Madeleine Ingelsten

Recruitment and activation of natural killer cells in vitro by a human dendritic cell vaccine.

Recruitment of circulating natural killer (NK) cells into inflamed lymph nodes is known to provide a potent, IFN-gamma-dependent boost for Th1-polarized immune responses in mouse models. Such NK cell recruitment into draining lymph nodes is induced by certain s.c. injected adjuvants, including mature vaccine dendritic cells (DC), and is mediated by a CXCR3-dependent pathway. Here, we show that monocyte-derived immature human DCs stimulated with polyinosinic acid:polycytidylic acid, IFN-alpha, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IFN-gamma, alpha-type 1-polarized DC (alpha DC1), secrete profuse amounts of the CXCR3 ligand CXCL9/MIG and substantial amounts of CXCL10/IP-10 and CXCL11/I-TAC after withdrawal of maturation stimuli. In sharp contrast, no measurable production of these chemokines was found in DCs after maturation with the current gold standard maturation cocktail for human DC-based cancer vaccines consisting of TNF-alpha, IL-1 beta, IL-6, and prostaglandin-E(2) (PGE(2)-DC). PGE(2)-DCs preferentially produced the Th2 and regulatory T-cell-attracting chemokines CCL17/TARC and CCL22/MDC, whereas only marginal levels of these chemokines were produced by alpha DC1s. Functional studies in vitro showed that supernatants from mature alpha DC1s actively recruited CD3(-)CD56(+) NK cells and that adding anti-CXCL9/MIG antibodies to the alpha DC1 supernatant substantially reduced this recruitment. Finally, alpha DC1s were able to induce IFN-gamma production when cocultured with resting autologous NK cells, but only if concurrent CD40 ligation was provided. These novel findings indicate that injected human alpha DC1-based vaccines have the potential to recruit and activate NK cells during their arrival to draining lymph nodes and that this feature may be of relevance for efficient priming of Th1 cells and CTLs.

Role of Glomerular Proteoglycans in IgA Nephropathy

Mesangial matrix expansion is a prominent feature of the most common form of glomerulonephritis, IgA nephropathy (IgAN). To find molecular markers and improve the understanding of the disease, the gene and protein expression of proteoglycans were investigated in biopsies from IgAN patients and correlated to clinical and morphological data. We collected and microdissected renal biopsies from IgAN patients (n = 19) and from healthy kidney donors (n = 14). Patients were followed for an average time of 4 years and blood pressure was according to target guidelines. Distinct patterns of gene expression were seen in glomerular and tubulo-interstitial cells. Three of the proteoglycans investigated were found to be of special interest and upregulated in glomeruli: perlecan, decorin and biglycan. Perlecan gene expression negatively correlated to albumin excretion and progress of the disease. Abundant decorin protein expression was found in sclerotic glomeruli, but not in unaffected glomeruli from IgAN patients or in controls. Transforming growth factor beta (TGF-β), known to interact with perlecan, decorin and biglycan, were upregulated both on gene and protein level in the glomeruli. This study provides further insight into the molecular mechanisms involved in mesangial matrix expansion in IgAN. We conclude that perlecan is a possible prognostic marker for patients with IgAN. In addition, the up-regulation of biglycan and decorin, as well as TGF-β itself, indicate that regulation of TGF-β, and other profibrotic markers plays a role in IgAN pathology.

Postischemic inflammatory response in an auxiliary liver graft predicts renal graft outcome in sensitized patients.

Background. The liver is considered a tolerogenic organ that favors the induction of peripheral tolerance and protects other organs from the same donor from rejection. This has been exploited in combined auxiliary liver-kidney transplantation, where a renal graft is transplanted against a positive crossmatch under the protection of a liver transplanted from the same donor. Methods. To elucidate mechanisms behind the liver protective effect, we studied early transcriptional changes of inflammatory mediators in the grafts during combined auxiliary liver-kidney transplantation using microarrays and real-time polymerase chain reaction. The results were correlated to clinical data. Results. Liver and kidney grafts both exhibited an upregulation of the leukocyte-recruiting chemokines CCL2, CCL3, and CCL4. Notably, liver grafts strongly upregulated CCL20, a dendritic cell, and T-cell recruiting chemokine. By comparing the gene expression in liver grafts with the clinical outcome, we found that 14 of 45 investigated inflammatory genes were expressed significantly higher in patients without early rejection when compared with those with early rejections. This included the above-mentioned chemokines and the T-cell-recruiting CX3CL1, NFKB1, and the tolerance-inducing gene indoleamine 2,3-dioxygenase. Conclusions. In this study, the protective role of the liver was associated with a proinflammatory reaction within this organ after ischemia-reperfusion. In particular, we found an increased expression of leukocyte-recruiting chemokines in patients without rejection, indicating a protective role of host inflammatory cells infiltrating the auxiliary liver graft in presensitized patients. Second, gene expression profiling of transplant biopsies shortly after reperfusion predicted the risk of early rejection in these patients.

Is Indoleamine 2,3-Dioxygenase Important for Graft Acceptance in Highly Sensitized Patients After Combined Auxiliary Liver-Kidney Transplantation?

