Camelid-derived Tcell engagers harnessing human γδ T cells as promising antitumor immunotherapeutic agents.

In the last decade, there has been a surge in developing immunotherapies to enhance the immune system's ability to eliminate tumor cells. Bispecific antibodies known as T cell engagers (TCEs) present an attractive strategy in this pursuit. TCEs aim to guide cytotoxic T cells toward tumor cells, thereby inducing a strong activation and subsequent tumor cell lysis. In this study, we investigated the activity of different TCEs on both conventional alpha-beta (αß) T cells and unconventional gamma delta (γδ) T cells. TCEs were built using camelid single-domain antibodies (VHHs) targeting the tumor-associated antigen CEACAM5 (CEA), together with T cell receptor chains or a CD3 domain. We show that Vγ9Vδ2 T cells display stronger in vitro antitumor activity than αß T cells when stimulated with a CD3xCEA TCE. Furthermore, restricting the activation of fresh human peripheral T cells to Vγ9Vδ2 T cells limited the production of protumor factors and proinflammatory cytokines, commonly associated with toxicity in patients. Taken together, our findings provide further insights that γδ T cell-specific TCEs hold promise as specific, effective, and potentially safe molecules to improve antitumor immunotherapies.

Increased endogenous antigen presentation in the periphery enhances susceptibility to inflammation-induced gastric autoimmunity in mice.

How the immune system maintains peripheral tolerance under inflammatory conditions is poorly understood. Here we assessed the fate of gastritogenic T cells following inflammatory activation in vivo. Self-reactive T cells (A23 T cells) specific for the gastric H+ /K+ ATPase α subunit (HKα) were transferred into immunosufficient recipient mice and immunised at a site distant to the stomach with adjuvant containing the cognate HKα peptide antigen. Activation of A23 T cells by immunisation did not impact on either immune tolerance or protection from gastric autoimmunity in wild-type BALB/c mice. However, increased presentation of endogenously derived HKα epitopes by dendritic cells (DCs) in the gastric lymph node of IE-H+ /K+ ß transgenic mice (IEß) reduces A23 T-cell tolerance to gastric antigens after inflammatory activation, with subsequent development of gastritis. While HKα-specific A23 T cells from immunised wild-type mice were poorly responsive to in vitro antigen specific activation, A23 T cells from immunised IEß transgenic mice were readily re-activated, indicating loss of T-cell anergy. These findings show that DCs of gastric lymph nodes can maintain tolerance of pathogenic T cells following inflammatory stimulation and that the density of endogenous antigen presented to self-reactive T cells is critical in the balance between tolerance and autoimmunity.

Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen.

Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.

Differential use of autophagy by primary dendritic cells specialized in cross-presentation.

Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed "cross-presentation." The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.

Transient Treg-cell depletion in adult mice results in persistent self-reactive CD4(+) T-cell responses.

Depletion of Foxp3(+) CD4(+) regulatory T cells (Treg) in adults results in chronic inflammation and autoimmune disease. However, the impact of transient Treg-cell depletion on self-reactive responses is poorly defined. Here, we studied the effect of transient depletion of Treg cells on CD4(+) T-cell responses to endogenous self-antigens. Short-term ablation of Treg cells in mice resulted in rapid activation of CD4(+) T cells, increased percentage of IFN-γ(+) and Th17 cells in lymphoid organs, and development of autoimmune gastritis. To track self-reactive responses, we analyzed the activation of naïve gastric-specific CD4(+) T cells. There was a dramatic increase in proliferation and acquisition of effector function of gastric-specific T cells in the stomach draining LNs of Treg-cell-depleted mice, compared with untreated mice, either during Treg-cell depletion or after Treg-cell reconstitution. Moreover, the hyperproliferation of gastric-specific T cells in the Treg-cell-ablated mice was predominantly antigen-dependent. Transient depletion of Treg cells resulted in a shift in the ratio of peripheral:thymic Treg cells in the reemerged Treg-cell population, indicating an altered composition of Treg cells. These findings indicate that transient Treg-cell depletion results in ongoing antigen-driven self-reactive T-cell responses and emphasize the continual requirement for an intact Treg-cell population.

Transient systemic inflammation does not alter the induction of tolerance to gastric autoantigens by migratory dendritic cells.

