Bjarne Bogen

Endocytic pathways regulate Toll‐like receptor 4 signaling and link innate and adaptive immunity

Immune responses are initiated when molecules of microbial origin are sensed by the Toll‐like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor‐mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin‐binding endosomal sorting protein hepatocyte growth factor‐regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS‐associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.

MHC class II—Restricted presentation of intracellular antigen

An endogenously produced immunoglobulin light chain (lambda 2(315] is processed and presented to T cells in association with major histocompatibility complex (MHC) class II molecules. Using transfectants producing variant forms of lambda 2(315) that are neither expressed on the cell surface nor secreted, we demonstrate that intracellular lambda 2(315), which has never been exported outside of the cell, is the source of processed lambda 2(315) idiotype. This challenges the currently accepted paradigm that endogenous antigens are only presented by MHC class I molecules. Variants of lambda 2(315) protein that are retained in the endoplasmic recticulum (ER) are also presented. Variants that are expressed in the cytosol as well as those that are transported into the nucleus rather than the ER are not presented. Thus, the ER is likely to be the processing compartment.

Inflammation driven by tumour-specific Th1 cells protects against B-cell cancer

The immune system can both promote and suppress cancer. Chronic inflammation and proinflammatory cytokines such as interleukin (IL)-1 and IL-6 are considered to be tumour promoting. In contrast, the exact nature of protective antitumour immunity remains obscure. Here, we quantify locally secreted cytokines during primary immune responses against myeloma and B-cell lymphoma in mice. Strikingly, successful cancer immunosurveillance mediated by tumour-specific CD4+ T cells is consistently associated with elevated local levels of both proinflammatory (IL-1α, IL-1β and IL-6) and T helper 1 (Th1)-associated cytokines (interferon-γ (IFN-γ), IL-2 and IL-12). Cancer eradication is achieved by a collaboration between tumour-specific Th1 cells and tumour-infiltrating, antigen-presenting macrophages. Th1 cells induce secretion of IL-1β and IL-6 by macrophages. Th1-derived IFN-γ is shown to render macrophages directly cytotoxic to cancer cells, and to induce macrophages to secrete the angiostatic chemokines CXCL9/MIG and CXCL10/IP-10. Thus, inflammation, when driven by tumour-specific Th1 cells, may prevent rather than promote cancer.

Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species.

Summary Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.

Cutting Edge: Expression of XCR1 Defines Mouse Lymphoid-Tissue Resident and Migratory Dendritic Cells of the CD8α+ Type

Subsets of dendritic cells (DCs) have been described according to their functions and anatomical locations. Conventional DC subsets are defined by reciprocal expression of CD11b and CD8α in lymphoid tissues (LT), and of CD11b and CD103 in non-LT (NLT). Spleen CD8α+ and dermal CD103+ DCs share a high efficiency for Ag cross-presentation and a developmental dependency on specific transcription factors. However, it is not known whether all NLT-derived CD103+ DCs and LT-resident CD8α+ DCs are similar despite their different anatomical locations. XCR1 was previously described as exclusively expressed on mouse spleen CD8α+ DCs and human blood BDCA3+ DCs. In this article, we showed that LT-resident CD8α+ DCs and NLT-derived CD103+ DCs specifically express XCR1 and are characterized by a unique transcriptional fingerprint, irrespective of their tissue of origin. Therefore, CD8α+ DCs and CD103+ DCs belong to a common DC subset which is unequivocally identified by XCR1 expression throughout the body.

Clonal deletion of specific thymocytes by an immunoglobulin idiotype.

We have investigated whether immunoglobulin can induce clonal deletion of thymocytes by employing two strains of transgenic mice. One strain is transgenic for an alpha/beta T cell receptor (TCR) which recognizes a processed idiotypic peptide of the lambda 2(315) light chain variable region, bound to the I‐Ed class II major histocompatibility complex molecule. The other mouse strain is transgenic for the lambda 2(315) gene. Double transgenic offspring from a TCR‐transgenic female mated with a lambda 2(315) transgenic male exhibit a pronounced clonal deletion of CD4+CD8+ thymocytes. Analysis of neonates from the reciprocal (lambda 2(315)‐transgenic female × TCR‐transgenic male) cross suggests that the deletion in double transgenic offspring most likely is caused by lambda 2(315) produced within the thymus rather than by maternally derived IgG, lambda 2(315). Nevertheless, IgG, lambda 2(315) can cause deletion of CD4+CD8+ thymocytes when injected in large amounts intraperitoneally into either adult or neonatal TCR‐transgenic mice. Deletion is evident 48 and 72 h after injection, but by day 7 the thymus has already regained its normal appearance. A serum concentration of several hundred microgram/ml is required for deletion to be observed. Therefore, the heterogeneous idiotypes of serum Ig are probably each of too low concentration to cause thymocyte deletion in normal animals.

