Holger Hackstein

DENDRITIC CELLS: EMERGING PHARMACOLOGICAL TARGETS OF IMMUNOSUPPRESSIVE DRUGS

Immunosuppressive drugs have revolutionized organ transplantation and improved the therapeutic management of autoimmune diseases. The development of immunosuppressive drugs and understanding of their action traditionally has been focused on lymphocytes, but recent evidence indicates that these agents interfere with immune responses at the earliest stage, targeting key functions of dendritic cells (DCs). Here, we review our present understanding of how classical and new immunosuppressive agents interfere with DC development and function. This knowledge might provide a rational basis for the selection of immunosuppressive drugs in different clinical settings and for the generation of tolerogenic DCs in the laboratory.

Rapamycin-Treated, Alloantigen-Pulsed Host Dendritic Cells Induce Ag-Specific T Cell Regulation and Prolong Graft Survival

Tolerogenic properties of dendritic cells (DC), particularly those in the immature state, and their therapeutic potential are increasingly being recognized. Among several distinct approaches to generate stably immature DC, pharmacologic manipulation stands out as a promising and clinically applicable option. We have shown recently that the immunophilin ligand rapamycin (Rapa) can inhibit DC maturation and their effector functions. Here, we examined the impact of Rapa exposure on subsequent alloantigen (Ag) presentation by myeloid DC via the indirect pathway. Rapa‐treated, allogeneic lysate‐pulsed host DC (Rapa‐DC) were inferior stimulators of syngeneic T cells, compared to lysate‐pulsed control DC. Rapa exposure did not block alloAg uptake by DC nor impair their in vivo homing to splenic T cell areas after adoptive transfer. T cells primed by Rapa‐treated, alloAg‐pulsed DC showed decreased capacity to produce IL‐2 and IFNγ, and were hyporesponsive to subsequent challenge via both the direct and indirect pathways, in an Ag‐specific manner. When infused 1 week before transplantation, these Rapa‐DC significantly prolonged alloAg‐specific heart graft survival. This effect was reversed by systemic IL‐2 administration but enhanced by either repeated infusion of the cells or a short post‐transplant course of FK506. These therapeutic effects, achieved by targeting both major pathways of allorecognition, provide the basis for a clinically applicable strategy to suppress graft rejection.

Low TLR4 Expression by Liver Dendritic Cells Correlates with Reduced Capacity to Activate Allogeneic T Cells in Response to Endotoxin

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8α− (myeloid) and CD11c+CD8α+ (“lymphoid-related”) DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-γ) and Th2 (IL-4) responses. When higher LPS concentrations (≥100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.

Testosterone Replacement Effectively Inhibits the Development of Experimental Autoimmune Orchitis in Rats: Evidence for a Direct Role of Testosterone on Regulatory T Cell Expansion

Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4+ T cells with a strong concomitant increase in the number of regulatory T cells (CD4+CD25+Foxp3+) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1–stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.

Continuous cytomegalovirus seroconversion in a large group of healthy blood donors.

Background and Objectives  Transmission of cytomegalovirus (CMV) to seronegative, immunocompromised recipients can cause serious and fatal complications. Although the seroprevalence of CMV is high, the risk of primary CMV infection among healthy blood donors has not yet been analysed in a large population.

A novel polymorphism in the 5′ promoter region of the human interleukin-4 receptor α-chain gene is associated with decreased soluble interleukin-4 receptor protein levels

Abstract. Interleukin (IL)-4 exerts its biological effects through binding to the IL-4 receptor (IL4R) complex, plays a central role in stimulating B-cell differentiation, and is crucial for the development of T helper 2 cells. Recently, a soluble form of the human IL4R α chain (sIL4Rα), which is produced by alternate mRNA splicing of exon 8, was discovered. sIL4R is thought to play an important role in either enhancing or inhibiting IL-4 signalling. We analyzed the 5′ promoter region of the human IL4R α-chain gene (IL4RA) of healthy volunteers by DNA sequencing and found three novel single-nucleotide polymorphisms (SNPs; T–890C, T–1914C, C–3223T) and one novel short tandem repeat [(CAAAA)5–7–3600]. The two common promoter region SNPs T–1914C and C–3223T as well as six known coding SNPs in the IL4RA gene were genotyped in healthy blood donors by PCR with sequence-specific primers; total sIL4R levels were measured by ELISA. Results revealed a highly significant association of the –3223T variant with lowered sIL4R levels (two-tailed t-test, P=0.0002). Results remained highly significant after Bonferroni adjustment for multiple comparisons (P=0.0017). Moreover, the C–3223T variant was found to be in strong linkage disequilibrium with the extracellular I50V variant (P<0.001), which was recently described to be associated with atopic asthma in a Japanese population. Since this novel IL4RA promoter region SNP is common (allele frequency 29.8%), we conclude that it may be of importance for the genetic regulation of the IL-4 signalling pathway.

