CTLA4-Ig Restores Rejection of MHC Class-II Mismatched Allografts by Disabling IL-2-Expanded Regulatory T Cells

Allograft acceptance and tolerance can be achieved by different approaches including inhibition of effector T cell responses through CD28-dependent costimulatory blockade and induction of peripheral regulatory T cells (Tregs). The observation that Tregs rely upon CD28-dependent signals for development and peripheral expansion, raises the intriguing possibility of a counterproductive consequence of CTLA4-Ig administration on tolerance induction. We have investigated the possible negative effect of CTLA4-Ig on Treg-mediated tolerance induction using a mouse model of single MHC class II-mismatched skin grafts in which long-term acceptance was achieved by short-term administration of IL-2/anti-IL-2 complex. CTLA4-Ig treatment was found to abolish Treg-dependent acceptance in this model, restoring skin allograft rejection and Th1 alloreactivity. CTLA4-Ig inhibited IL-2-driven Treg expansion, and prevented in particular the occurrence of ICOS(+) Tregs endowed with potent suppressive capacities. Restoring CD28 signaling was sufficient to counteract the deleterious effect of CTLA4-Ig on Treg expansion and functionality, in keeping with the hypothesis that costimulatory blockade inhibits Treg expansion and function by limiting the delivery of essential CD28-dependent signals. Inhibition of regulatory T cell function should therefore be taken into account when designing tolerance protocols based on costimulatory blockade. Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel β-strand interactions between enzyme and target is suggested.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Intestinal microbes affect phenotypes and functions of invariant natural killer T cells in mice

Invariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells.

Going against the tide--how encephalitogenic T cells breach the blood-brain barrier

During multiple sclerosis or its animal model, experimental autoimmune encephalomyelitis, circulating immune cells enter the central nervous system (CNS) causing neuroinflammation. Extravasation from the blood circulation across the vessel wall occurs through a multistep process regulated by adhesion and signal transducing molecules on the immune cells and on the endothelium. Since the CNS is shielded by the highly specialized blood-brain barrier (BBB), immune cell extravasation into the CNS requires breaching this particularly tight endothelial border. Consequently, travelling into the CNS demands unique adaptations which account for the extreme tightness of the BBB. Modern imaging tools have shown that after arresting on BBB endothelium, in vivo or in vitro encephalitogenic effector/memory T cells crawl for long distances, possibly exceeding 150 µm along the surface of the BBB endothelium before rapidly crossing the BBB. Interestingly, in addition to the distance of crawling, the preferred direction of crawling against the flow is unique for T cell crawling on the luminal surface of CNS microvessels. In this review, we will summarize the cellular and molecular mechanisms involved in the unique T cell behavior that is obviously required for finding a site permissive for diapedesis across the unique vascular bed of the BBB.

Inhibition of indoleamine 2,3-dioxygenase in mixed lymphocyte reaction affects glucose influx and enzymes involved in aerobic glycolysis and glutaminolysis in alloreactive T-cells

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. T-cell proliferation and differentiation to effector cells require increased glucose consumption, aerobic glycolysis and glutaminolysis. The effect of IDO on the above metabolic pathways was evaluated in alloreactive T-cells. Mixed lymphocyte reaction (MLR) in the presence or not of the IDO inhibitor, 1-DL-methyl-tryptophane (1-MT), was used. In MLRs, 1-MT decreased tryptophan consumption, increased cell proliferation, glucose influx and lactate production, whereas it decreased tricarboxylic acid cycle activity. In T-cells, from the two pathways that could sense tryptophan depletion, i.e. general control nonrepressed 2 (GCN2) kinase and mammalian target of rapamycin complex 1, 1-MT reduced only the activity of the GCN2 kinase. Additionally 1-MT treatment of MLRs altered the expression and/or the phosphorylation state of glucose transporter-1 and of key enzymes involved in glucose metabolism and glutaminolysis in alloreactive T-cells in a way that favors glucose influx, aerobic glycolysis and glutaminolysis. Thus in alloreactive T-cells, IDO through activation of the GCN2 kinase, decreases glucose influx and alters key enzymes involved in metabolism, decreasing aerobic glycolysis and glutaminolysis. Acting in such a way, IDO could be considered as a constraining factor for alloreactive T-cell proliferation and differentiation to effector T-cell subtypes.

Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.

IL-6 transsignalling modulates the early effector phase of EAE and targets the blood-brain barrier

Interleukin-6 (IL-6) plays a crucial role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). It exerts its cellular effects by a membrane-bound IL-6 receptor (IL-6R), or, alternatively, by forming a complex with the soluble IL-6R (sIL-6R), a process named IL-6 transsignalling. Here we investigate the role of IL-6 transsignalling in myelin basic protein (MBP)-induced EAE in the Lewis rat. In vivo blockade of IL-6 transsignalling by the injection of a specifically designed gp130-Fc fusion protein significantly delayed the onset of adoptively transferred EAE in comparison to control rats injected with PBS or isotype IgG. Histological evaluation on day 3 after immunization revealed reduced numbers of T cells and macrophages in the lumbar spinal cord of gp130-Fc treated rats. At the same time, blockade of IL-6 transsignalling resulted in a reduced expression of vascular cell adhesion molecule-1 on spinal cord microvessels while experiments in cell culture failed to show a direct effect on the regulation of endothelial adhesion molecules. In experiments including active EAE and T cell culture, inhibition of IL-6 transsignalling mildly increased T cell proliferation, but did not change severity of active MBP-EAE or regulate Th1/Th17 responses. We conclude that IL-6 transsignalling may play a role in autoimmune inflammation of the CNS mainly by regulating early expression of adhesion molecules, possibly via cellular networks at the blood-brain barrier.

Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells

Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, Fas ligand activity has to be tightly regulated, involving various transcriptional and post-transcriptional processes. For example, preformed Fas ligand is stored in secretory lysosomes of activated T cells, and rapidly released by degranulation upon reactivation. In this study, we analyzed the minimal requirements for activation-induced degranulation of Fas ligand. T cell receptor activation can be mimicked by calcium ionophore and phorbol ester. Unexpectedly, we found that stimulation with phorbol ester alone is sufficient to trigger Fas ligand release, whereas calcium ionophore is neither sufficient nor necessary. The relevance of this process was confirmed in primary CD4(+) and CD8(+) T cells and NK cells. Although the activation of protein kinase(s) was absolutely required for Fas ligand degranulation, protein kinase C or A were not involved. Previous reports have shown that preformed Fas ligand co-localizes with other markers of cytolytic granules. We found, however, that the activation-induced degranulation of Fas ligand has distinct requirements and involves different mechanisms than those of the granule markers CD63 and CD107a/Lamp-1. We conclude that activation-induced degranulation of Fas ligand in cytotoxic lymphocytes is differently regulated than other classical cytotoxic granule proteins.

Cathepsin W expressed exclusively in CD8+ T cells and NK cells, is secreted during target cell killing but is not essential for cytotoxicity in human CTLs

OBJECTIVE: Cathepsin W (CatW, lymphopain) is a putative cysteine protease with restricted expression to natural killer (NK) cells and CD8(+) T cells and so far unknown function and properties. Here, we characterize in detail, the regulation of human CatW during T-cell development in response to different stimuli and its functional involvement in cytotoxic lymphocyte effector function. MATERIALS AND METHODS: Western blots and real time polymerase chain reaction of sorted, unstimulated, and stimulated cell subsets (thymocytes, T cells, NK cells) and their culture supernatants were used to study regulation and expression of CatW. Primary CD8(+) T cells and short-term T-cell lines were transfected with small interfering RNA to study the involvement of CatW in effector function such as target cell killing and interferon-gamma production. RESULTS: Levels of CatW expression correlate closely with cytotoxic capacity both during development and in response to factors influencing cytotoxicity. Furthermore, CatW is secreted during specific target cell killing. However, knockdown of CatW expression by small interfering RNA neither influences target cell killing nor interferon-gamma production. CONCLUSION: Despite being expressed in the effector subset of CD8(+) and NK cells and of being released during target cell killing, our functional inhibition studies exclude an essential role of CatW in the process of cytotoxicity.

Pregnancy in patients with ankylosing spondylitis: do regulatory T cells play a role?

OBJECTIVE: To investigate the numerical and functional changes of CD4+CD25(high) regulatory T (Treg) cells during pregnancy and postpartum in patients with ankylosing spondylitis (AS). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 10 pregnant and 5 nonpregnant patients with AS as well as in 14 pregnant and 4 nonpregnant healthy controls. Pregnant individuals were investigated at the third trimester and 8 weeks postpartum. Treg cells and CD4+CD25- effector T (Teff) cells separated by fluorescence-activated cell sorting were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies, alone or in coculture, to investigate proliferation and cytokine secretion. RESULTS: The frequency of CD4+CD25(high) Treg cells was significantly higher during pregnancy than postpartum in both healthy control subjects and patients with AS. In contrast to Treg cells in healthy pregnant women, Treg cells in pregnant women with AS secreted only small amounts of interleukin-10 and showed lower suppression of tumor necrosis factor alpha and interferon-gamma secretion by CD4+CD25- Teff cells. At the postpartum time point, proinflammatory cytokine levels in the Treg/Teff cell cocultures and Teff cell monocultures were significantly higher in patients with AS than in healthy controls. CONCLUSION: Pregnancy influenced the expansion and cytokine secretion of Treg cells in both patients with AS and control subjects. However, the Treg cells of pregnant patients with AS failed to support an antiinflammatory cytokine milieu, thereby possibly contributing to the persistent disease activity of AS during pregnancy.

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells.

Protection against malaria can be achieved by induction of a strong CD8(+) T-cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long-lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8(+) T-cell response and sterile protection, which is short-lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite-specific antibodies that showed protective capacity. The numbers of CSP-specific CD8(+) T cells were not substantially enhanced by sporozoite infections; however, CSP-specific memory CD8(+) T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized-only mice. CD4(+) T cells contributed to protection as well; but CD8(+) memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8(+) effector-memory T cells with increased cytotoxic activity as well as CD4(+) memory T cells and neutralizing antibodies.

Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

Cytotoxic T cells induce proliferation of chronic myeloid leukemia stem cells by secreting interferon-γ

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasia arising from the oncogenic break point cluster region/Abelson murine leukemia viral oncogene homolog 1 translocation in hematopoietic stem cells (HSCs), resulting in a leukemia stem cell (LSC). Curing CML depends on the eradication of LSCs. Unfortunately, LSCs are resistant to current treatment strategies. The host’s immune system is thought to contribute to disease control, and several immunotherapy strategies are under investigation. However, the interaction of the immune system with LSCs is poorly defined. In the present study, we use a murine CML model to show that LSCs express major histocompatibility complex (MHC) and co-stimulatory molecules and are recognized and killed by leukemia-specific CD8+ effector CTLs in vitro. In contrast, therapeutic infusions of effector CTLs into CML mice in vivo failed to eradicate LSCs but, paradoxically, increased LSC numbers. LSC proliferation and differentiation was induced by CTL-secreted IFN-γ. Effector CTLs were only able to eliminate LSCs in a situation with minimal leukemia load where CTL-secreted IFN-γ levels were low. In addition, IFN-γ increased proliferation and colony formation of CD34+ stem/progenitor cells from CML patients in vitro. Our study reveals a novel mechanism by which the immune system contributes to leukemia progression and may be important to improve T cell–based immunotherapy against leukemia.

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

BACKGROUND: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. PRINCIPAL FINDINGS: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2. SIGNIFICANCE: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.