Transcriptional profiling reveals monocyte-related macrophages phenotypically resembling DC in human intestine.

The tissue dendritic cell (DC) compartment is heterogeneous, and the ontogeny and functional specialization of human tissue conventional DC (cDC) subsets and their relationship with monocytes is unresolved. Here we identify monocyte-related CSF1R+Flt3- antigen presenting cells (APCs) that constitute about half of the cells classically defined as SIRPα+ DCs in the steady-state human small intestine. CSF1R+Flt3- APCs express calprotectin and very low levels of CD14, are transcriptionally related to monocyte-derived cells, and accumulate during inflammation. CSF1R+Flt3- APCs show typical macrophage characteristics functionally distinct from their Flt3+ cDC counterparts: under steady-state conditions they excel at antigen uptake, have a lower migratory potential, and are inefficient activators of naïve T cells. These results have important implications for the understanding of the ontogenetic and functional heterogeneity within human tissue DCs and their relation to the monocyte lineage.

Pancreas transplant rejection episodes are not revealed by biopsies of the donor duodenum in a prospective study with paired biopsies.

The surgical technique with duodeno-duodenal enteroanastomosis of pancreas transplants allows for representative endoscopic ultrasound-guided needle biopsies of the donor duodenum and the pancreas graft. We assessed whether histological findings in transplanted donor duodenal biopsies can indicate rejection in the transplanted pancreas. Since September 2012, a duodeno-duodenal enteroanastomosis has been the default technique for pancreas transplantations at our center. In 67 recipients we prospectively examined 113 endoscopic ultrasound-guided procedures with representative biopsies from the duodenum grafts and the pancreas grafts (97 per protocol and 16 on indication). All graft biopsies were evaluated according to established rejection criteria. A total of 22 biopsy-proven pancreas rejections were detected, with 2 matching duodenal biopsies showing rejection. This gives a sensitivity of 9% for detection of a pancreas rejection by duodenal biopsies. The other matching duodenal biopsies were either normal (n = 13) or indeterminate (n = 7). Rejection of the donor duodenum was found in only 6/113 biopsies, with 2 concurrent pancreas rejections. In conclusion, the donor duodenum is not a useful reporter organ for rejection in the pancreas graft.

Outcomes in Pancreas Transplantation With Exocrine Drainage Through a Duodenoduodenostomy Versus Duodenojejunostomy.

Until recently, pancreas transplantation has mostly been performed with exocrine drainage via duodenojejunostomy (DJ). Since 2012, DJ was substituted with duodenoduodenostomy (DD) in our hospital, allowing endoscopic access for biopsies. This study assessed safety profiles with DD versus DJ procedures and clinical outcomes with the DD technique in pancreas transplantation. DD patients (n = 117; 62 simultaneous pancreas-kidney [SPKDD ] and 55 pancreas transplantation alone [PTADD ] with median follow-up 2.2 years) were compared with DJ patients (n = 179; 167 SPKDJ and 12 PTADJ ) transplanted in the period 1998-2012 (pre-DD era). Postoperative bleeding and pancreas graft vein thrombosis requiring relaparotomy occurred in 17% and 9% of DD patients versus 10% (p = 0.077) and 6% (p = 0.21) in DJ patients, respectively. Pancreas graft rejection rates were still higher in PTADD patients versus SPKDD patients (p = 0.003). Hazard ratio (HR) for graft loss was 2.25 (95% CI 1.00, 5.05; p = 0.049) in PTADD versus SPKDD recipients. In conclusion, compared with the DJ procedure, the DD procedure did not reduce postoperative surgical complications requiring relaparatomy or improve clinical outcomes after pancreas transplantation despite serial pancreatic biopsies for rejection surveillance. It remains to be seen whether better rejection monitoring in DD patients translates into improved long-term pancreas graft survival.

Pancreas transplantation with enteroanastomosis to native duodenum poses technical challenges--but offers improved endoscopic access for scheduled biopsies and therapeutic interventions.

To facilitate endoscopic access for rejection surveillance and stenting of the pancreas, we have abandoned the duodenojejunostomy (DJ) in favor of duodenoduodenostomy (DD) in pancreas transplantation (PTx). From September 2012 to September 2013 we performed 40 PTx with DD; 20 solitary-PTx (S-PTx) and 20 simultaneous pancreas and kidney transplantation (SPK). We compared the outcomes with results from 40 PTx-DJ (10 S-PTx and 30 SPK) from the preceding era. The DD-enteroanastomoses were performed successfully. Endoscopic pancreas biopsies (endoscopic ultrasound examination [EUS]) yielded representative material in half of the cases. One exocrine fistula was treated by endoscopic stenting. PTxs-DD were associated with a higher rate of thrombosis compared to PTx-DJ (23% vs. 5%) and reoperations (48% vs. 30%), as well as inferior graft survival (80% vs. 88%). Time on waiting list, HLA A + B mismatches and reoperations were associated with graft loss. Only recipient age remained an independent predictor of patient death in multivariate analysis. PTx-DD showed a higher rate of thrombosis and inferior results, but facilitated a protocol biopsy program by EUS that was feasible and safe. Given that technical difficulties can be solved, the improved endoscopic access might confer long-term benefits, yet this remains to be proven.

