as a marker. Six different genetic complementation groups were identified. Four of these, called AB, C, D, and E (of which AB is the largest), represent patients with clinical signs of AT. Patients having NBS fall into two groups, V1 and V2. An individual with clinical symptoms of both AT and NBS was found in group V2, indicating that the two disorders are closely related. In AT, any group-specific patterns with respect to clinical characteristics or ethnic origin were not apparent. In addition to the radiosensitive ATs, a separate category of patients exists, characterized by a relatively mild clinical course and weak radiosensitivity. It is concluded that a defect in one of at least six different genes may underlie inherited radiosensitivity in humans. To facilitate research on defined defects, a complete list of genetically characterized fibroblast strains is presented.

Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells

There is emerging interest in the application of mesenchymal stem cells (MSC) for the prevention and treatment of autoimmune diseases, graft‐versus‐host disease and allograft rejection. It is, however, unknown how inflammatory conditions affect phenotype and function of MSC. Adipose tissue‐derived mesenchymal stem cells (ASC) were cultured with alloactivated peripheral blood mononuclear cells (PBMC) (mixed lymphocyte reaction: MLR), with proinflammatory cytokines [interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6] or under control conditions, and their full genome expression and function examined. Proinflammatory cytokines mainly increased indoleamine‐2,3‐dioxygenase expression, whereas ASC cultured with MLR showed increased expression of COX‐2, involved in prostaglandin E2 production. Both conditions had a stimulatory, but differential, effect on the expression of proinflammatory cytokines and chemokines, while the expression of fibrotic factors was decreased only in response to proinflammatory cytokines. Functional analysis demonstrated that inflammatory conditions affected morphology and proliferation of ASC, while their differentiation capacity and production of trophic factors was unaffected. The immunosuppressive capacity of ASC was enhanced strongly under inflammatory conditions. In conclusion, ASC showed enhanced immunosuppressive capacity under inflammatory conditions, while their differentiation capacity was preserved. Therefore, in vitro preconditioning provides ASC with improved properties for immediate clinical immune therapy.

The Chemokine and Chemokine Receptor Profile of Infiltrating Cells in the Wall of Arteries With Cardiac Allograft Vasculopathy Is Indicative of a Memory T–Helper 1 Response

Background— Despite improvement in short-term patient survival after heart transplantation (HTx), long-term survival rates have not improved much, mainly because of cardiac allograft vasculopathy (CAV). Cytokines and chemokines are considered to play an important role in CAV development. Methods and Results— We focused on coronary arteries of HTx patients and made an inventory of the infiltrating cells and the expression of cytokines as well as chemokines and chemokine receptors (C+CR) in the different layers of the vessel wall with CAV. Tissue slides were stained for a variety of cell markers (CD3, CD4, CD8, CD20, CD68, CD79a), chemokines (monokine induced by interferon [MIG], interferon-inducible protein 10 [IP-10], interferon-inducible T cell-&agr; chemoattractant [ITAC], RANTES [regulated on activation normal T cell expressed and secreted], and fractalkine), and chemokine receptors (CXCR3, CCR5, and CX3CR1). In reference coronary arteries (not transplanted), almost no infiltrating cells were found, and in transplanted hearts with CAV (HTx+CAV), a large number of T cells were observed (CD4:CD8=2:1), mainly localized in the neointima and adventitia. Most of these T cells appeared to be activated (human leukocyte antigen DR positive). Coronary arteries from transplanted hearts without CAV (HTx−CAV), HTx+CAV, and references were also analyzed for cytokine and C+CR mRNA expression with the use of quantitative polymerase chain reaction. Interferon-&ggr; was highly expressed in HTx+CAV compared with HTx−CAV. Interleukin-4 and interleukin-10 were expressed at the same level in both HTx groups and references. In HTx+CAV, all C+CR, but especially the T–helper 1 (TH1) C+CR, were more abundant than in the HTx−CAV and references. However, TH2 CCR4 expression did not differ significantly between both HTx groups. Conclusions— In coronary arteries with CAV, most T cells are CD4+ and express human leukocyte antigen DR. These activated TH cells are mainly memory TH1 cells on the basis of their C+CR profile and cytokine expression.

Donor-derived mesenchymal stem cells suppress alloreactivity of kidney transplant patients.

