Hans W. Mages

Homozygous loss of ICOS is associated with adult-onset common variable immunodeficiency

No genetic defect is known to cause common variable immunodeficiency (CVID), a heterogeneous human disorder leading to adult-onset panhypogammaglobulinemia. In a search for CVID candidate proteins, we found four of 32 patients to lack ICOS, the “inducible costimulator” on activated T cells, due to an inherited homozygous deletion in the ICOS gene. T cells from these individuals were normal with regard to subset distribution, activation, cytokine production and proliferation. In contrast, naive, switched and memory B cells were reduced. The phenotype of human ICOS deficiency, which differs in key aspects from that of the ICOS−/− mouse, suggests a critical involvement of ICOS in T cell help for late B cell differentiation, class-switching and memory B cell generation.

Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells

In recent years, human dendritic cells (DCs) could be subdivided into CD304+ plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter encompassing the CD1c+, CD16+, and CD141+ DC subsets. To date, the low frequency of these DCs in human blood has essentially prevented functional studies defining their specific contribution to antigen presentation. We have established a protocol for an effective isolation of pDC and cDC subsets to high purity. Using this approach, we show that CD141+ DCs are the only cells in human blood that express the chemokine receptor XCR1 and respond to the specific ligand XCL1 by Ca2+ mobilization and potent chemotaxis. More importantly, we demonstrate that CD141+ DCs excel in cross-presentation of soluble or cell-associated antigen to CD8+ T cells when directly compared with CD1c+ DCs, CD16+ DCs, and pDCs from the same donors. Both in their functional XCR1 expression and their effective processing and presentation of exogenous antigen in the context of major histocompatibility complex class I, human CD141+ DCs correspond to mouse CD8+ DCs, a subset known for superior antigen cross-presentation in vivo. These data define CD141+ DCs as professional antigen cross-presenting DCs in the human.

Expression of ICOS In Vivo Defines CD4+ Effector T Cells with High Inflammatory Potential and a Strong Bias for Secretion of Interleukin 10

The studies performed to date analyzed the overall participation of the inducible costimulator (ICOS) in model diseases, but did not yield information on the nature and function of ICOS-expressing T cells in vivo. We examined ICOS+ T cells in the secondary lymphoid organs of nonmanipulated mice, in the context of an “unbiased” immune system shaped by environmental antigens. Using single cell analysis, ICOSlow cells were found to be loosely associated with the early cytokines interleukin (IL)-2, IL-3, IL-6, and interferon (IFN)-γ. ICOSmedium cells, the large majority of ICOS+ T cells in vivo, were very tightly associated with the synthesis of the T helper type 2 (Th2) cytokines IL-4, IL-5, and IL-13, and these cells exhibited potent inflammatory effects in vivo. In contrast, ICOShigh T cells were highly and selectively linked to the anti-inflammatory cytokine IL-10. Overall, these data seem to indicate that ICOS cell surface density serves as a regulatory mechanism for the release of cytokines with different immunological properties. Further in vivo functional experiments with in vitro–activated T cells strongly suggested that the ICOS+ population, although representing in vivo only around 10% of T cells bearing early or late activation markers, nevertheless encompasses virtually all effector T cells, a finding with major diagnostic and therapeutic implications.

Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand.

Human ICOS (huICOS) is a T cell‐specific molecule structurally related to CD28 and CTLA‐4 with potent co‐stimulatory activities on T cell proliferation, cytokine induction and T cell help for B cells. We have now cloned and characterized murine ICOS (muICOS). muICOS mRNA of 1.5 kb and 3.3 kb encodes a protein with a deduced molecular mass of 20.3 kDa, which is 71.7 % identical to huICOS. On the cell surface, muICOS is expressed as a disulfide‐linked, glycosylated homodimer of 47‐57 kDa, with subunits of approximately 26 kDa. With a panel of monoclonal antibodies we have determined the expression of muICOS in vitro and in vivo. Following activation of splenic T cells via CD3, muICOS became detectable at 12 h and reached a maximum of expression at around 48 h, thus exhibiting expression kinetics similar to huICOS. In vivo, muICOS was found to be substantially expressed in the thymic medulla and in the germinal centers and T cell zones of lymph nodes and Peyers patches. Non‐lymphoid tissue was ICOS negative. The muICOS gene was mapped to a region of chromosome 1 also harboring the CD28 and CTLA‐4 genes. Using recombinant chimeric muICOS‐Ig we determined that B7h, a recently cloned B7‐like molecule, is a ligand for muICOS.