Human naturally occurring and adaptive regulatory T cells secrete high levels of leukaemia inhibitory factor upon activation.

Leukaemia inhibitory factor (LIF) is a member of the IL-6 cytokine family which signals through cognate receptors and activates target genes involved in survival, apoptosis, proliferation, differentiation and suppression of differentiation in different cell types. Binding of LIF to the LIFRalpha/gp130 receptor complex has been shown to activate the Janus kinase-signal transducer and activator of transcription 3 pathway. Here we show that activation of naturally occurring and adaptive regulatory T cells leads to increased LIF expression which is abrogated by cyclic adenosine monophosphate. Furthermore, the LIF receptors gp130 and LIFRalpha are upregulated on the surface of activated T cells and signal transducer and activator of transcription 3 phosphorylation is increased. Interestingly, LIF was not required for suppressive function but rather appeared to have a stimulatory effect on T cells that served to modulate and counteract immunosuppression by regulatory T cells.

Molecular architecture of signal complexes regulating immune cell function.

Signals transmitted via multichain immunoreceptors control the development, differentiation and activation of hematopoetic cells. The cytoplasmic parts of these receptors contain immunoreceptor tyrosine-based activation motifs (ITAMs) that upon phosphorylation by members of the Src tyrosine kinase family orchestrate a complex set of signaling events involving tyrosine phosphorylation, generation of second messengers like DAG, IP3 and Ca2+, activation of effector molecules like Ras and MAPKs and the translocation and activation of transcription factors like NFAT, API and NF-kB. Spatial and temporal organization of these signaling events is essential both to connect the receptors to downstream cascades as well as to control the functional outcome of the immune activation. Throughout this process control and fine-tuning of the different signals are necessary both for effective immune function and in order to avoid inappropriate or exaggerated immune activation and autoimmunity. This control includes modulating mechanisms that set the threshold for activation and reset the activation status after an immune response has been launched. One immunomodulating pathway is the cAMP-protein kinase A-Csk pathway scaffolded by a supramolecular complex residing in lipid rafts with the A kinase-anchoring protein (AKAP) ezrin, the Csk-binding protein PAG and a linker between the two, EBP50. Failure of correct scaffolding and loss of spatiotemporal control can potentially have severe consequences, leading to immune failure or autoimmunity. The clinical relevance of supramolecular complexes specifically organized by scaffolding proteins in regulating immune activity and the specter of genetic diseases linked to different signaling components suggest that protein-protein contact surfaces can be potential targets for drug intervention. It is also of interest to note that different pathogens have evolved strategies to specifically modulate signal integration, thereby rewiring the signal in a way beneficial for their survival. In addition to demonstrating the importance of different signal processes, these adaptations are elegant illustrations of the potential for drug targeting of protein assembly. This chapter reviews some of the important scaffolding events downstream of immunoreceptors with focus on signaling transduction through the T-cell receptor (TCR).

PKAI as a potential target for therapeutic intervention.

We have mapped a molecular mechanism for the impaired T-cell function in HIV infection and common variable immunodeficiency (CVI). Protein kinase A type I (PKAI) has a key role as an inhibitor of immune function in T lymphocytes and is activated following antigen receptor triggering. T cells from patients with HIV infection and CVI have increased activation of PKAI. This inhibits immune function and proliferation of T cells. Selective antagonists that block cAMP action through PKAI improve the immune function of T cells from HIV-infected patients up to 300%. Furthermore, combination of cAMP antagonists with interleukin-2 normalized immune responses of T cells from all patients examined and stimulated immune function of T cells from HIV-infected patients up to 600%. In addition, in vitro experiments indicate that approximately 50% of patients with CVI have a T-cell dysfunction that might benefit from a treatment reversing PKAI hyperactivation. This outlines PKAI as a potentially attractive drug target for immunomodulating therapy in HIV infection, as well as for the treatment of other immunodeficiency disorders such as CVI.

Additive effects of IL-2 and protein kinase A type I antagonist on function of T cells from HIV-infected patients on HAART.