Human platelets target dendritic cell differentiation and production of proinflammatory cytokines

BACKGROUND:  Dendritic cells (DCs) are the most potent antigen‐presenting cells that initiate and regulate immune responses. They are unique in their feature to produce bioactive interleukin (IL)‐12, a major proinflammatory cytokine connecting innate and adaptive immunity. Platelets (PLTs) are highly reactive components of the circulatory system with fundamental importance in hemostasis and innate immunity. Recently, immunomodulatory capacities of single specific human PLT‐derived products on DC effector functions were identified. To improve the understanding of PLT‐DC interactions, this study investigates the influence of intact resting and activated PLTs on DC phenotype and key functions.

Common human Toll‐like receptor 9 polymorphisms and haplotypes: association with atopy and functional relevance

Background Toll‐like receptor 9 (TLR9) is a pattern‐recognition receptor that detects unmethylated CpG motifs prevalent in bacterial and viral DNA. TLR9 stimulation is a key event after bacterial infection, triggering innate immunity and T‐helper type 1 skewed adaptive immunity. Synthetic CpG‐oligodeoxynucleotides (CpG‐ODNs) represent a promising and novel class of immune adjuvants for allergy treatment, vaccination, and cancer therapy. However, common functional TLR9 gene variants could interfere with the clinical utilization of CpG‐ODN in immunotherapy. Recently, a possible association of TLR9 polymorphism C‐1237T with asthma has been reported.

Good manufacturing practice-compliant animal-free expansion of human bone marrow derived mesenchymal stroma cells in a closed hollow-fiber-based bioreactor

Mesenchymal stroma cells (MSC) are increasingly recognized for various applications of cell-based therapies such as regenerative medicine or immunomodulatory treatment strategies. Standardized large-scale expansions of MSC under good manufacturing practice (GMP)-compliant conditions avoiding animal derived components are mandatory for further evaluation of these novel therapeutic approaches in clinical trials. We applied a novel automated hollow fiber cell expansion system (CES) for in vitro expansion of human bone marrow derived MSC employing a GMP-compliant culture medium with human platelet lysate (HPL). Between 8 and 32 ml primary bone marrow aspirate were loaded into the hollow fiber CES and cultured for 15-27 days. 2-58 million MSC were harvested after primary culture. Further GMP-compliant cultivation of second passage MSC for 13 days led to further 10-20-fold enrichment. Viability, surface antigen expression, differentiation capacity and immunosuppressive function of MSC cultured in the hollow fiber CES were in line with standard criteria for MSC definition. We conclude that MSC can be enriched from primary bone marrow aspirate in a GMP-conform manner within a closed hollow fiber bioreactor and maintain their T lymphocyte inhibitory capacity. Standardized and reliable conditions for large scale MSC expansion pave the way for safe applications in humans in different therapeutic approaches.

Sepsis syndrome and death in trauma patients are associated with variation in the gene encoding tumor necrosis factor.

Objective:Patients encountering severe trauma are at risk of developing sepsis syndrome and subsequent multiple organ failure. This is often associated with fatal outcome despite survival of the initial injury. We postulate that variation of the gene coding for tumor necrosis factor (TNF)-α is associated with increased occurrence of sepsis syndrome and mortality in trauma patients. Design:Prospective cohort study; validation in an external replication sample. Setting:Tertiary academic medical center. Patients:We included 159 severely traumatized patients from a single center. Serial blood samples were analyzed for serum concentrations of TNF-α and lymphotoxin-α (LTA). We genotyped nine polymorphisms in the TNF gene and tested for an association with sepsis syndrome and outcome. Genetic associations were validated in an external replication sample (n = 76). We examined the peripheral blood transcriptome in 28 patients by whole genome-based profiling and validated the results. Interventions:None. Measurements and Main Results:Carriage of the TNF rs1800629 A allele was associated with higher TNF-α serum concentrations on the first day after trauma and during follow-up (two-sided p = 5.0 × 10−5), with development of sepsis syndrome (odds ratio 7.14, two-sided p = 1.2 × 10−6; external validation sample [n = 76]: odds ratio 3.3, one-sided p = .03), and with fatal outcome (odds ratio 7.65, two-sided p = 1.9 × 10−6). Carriage of the TNF rs1800629 A allele was associated with differential expression of genes representing stronger proinflammatory and apoptotic responses compared with carriage of the wild-type allele. Conclusions:Common TNF gene variants are associated with sepsis syndrome and death after severe injury. These findings are strongly supported by functional data and may be important for developing preemptive anti-inflammatory interventions in carriers of the risk-associated allele.