Günter J. Hämmerling

Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction

By generating two types of transgenic mice we have investigated how extrathymic events can contribute to self tolerance. The major histocompatibility complex class I gene Kb was expressed under the control of the glial fibrillary acidic protein promoter in cells of neuroectodermal origin outside the thymus. These mice were tolerant to Kb. When crossed to transgenic mice expressing a Kb-specific T cell receptor (TCR), clonotype+, CD8+CD4- mature T cells could be detected in normal numbers in the thymus of the double-transgenic mice but were strongly reduced in spleen and lymph nodes in comparison with TCR single-transgenic mice. After isolation of clonotype negative splenic T cells and activation in vitro, reappearance of the clonotype+, CD8+CD4- cells was observed. These results indicate that down-regulation of TCR and CD8 molecules on the antigen-specific T cells is a novel mechanism, by which peripheral tolerance to this antigen can occur.

T cell awareness of paternal alloantigens during pregnancy.

During pregnancy a semiallogeneic fetus survives despite the presence of maternal T cells specific for paternally inherited histocompatibility antigens. A mouse transgenic for a T cell receptor recognizing the major histocompatibility (MHC) antigen H-2Kb was used to follow the fate of T cells reactive to paternal alloantigens. In contrast to syngeneic and third-party allogeneic pregnancies, mice bearing a Kb-positive conceptus had reduced numbers of Kb-reactive T cells and accepted Kb-positive tumor grafts. T cell phenotype and responsiveness were restored after delivery. Thus, during pregnancy maternal T cells acquire a transient state of tolerance specific for paternal alloantigens.

Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response

While the initiation of the adaptive and innate immune response is well understood, less is known about cellular mechanisms propagating inflammation. The receptor for advanced glycation end products (RAGE), a transmembrane receptor of the immunoglobulin superfamily, leads to perpetuated cell activation. Using novel animal models with defective or tissue-specific RAGE expression, we show that in these animal models RAGE does not play a role in the adaptive immune response. However, deletion of RAGE provides protection from the lethal effects of septic shock caused by cecal ligation and puncture. Such protection is reversed by reconstitution of RAGE in endothelial and hematopoietic cells. These results indicate that the innate immune response is controlled by pattern-recognition receptors not only at the initiating steps but also at the phase of perpetuation.

Vascular normalization in Rgs5-deficient tumours promotes immune destruction

The vasculature of solid tumours is morphologically aberrant and characterized by dilated and fragile vessels, intensive vessel sprouting and loss of hierarchical architecture. Constant vessel remodelling leads to spontaneous haemorrhages and increased interstitial fluid pressure in the tumour environment. Tumour-related angiogenesis supports tumour growth and is also a major obstacle for successful immune therapy as it prevents migration of immune effector cells into established tumour parenchyma. The molecular mechanisms for these angiogenic alterations are largely unknown. Here we identify regulator of G-protein signalling 5 (Rgs5) as a master gene responsible for the abnormal tumour vascular morphology in mice. Loss of Rgs5 results in pericyte maturation, vascular normalization and consequent marked reductions in tumour hypoxia and vessel leakiness. These vascular and intratumoral changes enhance influx of immune effector cells into tumour parenchyma and markedly prolong survival of tumour-bearing mice. This is the first demonstration, to our knowledge, of reduced tumour angiogenesis and improved immune therapeutic outcome on loss of a vascular gene function and establishes a previously unrecognized role of G-protein signalling in tumour angiogenesis.

Low-Dose Irradiation Programs Macrophage Differentiation to an iNOS+/M1 Phenotype that Orchestrates Effective T Cell Immunotherapy

Inefficient T cell migration is a major limitation of cancer immunotherapy. Targeted activation of the tumor microenvironment may overcome this barrier. We demonstrate that neoadjuvant local low-dose gamma irradiation (LDI) causes normalization of aberrant vasculature and efficient recruitment of tumor-specific T cells in human pancreatic carcinomas and T-cell-mediated tumor rejection and prolonged survival in otherwise immune refractory spontaneous and xenotransplant mouse tumor models. LDI (local or pre-adoptive-transfer) programs the differentiation of iNOS⁺ M1 macrophages that orchestrate CTL recruitment into and killing within solid tumors through iNOS by inducing endothelial activation and the expression of TH1 chemokines and by suppressing the production of angiogenic, immunosuppressive, and tumor growth factors.

A Viral ER-Resident Glycoprotein Inactivates the MHC-Encoded Peptide Transporter

Human cytomegalovirus inhibits peptide import into the endoplasmic reticulum (ER) by the MHC-encoded TAP peptide transporter. We identified the open reading frame US6 to mediate this effect. Expression of the 21 kDa US6 glycoprotein in human cytomegalovirus-infected cells correlates with the inhibition of peptide transport during infection. The subcellular localization of US6 is ER restricted and is identical with TAP. US6 protein is found in complexes with TAP1/2, MHC class I heavy chain, beta2-microglobulin, calnexin, calreticulin, and tapasin. TAP inhibition, however, is independent of the presence of class I heavy chain and tapasin. The results establish a new mechanism for viral immune escape and a novel role for ER-resident proteins to regulate TAP via its luminal face.

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell–T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell–mediated immunity.

Resistance of young gelatinase B-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions.

Regulated expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) plays a role in various physiological processes. To determine in vivo how unbalanced expression of these factors can promote or affect the course of pathologies, we knocked out the mouse gelatinase B gene by replacing the catalytic and zinc-binding domains with an antisense-oriented neomycin resistance gene. Adult gelatinase B-deficient mice and wild-type controls could be induced to develop experimental autoimmune encephalomyelitis (EAE) with similar scores for neurologic disease, blood-brain barrier permeability, and central nervous system histopathology. However, whereas diseased control animals showed necrotizing tail lesions with hyperplasia of osteocartilaginous tissue, adult gelatinase B-deficient mice were resistant to this tail pathology. Gelatinase B-deficient mice younger than 4 weeks of age were significantly less susceptible to the development of EAE than were age matched controls and, even as they aged, they remained resistant to tail lesions. These data illustrate that gelatinase B expression plays a role in the development of the immune system and that, in ontogenesis, the propensity to develop autoimmunity is altered by the absence of this MMP.

Editing of the HLA-DR-peptide repertoire by HLA-DM

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non‐classical MHC class II molecule HLA‐DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self‐peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA‐DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self‐peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high‐performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR‐associated self‐peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high‐stability MHC class II‐peptide complexes by kinetic proofreading before these complexes are presented to T cells.

Combination of T-Cell Therapy and Trigger of Inflammation Induces Remodeling of the Vasculature and Tumor Eradication

In a transgenic mouse model of multistep carcinogenesis, highly angiogenic insulinomas contain an irregular vascular network and develop an intrinsic resistance to leukocyte infiltration and effector function. Even persistently high levels of activated tumor-specific T lymphocytes fail to eradicate the tumors. In contrast, we show that irradiation before adoptive transfer results in complete macroscopic tumor regression. Thus, effective tumor therapy requires a proinflammatory microenvironment that permits T cells to extravasate and to destroy the tumor. Early after initiation of the irradiation/adoptive transfer therapy, the capillary network reacquires an almost normal appearance, a likely consequence of strong induction of the chemokines monokine induced by IFN-gamma (Mig) and IFN-inducible protein 10 (IP10). This remodeling of the vasculature in a proinflammatory environment may directly affect lymphocyte extravasation and effector function. Therefore, irradiation/adoptive transfer therapy combines antigen-driven tumor cell eradication with anti-angiogenic effects on tumor endothelium, a powerful synergy that has not been previously appreciated.