Background. In the clinical setting, transplanted liver seems to protect other grafts from the same donor from rejection. Our previous findings suggest that an auxiliary liver transplantation a few hours before a renal transplantation not only inhibits hyperacute antibody-mediated rejection but also improves long-term kidney graft survival in sensitized recipients. Here, we investigated indoleamine 2,3-dioxygenase (IDO) activity, as one potential mechanism for liver-induced long-term acceptance of kidney grafts. Methods. Tryptophan degradation was measured to estimate IDO activity in patient sera and cell culture supernatants with high performance liquid chromatography. Gene expression in the grafted organs and cell lysates was studied using real time polymerase chain reaction analysis. Results. Tryptophan degradation increased in peripheral blood from patients undergoing combined auxiliary liver-kidney transplantation, whereas it decreased in patients after regular renal transplantation. A 100-fold increase in IDO mRNA, preceded by upregulation of the IDO-inducing cytokines tumor necrosis factor-α, interleukin-1β, and interferon-&ggr;, was observed in the transplanted organs after graft reperfusion in patients undergoing combined graft transplantation. Subsequent studies in vitro revealed that immature dendritic cells, but not hepatocytes, strongly activated IDO on maturation with tumor necrosis factor-α, interleukin-1β, and interferon-&ggr;. Finally, serum from liver-transplanted patients elicited an even stronger IDO-activity in such cytokine-stimulated dendritic cells. Conclusions. Taken together these findings suggest that the liver-induced long-term acceptance seen in human combined auxiliary liver and kidney transplantation is at least partly mediated by IDO activity.

Rapid increase of interleukin-10 plasma levels after combined auxiliary liver-kidney transplantation in presensitized patients.

Background After transplantation, donor dendritic cells (DCs) in the grafted organ are activated by an ischemia/reperfusion–induced inflammatory process that induces their migration to the recipient’s secondary lymphoid tissues. The subsequent interaction between migrated and mature donor DCs, recipient T cells, and natural killer (NK) cells is proposed to be crucial in directing host immune reactions toward allograft rejection. A liver transplant is less prone to induce rejection compared with most other solid organ transplants, and simultaneous transplantation of liver and kidney is known to improve the clinical outcome of kidney transplantation. Methods and Results Here we show that liver as well as combined auxiliary liver-kidney transplantation in patients induces a rapid increase in plasma interleukin-10 (IL-10) to levels that are significantly higher than those seen after standard kidney transplantation. Addition of IL-10 during in vitro maturation of human monocyte–derived DCs with ischemia/reperfusion-associated factors was found to affect phenotypic DC maturation significantly. Addition of IL-10 inhibited DC production of the NK cell- and T cell-recruiting chemokines CXCL9, CXCL10 and CXCL11. Conclusion Our findings indicate that liver transplantation induces a substantial systemic release of IL-10, which may inhibit T cell- and NK cell-mediated rejection processes toward the transplanted liver and concurrently transplanted kidney.

Eosinophils from Hematopoietic Stem Cell Recipients Suppress Allogeneic T Cell Proliferation

Eosinophilia has been associated with less severe graft-versus-host disease (GVHD), but the underlying mechanism is unknown. We hypothesized that eosinophils diminish allogeneic T cell activation in patients with chronic GVHD. The capacity of eosinophils derived from healthy subjects and hematopoietic stem cell (HSC) transplant recipients, with or without chronic GVHD, to reduce allogeneic T cell proliferation was evaluated using a mixed leukocyte reaction. Eosinophil-mediated inhibition of proliferation was observed for the eosinophils of both healthy subjects and patients who underwent HSC transplantation. Eosinophils from patients with and without chronic GVHD were equally suppressive. Healthy eosinophils required cell-to-cell contact for their suppressive capacity, which was directed against CD4(+) T cells and CD8(+) T cells. Neither eosinophilic cationic protein, eosinophil-derived neurotoxin, indoleamine 2,3-dioxygenase, or increased numbers of regulatory T cells could account for the suppressive effect of healthy eosinophils. Real-time quantitative PCR analysis revealed significantly increased mRNA levels of the immunoregulatory protein galectin-10 in the eosinophils of both chronic GVHD patients and patients without GVHD, as compared with those from healthy subjects. The upregulation of galectin-10 expression in eosinophils from patients suggests a stimulatory effect of HSC transplantation in itself on eosinophilic galectin-10 expression, regardless of chronic GVHD status. To conclude, eosinophils from HSC transplant recipients and healthy subjects have a T cell suppressive capacity.

Indoleamine 2,3‐dioxygenase Expression and Functional Activity in Dendritic Cells Exposed to Cholera Toxin

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan‐metabolizing enzyme expressed by dendritic cells (DC), has the potential to inhibit T cell responses and to promote tolerance. In contrast, cholera toxin (CT), the enterotoxin produced by Vibrio cholerae, promotes T cell responses, partly through its ability to induce DC maturation and promote antigen presentation. We hypothesized that the adjuvant activity of CT is associated with a lack of induction of IDO in DC. To test this hypothesis, monocyte‐derived DC were pulsed with CT, and the IDO mRNA expression, IDO functional activity and cytokine production were measured as well as the ability of DC to induce T cell responses in vitro. Cholera toxin exposure induced enhanced levels of IDO mRNA in DC but no functional IDO protein activity. Cholera toxin pulsing however primed DC for CD40L‐induced IDO protein activity. CD40L stimulation of CT‐pulsed DC induced a modest IL‐12p40 production, but not IL‐12p70 or IL‐23 secretion. Furthermore, CT‐pulsed DC induced strong allogeneic and autologous T cell responses in vitro, which were not affected by the IDO‐specific inhibitor 1‐methyl tryptophan. Our results show that CT per se does not induce the expression of functional IDO protein, although it primes DC for CD40L‐mediated IDO production and IL‐12p40 secretion. Furthermore, CT‐treated DC were equally powerful in their T cell stimulatory capacity as cytokine‐matured DC.