Selective Expression of the Chemokine Receptor XCR1 on Cross-presenting Dendritic Cells Determines Cooperation with CD8+ T Cells

The expression of the chemokine receptor XCR1 and the function of its ligand XCL1 (otherwise referred to as ATAC, lymphotactin, or SCM-1) remained elusive to date. In the present report we demonstrated that XCR1 is exclusively expressed on murine CD8(+) dendritic cells (DCs) and showed that XCL1 is a potent and highly specific chemoattractant for this DC subset. CD8(+) T cells abundantly secreted XCL1 8-36 hr after antigen recognition on CD8(+) DCs in vivo, in a period in which stable T cell-DC interactions are known to occur. Functionally, XCL1 increased the pool of antigen-specific CD8(+) T cells and their capacity to secrete IFN-gamma. Absence of XCL1 impaired the development of cytotoxicity to antigens cross-presented by CD8(+) DCs. The XCL1-XCR1 axis thus emerges as an integral component in the development of efficient cytotoxic immunity in vivo.

Induction, binding specificity and function of human ICOS

Recently, we have identified the inducible co‐stimulator (ICOS), an activation‐dependent, T cell‐specific cell surface molecule related to CD28 and CTLA‐4. Detailed analysis of human ICOS presented here shows that it is a 55‐60‐kDa homodimer with differently N‐glycosylated subunits of 27 and 29 kDa. ICOS requires both phorbol 12‐myristate 13‐acetate and ionomycin for full induction, and is sensitive to Cyclosporin A. ICOS is up‐regulated early on all T cells, including the CD28– subset, and continues to be expressed into later phases of T cell activation. On stimulation of T cells by antigen‐presenting cells, the CD28/B7, but not the CD40 ligand/CD40 pathway is critically involved in the induction of ICOS. ICOS does not bind to B7‐1 or B7‐2, and CD28 does not bind to ICOS ligand; thus the CD28 and ICOS pathways do not cross‐interact on the cell surface. In vivo, ICOS is expressed in the medulla of the fetal and newborn thymus, in the T cell zones of tonsils and lymph nodes, and in the apical light zones of germinal centers (predominant expression). Functionally, ICOS co‐induces a variety of cytokines including IL‐4, IL‐5, IL‐6, IFN‐γ, TNF‐α, GM‐CSF, but not IL‐2, and superinduces IL‐10. Furthermore, ICOS co‐stimulation prevents the apoptosis of pre‐activated T cells. The human ICOS gene maps to chromosome 2q33 – 34.

ICOS-ligand, expressed on human endothelial cells, costimulates Th1 and Th2 cytokine secretion by memory CD4+ T cells

Endothelial cells (EC) play a central role in inflammatory immune responses and efficiently induce effector functions in T cells, despite lacking the classical costimulatory ligands CD80 and CD86. By using the mAb HIL-131 we now demonstrate that human inducible costimulator-ligand (ICOS-L), a molecule related to CD80/CD86, is constitutively expressed on human EC in vivo. In vitro, ICOS-L expression was strongly enhanced on human umbilical vein EC and microvascular EC by the inflammatory cytokines tumor necrosis factor α and IL-1β, and to a lower extent by stimulation of EC by CD40 or lipopolysaccharide. Coculture of MHC class II+ EC with resting memory CD4+ T cells in the presence of superantigen led to a marked up-regulation of ICOS on T cells and to the production of Th1 (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10, IL-13). When these cocultures were performed in the presence of the inhibitory mAb HIL-131, secretion of all cytokines was reduced by about 50–80%, indicating that ICOS-L is a major costimulator in EC-mediated T cell activation. Taken together, our data suggest an important physiological role of ICOS-L in the reactivation of effector/memory T cells on the endothelium controlling the entry of immune cells into inflamed tissue.