OBJECTIVE: To explore the basis for a possible immunomodulatory combination therapy with IL-2 and agents inhibiting protein kinase A (PKA) type I. DESIGN: Highly active antiretroviral therapy (HAART) has dramatically improved HIV therapy, but fails to eradicate the virus, and the persistence of HIV-associated immunodeficiency demonstrates the need for additional immunomodulating therapies. We have previously shown that hyperactivation of PKA type I inhibits the function of HIV-infected patient T cells. The separate and combined effect of a PKA type I-selective antagonist (Rp-8-Br-cAMPS) and Interleukin (IL)-2 on the function of T cells from HIV-infected patients on HAART was examined. METHODS: The effect of Rp-8-Br-cAMPS on anti-CD3 stimulated proliferation and IL-2 production and the combined effect with exogenous IL-2 was studied in vitro with cells from 13 HIV-infected patients on HAART and six uninfected controls. RESULTS: The PKA type I-selective antagonist improved cell proliferation (median 1.5-fold, maximal 2.8-fold) and IL-2 production (median 1.5-fold, maximal 2.4-fold) in T cells from HIV-infected patients on HAART, but not in controls. The addition of IL-2 enhanced proliferation of T cells from HIV-infected patients (approximately 1.9-fold) and that of controls (approximately 1.4-fold), but IL-2 had no effect at the concentrations produced by treatment with PKA type I antagonist. However, the combined effect of IL-2 and PKA type I antagonist was additive and resulted in a further increase in T-cell proliferation (median 2.5-fold, maximal 5.8-fold), reaching levels comparable with those of uninfected controls in most of the patients. CONCLUSION: Our findings suggest a basis for a novel strategy in treatment of HIV infection by combining IL-2 therapy and treatment modalities counteracting PKA type I activity with HAART.

Increased activation of protein kinase A type I contributes to the T cell deficiency in common variable immunodeficiency.

The molecular mechanisms underlying the T cell dysfunction often present in common variable immunodeficiency (CVI) are not established. cAMP-dependent protein kinase A type I (PKAI) is an important inhibitor of T cell proliferation after Ag stimulation. We therefore investigated the possibility that activation of PKAI may be involved in the development of T cell dysfunction in CVI. An exogenously added PKAI-selective antagonist (Rp-8-Br-cAMPS) induced a significant increase in anti-CD3-stimulated PBMC proliferation in 20 CVI patients compared with no effect in 15 controls. Purified T cells from 7 CVI patients with strictly defined T cell deficiency had elevated endogenous cAMP levels compared with controls. Treatment of T cells from these CVI patients with Rp-8-bromo-cAMP-phosphorothioate markedly improved anti-CD3-stimulated proliferation (up to 3.7-fold), particularly in CD4+ lymphocytes, reaching proliferation levels comparable to control values. No effect of cAMP antagonist on T cell proliferation was seen in controls. In these CVI patients, cAMP antagonist also increased IL-2 production in anti-CD3-stimulated T cells. However, exogenously added IL-2 at concentrations comparable to the achieved increase in IL-2 levels after addition of cAMP antagonist had no effect on T cell proliferation. Furthermore, the stimulatory effects of exogenously added IL-2 at higher concentrations and cAMP antagonist on T cell proliferation were additive. Our findings indicate that increased PKAI activation may be an important molecular basis for the T cell defect in CVI and suggest that the cAMP/PKAI system may be a potential molecular target for immunomodulating therapy in these patients.

Isozymes of cyclic AMP-dependent protein kinases (PKA) in human lymphoid cell lines: levels of endogenous cAMP influence levels of PKA subunits and growth in lymphoid cell lines.

Activation of the cAMP signaling pathway in lymphoid cells is known to inhibit cell proliferation of T and B cells as well as cytotoxicity of natural killer (NK) cells. In order to find suitable model systems to study cAMP-mediated processes, we have examined the expression of cAMP-dependent protein kinase (PKA), endogenous levels of cAMP, and cell proliferation in eight cell lines of B lineage origin, four cell lines of T lineage origin, and normal human B and T cells. We demonstrated that the expression of mRNA and protein for one of the regulatory (R) subunits of PKA (RIalpha) was present in all the cells investigated, in contrast to the other R subunits (RIbeta, RIIalpha, and RIIbeta). Furthermore, three T cell lines and one B cell line expressed only RIalpha and C, implying these cells to contain solely PKA type I. Moreover, for the RI subunit, we observed an apparent reciprocal relationship between levels of mRNA and protein. Generally, RIalpha protein was low in cell lines where mRNA was elevated and vice versa. This was not the case for the RII subunits, where high levels of mRNA were associated with elevated levels of protein. Interestingly, we demonstrated an inverse correlation between levels of endogenous cAMP and cell growth as determined by [3H]-thymidine incorporation and cell-doubling rate (P < 0.05). Taken together, our results demonstrate great differences in PKA isozyme composition, which should be taken into consideration when using lymphoid cell lines as model system for cAMP/PKA effects in normal lymphocytes.

Protein kinase A type I antagonist restores immune responses of T cells from HIV-infected patients.

Cyclic AMP-dependent protein kinase A (PKA) type I has been established as an acute inhibitor of T cell activation. For this reason, we investigated the possible role of PKA type I in HIV-induced T cell dysfunction. T cells from HIV-infected patients have increased levels of cAMP and are more sensitive to inhibition by cAMP analog than are normal T cells. A PKA type I-selective antagonist increases the impaired proliferation of T cells from HIV-infected patients to normal or subnormal levels (up to 2.8-fold). Follow-up of patients after initiation of highly active antiretroviral treatment revealed that a majority of patients have a persistent T cell dysfunction that is normalized by incubation of T cells with Rp-8-Br-cAMPS. These observations imply that increased activation of PKA type I may contribute to the progressive T cell dysfunction in HIV infection and that PKA type I may be a potential target for immunomodulating therapy.