It has been proposed that activation of dendritic cells (DCs) presenting self-antigens during inflammation may lead to activation of autoreactive T cells and the development of autoimmunity. To test this hypothesis, we examined the presentation of the autoantigen recognized in autoimmune gastritis, gastric H(+)/K(+) ATPase, which is naturally expressed in the stomach and is constitutively presented in the stomach-draining lymph nodes. Systemic administration to mice of the TLR9 agonist CpG DNA, agonist anti-CD40 Ab, or TLR4 agonist LPS all failed to abrogate the process of peripheral clonal deletion of H(+)/K(+) ATPase-specific CD4 T cells or promote the development of autoimmune gastritis. We demonstrated that migratory DCs from the stomach-draining lymph nodes are the only DC subset capable of constitutively presenting the endogenous gastric H(+)/K(+) ATPase autoantigen in its normal physiological context. Analysis of costimulatory molecules indicated that, relative to resident DCs, migratory DCs displayed a partially activated phenotype in the steady state. Furthermore, migratory DCs were refractory to stimulation by transient exposure to TLR agonists, as they failed to upregulate costimulatory molecules, secrete significant amounts of inflammatory cytokines, or induce differentiation of effector T cells. Together, these data show that transient systemic inflammation failed to break tolerance to the gastric autoantigen, as migratory DCs presenting the gastric autoantigen remain tolerogenic under such conditions, demonstrating the robust nature of peripheral tolerance.

Helios defines T cells being driven to tolerance in the periphery and thymus.

The expression of the Ikaros transcription factor family member, Helios, has been shown to be associated with T-cell tolerance in both the thymus and the periphery. To better understand the importance of Helios in tolerance pathways, we have examined the expression of Helios in TCR-transgenic T cells specific for the gastric H(+) /K(+) ATPase, the autoantigen target in autoimmune gastritis. Analysis of H(+) /K(+) ATPase-specific T cells in mice with different patterns of H(+) /K(+) ATPase expression revealed that, in addition to the expression of Helios in CD4(+) Foxp3(+) regulatory T (Treg) cells, Helios is expressed by a large proportion of CD4(+) Foxp3(-) T cells in both the thymus and the paragastric lymph node (PgLN), which drains the stomach. In the thymus, Helios was expressed by H(+) /K(+) ATPase-specific thymocytes that were undergoing negative selection. In the periphery, Helios was expressed in H(+) /K(+) ATPase-specific CD4(+) T cells following H(+) /K(+) ATPase presentation and was more highly expressed when T-cell activation occurred in the absence of inflammation. Analysis of purified H(+) /K(+) ATPase-specific CD4(+) Foxp3(-) Helios(+) T cells demonstrated that they were functionally anergic. These results demonstrate that Helios is expressed by thymic and peripheral T cells that are being driven to tolerance in response to a genuine autoantigen.

Pathogenic T cells persist after reversal of autoimmune disease by immunosuppression with regulatory T cells.

Autoimmune disease can be prevented with immunosuppressive agents; however, the effectiveness of these treatments in advanced stage of disease and the fate of pathogenic T cells following such treatments are not clear. In this study we demonstrate that a single dose of in vitro-induced Treg cells (iTreg cells) resulted in the functional repair and restitution of stomach tissue that had been severely damaged in advanced autoimmune gastritis. iTreg cells caused depletion or inactivation of autoreactive naïve T cells that were antigen inexperienced, however, autoreactive effector/memory T cells persisted in treated mice, resulting in residual cellular infiltrates within the repaired stomach tissue. The persisting autoreactive T cells were able to rapidly cause autoimmune disease if iTreg cells were removed. Similar data were obtained from mice treated continuously with corticosteroid, in that there was substantial restitution of the gastric mucosa; however, effector T cells persisted and rapidly caused pathology following drug removal. Therefore, iTreg cells or corticosteroid can suppress pathogenic autoreactive cells in advanced autoimmune disease, reversing tissue damage and improving tissue function. However, the persistence of pathogenic T cells represents a disease risk.

Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport.

Regulation of membrane transport is controlled by small G proteins, which include members of the Rab and Arf families. Whereas the role of the classic Arf family members are well characterized, many of the Arf-like proteins (Arls) remain poorly defined. Here we show that Arl5a and Arl5b are localised to the trans-Golgi in mammalian cells, and furthermore have identified a role for Arl5b in the regulation of retrograde membrane transport from endosomes to the trans-Golgi network (TGN). The constitutively active Arl5b (Q70L)-GFP mutant was localised efficiently to the Golgi in HeLa cells whereas the dominant-negative Arl5b (T30N)-GFP mutant was dispersed throughout the cytoplasm and resulted in perturbation of the Golgi apparatus. Stable HeLa cells expressing GFP-tagged Arl5b (Q70L) showed an increased rate of endosome-to-Golgi transport of the membrane cargo TGN38 compared with control HeLa cells. Depletion of Arl5b by RNAi resulted in an alteration in the intracellular distribution of mannose-6-phosphate receptor, and significantly reduced the endosome-to-TGN transport of the membrane cargo TGN38 and of Shiga toxin, but had no affect on the anterograde transport of the cargo E-cadherin. Collectively these results suggest that Arl5b is a TGN-localised small G protein that plays a key role in regulating transport along the endosome-TGN pathway.

Shaping the T-cell repertoire in the periphery.

Selection of T cells does not end with events in the thymus, but continues in extrathymic tissues and for the life of the organism. In this review, we examine how self-reactive T cells are rendered harmless and the processes that select for T cells that are most efficient at combating pathogens. The implications of peripheral T-cell selection for the immune response as animals age are discussed as is the critical role of dendritic cells in directing T-cell differentiation.

Cutting edge: pulmonary Legionella pneumophila is controlled by plasmacytoid dendritic cells but not type I IFN.

Plasmacytoid dendritic cells (pDCs) are well known as the major cell type that secretes type I IFN in response to viral infections. Their role in combating other classes of infectious organisms, including bacteria, and their mechanisms of action are poorly understood. We have found that pDCs play a significant role in the acute response to the intracellular bacterial pathogen Legionella pneumophila. pDCs were rapidly recruited to the lungs of L. pneumophila-infected mice, and depletion of pDCs resulted in increased bacterial load. The ability of pDCs to combat infection did not require type I IFN. This study points to an unappreciated role for pDCs in combating bacterial infections and indicates a novel mechanism of action for this cell type.

Antimyeloperoxidase antibodies rapidly induce alpha-4-integrin-dependent glomerular neutrophil adhesion.

Patients with antineutrophil cytoplasmic antibodies (ANCAs) frequently develop severe vasculitis and glomerulonephritis. Although ANCAs, particularly antimyeloperoxidase (anti-MPO), have been shown to promote leukocyte adhesion in postcapillary venules, their ability to promote adhesion in the glomerular vasculature is less clear. We used intravital microscopy to examine glomerular leukocyte adhesion induced by anti-MPO. In mice pretreated with LPS, 50 microg anti-MPO induced LFA-1-dependent adhesion in glomeruli. In concert with this finding, in mice pretreated with LPS, more than 80% of circulating neutrophils bound anti-MPO within 5 minutes of intravenous administration. However, even in the absence of LPS, more than 40% of circulating neutrophils bound anti-MPO in vivo, a response not seen in MPO(-/-) mice. In addition, a higher dose of anti-MPO (200 microg) induced robust glomerular leukocyte adhesion in the absence of LPS. The latter response was beta2-integrin independent, instead requiring the alpha4-integrin, which was up-regulated on neutrophils in response to anti-MPO. These data indicate that anti-MPO antibodies bind to circulating neutrophils, and can induce glomerular leukocyte adhesion via multiple pathways. Lower doses induce adhesion only after an infection-related stimulus, whereas higher doses are capable of inducing responses in the absence of an additional inflammatory stimulus, via alternative adhesion mechanisms.

Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappaB and NFAT.

Although the transcription factor Foxp3 is implicated in regulating glucocorticoid-induced TNF receptor (GITR) expression in the T regulatory cell lineage, little is known about how GITR is transcriptionally regulated in conventional T cells. In this study, we provide evidence that TCR-mediated GITR expression depends on the ligand affinity and the maturity of conventional T cells. A genetic dissection of GITR transcriptional control revealed that of the three transcription factors downstream of the classical NF-kappaB pathway (RelA, cRel, and NF-kappaB1), RelA is a critical positive regulator of GITR expression, although cRel and NF-kappaB1 also play a positive regulatory role. Consistent with this finding, inhibiting NF-kappaB using Bay11-7082 reduces GITR up-regulation. In contrast, NFAT acts as a negative regulator of GITR expression. This was evidenced by our findings that agents suppressing NFAT activity (e.g., cyclosporin A and FK506) enhanced TCR-mediated GITR expression, whereas agents enhancing NFAT activity (e.g., lithium chloride) suppressed TCR-mediated GITR up-regulation. Critically, the induction of GITR was found to confer protection to conventional T cells from TCR-mediated apoptosis. We propose therefore that two major transcriptional factors activated downstream of the TCR, namely, NF-kappaB and NFAT, act reciprocally to balance TCR-mediated GITR expression in conventional T cells, an outcome that appears to influence cell survival.