Cutting Edge: Link Between Innate and Adaptive Immunity: Toll-Like Receptor 2 Internalizes Antigen for Presentation to CD4+ T Cells and Could Be an Efficient Vaccine Target

An ideal vaccine for induction of CD4+ T cell responses should induce local inflammation, maturation of APC, and peptide loading of MHC class II molecules. Ligation of Toll-like receptor (TLR) 2 provides the first two of these three criteria. We have studied whether targeting of TLR2 results in loading of MHC class II molecules and enhancement of CD4+ T cell responses. To dissociate MHC class II presentation from APC maturation, we have used an antagonistic, mouse anti-human TLR2 mAb (TL2.1) as ligand and measured proliferation of a mouse Cκ-specific human CD4+ T cell clone. TL2.1 mAb was 100-1000 times more efficiently presented by APC compared with isotype-matched control mAb. Moreover, TL2.1 mAb was internalized into endosomes and processed by the conventional MHC class II pathway. This novel function of TLR2 represents a link between innate and adaptive immunity and indicates that TLR2 could be a promising target for vaccines.

Minimum length of an idiotypic peptide and a model for its binding to a major histocompatibility complex class II molecule.

We have defined the minimum length of a synthetic peptide which can activate I‐Ed‐restricted BALB/c T cell clones specific for a mutated self‐antigen: an idiotope on the syngeneic lambda 2315 immunoglobulin light chain. A peptide comprising residues 91‐101 of the lambda 2315 sequence had full stimulatory potency. Surprisingly, a peptide analogue in which His97 was deleted was almost fully active. Truncated, deleted or substituted peptide analogues did not distinguish between seven T cell clones that use different alpha/beta T cell receptors. The 91‐101 region in the lambda 2315 light chain does not form an amphipathic helix even though such a helix has been suggested to be important for T cell epitopes. Further, a motif proposed by Rothbard and Taylor as being common to T cell immunogenic peptides is not necessary for the lambda 2315 idiotypic peptide. Comparison with seven other I‐Ed‐restricted peptides revealed that the peptides are generally positively charged and have two basic amino acids clustered around the centre. On the basis of a model of the class II molecule peptide binding site, we suggest that these positively charged residues may interact with the negatively charged residues at positions 114(Glu) and 155(Asp) of the E beta d chain.

Processing and presentation of idiotypes to MHC-restricted T cells.

Among the self antigens, immunoglobulins, and in particular idiotypes, are of special interest because of their extreme sequence heterogeneity and their postulated involvement in regulatory interactions in the immune system. We have therefore studied antigen processing and presentation of variable region peptides, processed idiotypes, to MHC class II molecule-restricted T cells. The immunoglobulin used has been the lambda 2(315) light chain produced by the BALB/c MOPC 315 plasmacytoma (alpha, lambda 2). The minimum length of a stimulatory synthetic idiotypic peptide comprises residues 91-101 of lambda 2(315) and is presented by the I-E(d) molecule to CD4+ T cells. T cell clones with specificity for the 91-101(lambda 2(315))/I-E(d) complex utilize a limited TCR repertoire and are of both Th1 and Th2 type. For presentation, extracellular lambda 2(315) requires endocytosis and processing, as previously described for conventional exogenous antigens. In addition, a B lymphoma cell can process and present its own endogenous lambda 2(315). This was shown by transfecting manipulated lambda 2(315) gene variants into B lymphoma cells, followed by evaluation of the APC function of the transfectants. These studies demonstrated that surface expression or secretion of lambda 2(315) is not necessary for presentation and suggested that the endoplasmic reticulum may be a processing compartment. To extend our findings to naive Id+ B cells and anti-Id T cells, we have generated lambda 2(315)-transgenic as well as TCR-transgenic mice. A model is presented for a T-B cell interaction based on presentation of processed idiotypes.

How Do CD4+ T Cells Detect and Eliminate Tumor Cells That Either Lack or Express MHC Class II Molecules?

CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR) transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor-specific antigen by host antigen-presenting cells (APCs) appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315), where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR-transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-γ stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed.