Christine Appelt

Permanent CNI Treatment for Prevention of Renal Allograft Rejection in Sensitized Hosts Can Be Replaced by Regulatory T Cells

Recent data suggest that donor‐specific memory T cells (Tmem) are an independent risk factor for rejection and poor graft function in patients and a major challenge for immunosuppression minimizing strategies. Many tolerance induction protocols successfully proven in small animal models e.g. costimulatory blockade, T cell depletion failed in patients. Consequently, there is a need for more predictive transplant models to evaluate novel promising strategies, such as adoptive transfer of regulatory T cells (Treg).

Anti-CD4-mediated selection of Treg in vitro – in vitro suppression does not predict in vivo capacity to prevent graft rejection

Regulatory T cells (Treg) have been shown to play a role in the prevention of autoimmune diseases and transplant rejection. Based on an established protocol known to generate alloantigen reactive Treg in vivo, we have developed a strategy for the in vitro selection of Treg. Stimulation of unfractionated CD4+ T cells from naive CBA.Ca (H2k) mice with C57BL/10 (H2b) splenocytes in the presence of an anti‐CD4 antibody, YTS 177, resulted in the selection of Treg able to inhibit proliferation of naive T cells. In vivo, the cells were able to prevent rejection of 80% C57BL/10 skin grafts when co‐transferred to CBA.Rag–/– mice together with naive CD45RBhighCD4+ cells. Purification of CD62L+CD25+CD4+ cells from the cultures enriched for cells with regulatory activity; as now 100% survival of C57BL/10 skin grafts was achieved. Furthermore, differentiation of Treg could be also achieved when using purified CD25–CD4+ naive T cells as a starting population. Interestingly, further in vitro expansion resulted in a partial loss of CD4+ cells expressing both CD62L and CD25 and abrogation of their regulatory activity in vivo. This study shows that alloantigen stimulation in the presence of anti‐CD4 in vitro provides a simple and effective strategy to generate alloreactive Treg.

Prevention of Graft-versus-Host Disease by Adoptive T Regulatory Therapy Is Associated with Active Repression of Peripheral Blood Toll-Like Receptor 5 mRNA Expression

Acute graft-versus-host disease (GVHD) occurs in 40% to 60% of recipients of partially matched umbilical cord blood transplantation (UCBT). In a phase I study, adoptive transfer of expanded CD4(+)CD25(+)Foxp3(+) natural regulatory T cells (nTregs) resulted in a reduced incidence of grade II-IV acute GVHD. To investigate potential mechanisms responsible for the reduced GVHD risk, we analyzed peripheral blood mononuclear cell mRNA expression of a tolerance gene set previously identified in operation- tolerant kidney transplant recipients, comparing healthy controls and patients who received nTregs and those who did not receive nTregs with and without experiencing GVHD. Samples from patients receiving nTregs regardless of GVHD status showed increased expression of Foxp3 expression, as well as B cell-related tolerance marker. This was correlated with early B cell recovery, predominately of naïve B cells, and nearly normal T cell reconstitution. CD8(+) T cells showed reduced signs of activation (HLA-DR(+) expression) compared with conventionally treated patients developing GVHD. In contrast, patients with GVHD had significantly increased TLR5 mRNA expression, whereas nTreg-treated patients without GVHD had reduced TLR5 mRNA expression. We identified Lin(-)HLADR(-)CD33(+)CD16(+) cells and CD14(++)CD16(-) monocytes as the main TLR5 producers, especially in samples of conventionally treated patients developing GVHD. Taken together, these data reveal interesting similarities and differences between tolerant organ and nTreg-treated hematopoietic stem cell transplantation recipients.

Generation of highly effective and stable murine alloreactive Treg cells by combined anti-CD4 mAb, TGF-β, and RA treatment.

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4+ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25+Foxp3+‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25+Foxp3+ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.

Elevation of CD4+ differentiated memory T cells is associated with acute cellular and antibody-mediated rejection after liver transplantation.

Background It is now well known that the outcome after allogeneic transplantation, such as incidence of acute rejections, very much depends on the individual’s immune reactivity status. There is also increasing evidence that the presence of preexisting memory T cells can affect antigraft immune responses. Methods In a prospective study, we monitored peripheral CD4+ and CD8+ central memory, effector memory, and terminal differentiated effector memory (TEMRA) T cells in 55 patients who underwent deceased liver transplantation and received conventional immunosuppressive treatment with or without basiliximab induction. The primary endpoint of the study was acute allograft rejection during a 1-year follow-up period. Results We observed significantly increased proportions of CD4+ and CD8+ TEMRA cells in patients before transplantation compared with healthy controls (P=0.006 and 0.009, respectively). This characteristic was independent of the underlying disease. In patients with no signs of acute rejection, we observed an immediate reduction of CD4+ TEMRA cells. In contrast, patients who experienced acute cellular rejection, and especially antibody-mediated rejection, displayed persistent elevated TEMRA cells (P=0.017 and 0.027, respectively). Basiliximab induction therapy did not influence CD4+ and CD8+ TEMRA numbers. Conclusions Conventional immunosuppressive or basiliximab treatment cannot control the persistence of TEMRA T cells, which may contribute to acute cellular rejection and antibody-mediated rejection after liver transplantation. In the future, specific targeting of TEMRA cells in selected patients may prevent the occurrence of difficult to treat steroid-resistant rejections, thereby leading to improved patient outcome.

Differential expression and function of α-mannosidase I in stimulated naive and memory CD4+ T cells.

N-linked protein glycosylation represents an important cellular process for modifying protein properties. It resembles a cascade of various enzymatic reactions, in which class I &agr;-mannosidases play a central role. We and others have recently shown that N-glycosylation plays a major role for immune functions. We now analyzed the expression and function of &agr;-mannosidase I in CD4+ naive and memory T cells studying human and murine T cells. Alpha-mannosidase I function was altered by (i) treatment with Kifunensine, a specific inhibitor class I &agr;-mannosidases, (ii) synthetic inhibitory RNA, and (iii) overexpression by retroviral gene transfer. T-cell activation was evaluated by CD69 expression, cytokine production and proliferation. Our results demonstrate (i) that &agr;-mannosidase I transcription is transiently downregulated after T-cell activation with either polyclonal anti-CD3/CD28 antibodies or allogeneic CD19+ B cells, and (ii) that &agr;-mannosidase I exerts an inhibitory effect on T-cell activation. It is interesting to note that the inhibitory effect was restricted to naive CD4+ T cells in both systems, human T cells and murine transgenic CD4+OT-II cells, whereas human memory T cells and primed CD4+OT-II cells remained unaffected. Alpha-mannosidase I inhibition reduced the activation threshold for naive but not already primed CD4+OT-II cells as the cells were able to respond to lower ovalbumin peptide concentrations and increased the rejection potential of alloreactive T cells in vivo. Thus, complex N-glycans generated by enzymes such as &agr;-mannosidase I inhibit the activation of naive T cells. These findings could be used to improve the ex vivo priming of naive T cells for adaptive T-cell therapies.

The mitochondrial protein TCAIM regulates activation of T cells and thereby promotes tolerance induction of allogeneic transplants.

Primary T cell activation and effector cell differentiation is required for rejection of allogeneic grafts in naïve recipients. It has become evident, that mitochondria play an important role for T cell activation. Expression of several mitochondrial proteins such as TCAIM (T cell activation inhibitor, mitochondrial) is down‐regulated upon T cell receptor triggering. Here we report that TCAIM inhibited spontaneous development of memory and effector T cells. CD4+ T cells from Tcaim knock‐in (KI) mice showed reduced activation, cytokine secretion and proliferation in vitro. Tcaim KI T cells tolerated allogeneic skin grafts upon transfer into Rag‐1 KO mice. CD4+ and CD8+ T cells from these mice did not infiltrate skin grafts and kept a naïve or central memory phenotype, respectively. They were unable to acquire effector phenotype and functions. TCAIM altered T cell activation‐induced mitochondrial distribution and reduced mitochondrial reactive oxygen species (mROS) production. Thus, TCAIM controls T cell activation and promotes tolerance induction probably by regulating TCR‐mediated mitochondrial distribution and mROS production.

TCAIM Decreases T Cell Priming Capacity of Dendritic Cells by Inhibiting TLR-Induced Ca2+ Influx and IL-2 Production