Background. Human mesenchymal stem cells (MSC) have immunosuppressive capacities. Although their efficacy is currently studied in graft-versus-host disease, their effect on alloreactivity in solid organ transplant patients is unknown. In this study, the immunosuppressive effect of MSC on recipient anti-donor reactivity was examined before and after clinical kidney transplantation. Methods. Anti-donor reactivity was established in pretransplant and posttransplant mixed lymphocyte reactions (MLR) of 14 living-kidney donor-recipient pairs. MSC from donors and third-party controls were added to the MLR in a ratio of 1:5. Results. MSC were isolated from donor perirenal fat and showed multilineage differentiation potential and the capacity to inhibit lymphocyte proliferation. The immunosuppressive effect of MSC was dose dependent and mediated by cell-membrane contact and soluble factors, including interleukin-10 and indoleamine 2,3-dioxygenase. Donor-derived MSC significantly inhibited the recipient anti-donor reactivity before and 1 month after transplantation. This effect was independent of human leukocyte antigen background of MSC. Flow cytometric analysis showed that MSC inhibited the proliferation of CD4+ and CD8+ T-lymphocyte subsets in pretransplant and posttransplant donor-directed MLR, whereas MSC had no effect on B- or natural killer-cell proliferation. Conclusion. Donor MSC significantly inhibited the proliferation of alloactivated recipient T cells before and after kidney transplantation. We believe these findings should encourage MSC-based intervention in clinical organ transplantation.

Impact of immunosuppressive drugs on CD4+CD25+FOXP3+ regulatory T cells: does in vitro evidence translate to the clinical setting?

Success of solid-organ transplantation requires the continuous administration of immunosuppressive drugs to prevent graft rejection. The currently prescribed immunosuppressive medication targets the immune system in a nonspecific fashion, leading to debilitating side effects that diminish patient survival and quality of life. Therefore, it is important to minimize immunosuppression, but this requires the development of alternative therapeutic strategies to induce and maintain transplant tolerance. One such strategy would be to allow and facilitate the induction of alloantigen-specific immune regulation by regulatory T cells (Treg). Recent experimental studies indicate that several commonly used immunosuppressive drugs have detrimental effects on the induction and function of Treg, whereas other drugs appear to spare these cells or may even be beneficial. These differential effects may be explained by differences in signaling pathways between Treg and effector T cells. In this review, we provide a comprehensive overview of the current literature on the effects of immunosuppressive drugs on CD4+CD25+FOXP3+ Treg and discuss whether these in vitro data are substantiated by in vivo evidence from the clinic. A greater understanding of the impact of immunosuppression on Treg may help to create future opportunities to manipulate the host allo-immune response and achieve operational tolerance in transplantation.

Human Adipose Tissue-Derived Mesenchymal Stem Cells Induce Explosive T-Cell Proliferation

Mesenchymal stem cells (MSCs) inhibit the proliferation of allo-activated lymphocytes. This effect is primarily dependent on the secretion of anti-inflammatory factors by MSCs and is enhanced under inflammatory conditions. MSCs, however, also produce factors that can potentially activate resting immune cells. Full understanding of the behavior of MSCs under inflammatory and noninflammatory conditions is crucial when clinical application of MSCs is considered. Human adipose tissue-derived MSCs were cultured with nonactivated peripheral blood mononuclear cells (PBMCs) and the activation, proliferation, and function of PBMCs were examined. Seven days of coculture with autologous or allogeneic MSCs significantly increased the proliferation of PBMCs (3-fold). This effect was observed in both direct and transwell coculture systems. MSCs cocultured with PBMCs showed increased mRNA expression of the proinflammatory mediators interleukin-6 (IL-6), IL-8, tumor necrosis factor-α, the growth factors basic fibroblast growth factor and vascular endothelial growth factor-α, and the anti-inflammatory factor indoleamine 2,3-dioxygenase. After removal of MSCs, PBMCs showed a spectacular further increase in proliferation, with a maximum of 25-fold after 7 days. This increase in proliferation was not seen when PBMCs were kept in the presence of MSCs. The proliferating fraction of PBMCs largely consisted of CD4(+) T-cells with high CD25 expression and the proportion of CD127(neg)FoxP3(+) regulatory T-cells significantly increased from 5.0% to 8.5% of total CD4(+) T-cells. The expanded T-cells demonstrated normal responses to mitogen or alloantigen stimulation. The CD25(positive) fraction of these cells had immunosuppressive capacity. In conclusion, MSCs can stimulate the activation and proliferation of resting T-cells and generate regulatory T-cells. These findings are important when MSCs are applied in the clinic.