Sequential involvement of NFAT and Egr transcription factors in FasL regulation.

The critical function of NFAT proteins in maintaining lymphoid homeostasis was revealed in mice lacking both NFATp and NFAT4 (DKO). DKO mice exhibit increased lymphoproliferation, decreased activation-induced cell death, and impaired induction of FasL. The transcription factors Egr2 and Egr3 are potent activators of FasL expression. Here we find that Egr2 and Egr3 are NFAT target genes. Activation of FasL occurs via the NFAT-dependent induction of Egr3, as demonstrated by the ability of exogenously provided NFATp to restore Egr-dependent FasL promoter activity in DKO lymph node cells. Further, Egr3 expression is enriched in Th1 cells, suggesting a molecular basis for the known preferential expression of FasL in the Th1 versus Th2 subset.

ICOS Controls the Pool Size of Effector-Memory and Regulatory T Cells

ICOS is an important regulator of T cell effector function. ICOS-deficient patients as well as knockout mice show severe defects in T cell-dependent B cell responses. Several in vitro and in vivo studies attributed this phenomenon to impaired up-regulation of cell surface communication molecules and cytokine synthesis by ICOS-deficient T cells. However, we now could show with Ag-specific T cells in a murine adoptive transfer system that signaling via ICOS does not significantly affect early T cell activation. Instead, ICOS substantially contributes to the survival and expansion of effector T cells upon local challenge with Ag and adjuvant. Importantly, the observed biological function of ICOS also extends to FoxP3+ regulatory T cells, as can be observed after systemic Ag delivery without adjuvant. In line with these findings, absence of ICOS under homeostatic conditions of nonimmunized mice leads to a reduced number of both effector-memory and FoxP3+ regulatory T cells. Based on these results, we propose a biological role for ICOS as a costimulatory, agonistic molecule for a variety of effector T cells with differing and partly opposing functional roles. This concept may reconcile a number of past in vivo studies with seemingly contradictory results on ICOS function.

Defective Expression of CD40 Ligand on T Cells Causes “X‐Linked Immunodeficiency with Hyper‐IgM (HIGM1)”

X-linked immunodeficiency with hyper-IgM (HIGM1) is a rare disorder, characterized by recurrent infections associated with very low or absent IgG and IgA, and normal to increased IgM serum levels. The disease has been earlier mapped to the q26-27 region of the X-chromosome. We have identified a novel molecule expressed on the surface of activated T cells, which was designated TRAP (Tumor necrosis factor Related Activation Protein), and could demonstrate that TRAP is a ligand for the CD40 receptor expressed on B cells. Our mapping of the TRAP gene to the Xq26.3-27.1 region suggested a causal relationship to HIGM1. Further work revealed that various mutations of the TRAP/CD40 ligand (CD40L) gene may lead to a defective expression of the TRAP/CD40L molecule on the T-cell surface in HIGM1 patients. A combination of structural and functional analyses finally demonstrated that the failure of TRAP/CD40L on T cells to interact with CD40 on B cells is responsible for the inefficient T-cell help for B cells observed in HIGM1. The observations made in HIGM1 allowed us to conclude that TRAP/CD40L is not required for IgM synthesis. In contrast, functional expression of TRAP is a prerequisite for effective immunoglobulin isotype switching and subsequent production of IgG, IgA and IgE by B cells in vivo. The interaction of TRAP/CD40L with CD40 thus provides a very critical link between the cellular and the humoral part of the immune system. The knowledge of TRAP/CD40L cDNA sequence, the availability of various reagents for the testing of expression and function of TRAP/CD40L, and our recent elucidation of the exon-intron structure of the TRAP/CD40L gene now provide all necessary tools for early diagnosis of affected patients and the detection of female carriers of HIGM1. The available information will also provide a basis for future attempts at gene therapy in this disease.