New insights into the dual recruitment of IgA+ B cells in the developing mammary gland.

In monogastric mammals, transfer of passive immunity via milk and colostrum plays an important role in protecting the neonate against mucosal infections. Here we analyzed the hypothesis that during gestation/lactation IgA+ plasmablasts leave the intestinal and respiratory surfaces towards the mammary gland (MG). We compared the recruitment of lymphocytes expressing homing receptors alpha4beta1 and alpha4beta7 to expression of their vascular counter-receptors, VCAM-1 and MAdCAM-1. Furthermore, the expression of the chemokines responsible for the recruitment of IgA+ plasmablasts was analyzed. Data confirmed that expressions of CCL28 and MAdCAM-1 in the MG increased during pregnancy and alpha4beta1+ and alpha4beta7+/IgA+ cell recruitment in lactation correlated with increase of CCL28 expression. Interestingly, VCAM-1 expression was found in small blood vessels of the lactating porcine MG, while in mice VCAM-1 was expressed in large blood vessels within the MG. Thus, our results indicate that the recruitment of IgA+ plasmablasts to MG is mediated by VCAM-1/alpha4beta1 and MAdCAM-1/alpha4beta7 in conjunction with CCL28/CCR10. They support the existence of a functional link between entero- and upper respiratory surfaces and MG, thereby, conferring protection against aero-digestive pathogens in the newborn.

Targeting the gut vascular endothelium induces gut effector CD8 T cell responses via cross-presentation by dendritic cells.

Systemic delivery of Ag usually induces poor mucosal immunity. To improve the CD8 T cell response at mucosal sites, we targeted the Ag to MAdCAM-1, a mucosal addressin cell adhesion molecule expressed mainly by high endothelial venules (HEV) in mesenteric lymph nodes (MLN) and Peyer's patches of gut-associated lymphoid tissue. When chemical conjugates of anti-MAdCAM-1 Ab and model Ag OVA were injected i.v., a greatly enhanced proliferative response of Ag-specific OT-I CD8 T cells was detected in MLN. This was preceded by prolonged accumulation, up to 2 wk, of the anti-MAdCAM OVA conjugate on HEV of Peyer's patches and MLN. In contrast, nontargeted OVA conjugate was very inefficient in inducing OT-I CD8 T cell proliferation in MLN and required at least 20-fold more Ag to induce a comparable response. In addition, MAdCAM targeting elicits an endogenous OVA-specific CD8 T cell response, evident by IFN-gamma production and target killing. Induced response offers protection against an OVA-expressing B cell lymphoma. We propose that the augmentation of gut CD8 T cell responses by MAdCAM targeting is due to both accumulation of Ag in the HEV and conversion of a soluble Ag to a cell-associated one, allowing cross-presentation by DCs.

Differential expression of adhesion molecules and chemokines between nasal and small intestinal mucosae: implications for T- and sIgA+ B-lymphocyte recruitment.

Nasal and small intestinal mucosae are the first sites of contact with infectious agents and the sites of T-cell-mediated and secreted immunoglobulin A (IgA)-mediated defences against pathogens. We investigated the factors controlling the infiltration of CD3(+) T lymphocytes and surface IgA(+) (sIgA(+)) B lymphocytes into swine epithelium and lamina propria (LP) within and between these two mucosal effector sites. Vascular addressins, vascular cell adhesion molecule 1 and mucosal addressin cell adhesion molecule-1 were reciprocally expressed in both mucosae. Strong expression of alpha(4)beta(1) relative to alpha(4)beta(7) was characteristic of CD3(+) T cells in nasal mucosa LP and epithelium and of sIgA(+) cells in nasal mucosa epithelium. The same profile was observed on corresponding blood cells. Conversely, higher levels of integrins beta(7) and alpha(4)beta(7) than alpha(4)beta(1) were characteristic of CD3(+) T cells and sIgA(+) cells in the small intestine. However, about 40% of the LP-activated sIgA(+) cells displayed sIgA(high), integrin alpha(4) and integrin alpha(4) expression. Whereas CCL19, CXCL12, CCL21 and CCL28 messenger RNAs were similarly expressed in both mucosae, CCL25 messenger RNA was only expressed in the small intestine. Thus, the nasal and small intestine mucosae represent separate compartments for infiltration by CD3(+) T cells and sIgA(+) effector cells, with the exception of a population of small intestine activated sIgA(+) cells, which may gain access to both mucosae.

The origin of thymic CD4+CD25+ regulatory T cells and their co-stimulatory requirements are determined after elimination of recirculating peripheral CD4+ cells.

Studies on the thymic ontogeny of naturally arising CD4(+)CD25(+) regulatory T cells (TR cells) are complicated by the contamination of recirculating cells from the periphery (both activated CD4(+) T and TR cells). We investigated TR cells in anti-CD4 antibody transgenic (Tg) (GK) mice that continuously deplete peripheral CD4 T cells but not thymocytes so that the generation of thymic TR cells and their developmental requirement can be accurately assessed. We show that in the thymuses of mice that lack peripheral CD4(+) cells, TR cells were present but were fewer in number compared with wild-type (WT) mice. Therefore, we show that peripheral TR cells do re-enter the thymus, comprising 20% of TR cells in the normal thymus. TR cells from both WT and GK mice expressed Foxp3 and GITR, and suppressed the proliferation of CD25(-)CD4(+) T cells. Furthermore, the co-stimulation requirements for TR generation were evaluated in mice with or without peripheral CD4 cells. Splenic TR cells in CD40L(-/-) mice and CTLA4Ig Tg mice were fewer compared with WT mice. Mice deficient in both co-stimulatory pathways had further reduction in splenic TR cells. Unlike the periphery, the reduction in thymic TR cells was only seen for CD40L(-/-) but not for CTLA4Ig Tg mice. Therefore, we found that the co-stimulation requirements for the thymic development of TR cells differed from those for peripheral homeostasis.

Milk IgA responses are augmented by antigen delivery to the mucosal addressin cellular adhesion molecule 1.

The mucosal addressin cellular adhesion molecule 1 (MAdCAM) is expressed on the venules of the gut associated lymphoid tissue (GALT); it is also expressed on the venules of the lobules of the mammary gland. We have previously found that MAdCAM-targeting using a rat anti-MAdCAM monoclonal Ab as both antigen and targeting moiety resulted in an enhanced local IgA gut response. We therefore surmised that such targeting may also enhance IgA responses in the mammary gland. We show that our model antigen localizes to the lobules of the mammary glands as well as the GALT, but not to the draining lymph nodes and that targeting MAdCAM results in secretory IgA responses in the milk. We provide evidence that this milk IgA Ab is of a secretory nature and is consistent with derivation from gut plasmablasts that have migrated to the mammary gland. Targeting MAdCAM may be a way for a novel vaccine strategy that affords protection to the mammary gland and the suckling neonate.

Role of Bordetella bronchiseptica adenylate cyclase in nasal colonization and in development of local and systemic immune responses in piglets.

Two Bordetella bronchiseptica mutants, lacking the adenylate cyclase (Cya) or both Cya and pertactin (Prn), were compared with their parental strain NL1013 in their abilities to colonize the nose of neonate piglets and to induce local and systemic antibody responses against filamentous hemagglutinin (FHA) after intranasal (i.n.) inoculation. The number of bacteria recovered and the duration of infection in the nasal secretions were greater for the wild-type parent strain than for the Cya-deficient mutant, indicating that Cya plays an important role during B. bronchiseptica colonization of the nasal cavity. The double mutant did not colonize the nasal cavity and was less able to adhere to epithelial cells in vitro than the other two strains, supporting the hypothesis that Prn plays a major role in cell adhesion. In piglets inoculated with the wild type strain, anti-FHA IgM was found in the nasal secretions one week after inoculation, followed two weeks later by anti-FHA IgA; their presence was concomitant with decreases in bacterial counts. Anti-FHA IgG appeared at six weeks after infection in the serum. In contrast, i.n. inoculation with either mutant failed to induce a nasal secretory antibody response but did induce an earlier and higher IgM response in the serum than inoculation with the wild type strain. However, only the Cya-deficient mutant was able to prime the piglets for the development of a secondary nasal IgM and serum IgG response to FHA after intranasal inoculation with the wild type B. bronchiseptica.