We previously showed that the T cell activation inhibitor, mitochondrial (Tcaim) is highly expressed in grafts of tolerance-developing transplant recipients and that the encoded protein is localized within mitochondria. In this study, we show that CD11c+ dendritic cells (DCs), as main producers of TCAIM, downregulate Tcaim expression after LPS stimulation or in vivo alloantigen challenge. LPS-stimulated TCAIM-overexpressing bone marrow–derived DC (BMDCs) have a reduced capacity to induce proliferation of and cytokine expression by cocultured allogeneic T cells; this is not due to diminished upregulation of MHC or costimulatory molecules. Transcriptional profiling also revealed normal LPS-mediated upregulation of the majority of genes involved in TLR signaling. However, TCAIM BMDCs did not induce Il2 mRNA expression upon LPS stimulation in comparison with Control-BMDCs. In addition, TCAIM overexpression abolished LPS-mediated Ca2+ influx and mitochondrial reactive oxygen species formation. Addition of IL-2 to BMDC–T cell cocultures restored the priming capacity of TCAIM BMDCs for cocultured allogeneic CD8+ T cells. Furthermore, BMDCs of IL-2–deficient mice showed similarly abolished LPS-induced T cell priming as TCAIM-overexpressing wild type BMDCs. Thus, TCAIM interferes with TLR4 signaling in BMDCs and subsequently impairs their T cell priming capacity, which supports its role for tolerance induction.

Differences in CD44 Surface Expression Levels and Function Discriminates IL-17 and IFN-γ Producing Helper T Cells

CD44 is a prominent activation marker which distinguishes memory and effector T cells from their naïve counterparts. It also plays a role in early T cell signaling events as it is bound to the lymphocyte-specific protein kinase and thereby enhances T cell receptor signalling. Here, we investigated whether IFN-γ and IL-17 producing T helper cells differ in their CD44 expression and their dependence of CD44 for differentiation. Stimulation of CD4+ T cells with allogeneic dendritic cells resulted in the formation of three distinguishable populations: CD44+, CD44++ and CD44+++. In vitro and in vivo generated allo-reactive IL-17 producing T helper cells were mainly CD44+++ as compared to IFN-γ+ T helper cells, which were CD44++. This effect was enhanced under polarizing conditions. T helper 17 polarization led to a shift towards the CD44+++ population, whereas T helper 1 polarization diminished this population. Furthermore, blocking CD44 decreased IL-17 secretion, while IFN-γ was barely affected. Titration experiments revealed that low T cell receptor and CD28 stimulation supported T helper 17 rather than T helper 1 development. Under these conditions CD44 could act as a co-stimulatory molecule and replace CD28. Indeed, rested CD44+++CD4+ T cells contained already more total and especially phosphorylated zeta-chain-associated protein kinase 70 as compared to CD44++ cells. Our results support the notion, that CD44 enhances T cell receptor signaling strength by delivering lymphocyte-specific protein kinase, which is required for induction of IL-17 producing T helper cells.

Control of TNF-Induced Dendritic Cell Maturation by Hybrid-Type N-Glycans

The activity of α-1,2-mannosidase I is required for the conversion of high-mannose to hybrid-type (ConA reactive) and complex-type N-glycans (Phaseolus vulgaris-leukoagglutinin [PHA-L] reactive) during posttranslational protein N-glycosylation. We recently demonstrated that α-1,2-mannosidase I mRNA decreases in graft-infiltrating CD11c+ dendritic cells (DCs) prior to allograft rejection. Although highly expressed in immature DCs, little is known about its role in DC functions. In this study, analysis of surface complex-type N-glycan expression by lectin staining revealed the existence of PHA-Llow and PHA-Lhigh subpopulations in murine splenic conventional DCs, as well as in bone marrow-derived DC (BMDCs), whereas plasmacytoid DCs are nearly exclusively PHA-Lhigh. Interestingly, all PHA-Lhigh DCs displayed a strongly reduced responsiveness to TNF-α–induced p38-MAPK activation compared with PHA-Llow DCs, indicating differences in PHA-L–binding capacities between DCs with different inflammatory properties. However, p38 phosphorylation levels were increased in BMDCs overexpressing α-1,2-mannosidase I mRNA. Moreover, hybrid-type, but not complex-type, N-glycans are required for TNF-α–induced p38-MAPK activation and subsequent phenotypic maturation of BMDCs (MHC-II, CD86, CCR7 upregulation). α-1,2-mannosidase I inhibitor-treated DCs displayed diminished transendothelial migration in response to CCL19, homing to regional lymph nodes, and priming of IFN-γ–producing T cells in vivo. In contrast, the activity of α-1,2-mannosidase I is dispensable for LPS-induced signaling, as well as the DCs’ general capability for phenotypic and functional maturation. Systemic application of an α-1,2-mannosidase I inhibitor was able to significantly prolong allograft survival in a murine high-responder corneal transplantation model, further highlighting the importance of N-glycan processing by α-1,2-mannosidase I for alloantigen presentation and T cell priming.