The polymorphic HCMV glycoprotein UL20 is targeted for lysosomal degradation by multiple cytoplasmic dileucine motifs.

Human cytomegalovirus (HCMV) is a widespread and persistent beta-herpesvirus. The large DNA genome of HCMV encodes many proteins that are non-essential for viral replication including numerous proteins subverting host immunosurveillance. One of them is the barely characterized UL20, which is encoded adjacent to the well-defined immunoevasins UL16 and UL18. UL20 is a type I transmembrane glycoprotein with an immunoglobulin-like ectodomain that is highly polymorphic among HCMV strains. Here, we show that the homodimeric UL20, by virtue of its cytoplasmic domain, does not reach the cell surface but is targeted to endosomes and lysosomes. Accordingly, UL20 exhibits a short half-life because of rapid lysosomal degradation. Trafficking of UL20 to lysosomes is determined by several, independently functioning dileucine-based sorting motifs in the cytoplasmic domain of UL20 and involves the adaptor protein (AP) complex AP-1. Combined substitution of three dileucine motifs allowed strong cell surface expression of UL20 comparable to UL20 mutants lacking the cytoplasmic tail. Finally, we show that the intracellularly located UL20 also is subject to lysosomal degradation in the context of viral infection. Altogether, from these data, we hypothesize that UL20 is destined to efficiently sequester yet-to-be defined cellular proteins for degradation in lysosomes.

Immunology and immunotherapeutic approaches in multiple myeloma.

Immunotherapy for patients suffering from multiple myeloma is a lively and emerging field in cancer research. Immunotherapeutic approaches offer unique treatment opportunities for this, to date, mostly incurable disease. Respective basic findings and recent clinical approaches are introduced and discussed. Although several obstacles still need to be overcome, it appears that clinically efficient immunotherapies will become available for multiple myeloma patients in the future.

Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses.

Mutated proteins arising from somatic mutations in tumors are promising targets for cancer immunotherapy. They represent true tumor-specific antigens (TSAs) as they are exclusively expressed in tumors, reduce the risk of autoimmunity and are more likely to overcome tolerance compared to wild-type (wt) sequences. Hence, we designed a panel of long peptides (LPs, 28-35 aa) comprising driver gene mutations in TP35 and KRAS frequently found in gastrointestinal tumors to test their combined immunotherapeutic potential. We found increased numbers of T cells responsive against respective mutated and wt peptides in colorectal cancer patients that carry the tested mutations in their tumors than patients with other mutations. Further, active immunization of HLA(-A2/DR1)-humanized mice with mixes of the same mutated LPs yielded simultaneous, polyvalent CD8+/CD4+ T cell responses against the majority of peptides. Peptide-specific T cells possessed a multifunctional cytokine profile with CD4+ T cells showing a TH1-like phenotype. Two mutated peptides (Kras[G12V], p53[R248W]) induced significantly higher T cell responses than corresponding wt sequences and comprised HLA-A2/DR1-restricted mutated epitopes. However, vaccination with the same highly immunogenic LPs strongly increased systemic regulatory T cells (Treg) numbers in a syngeneic sarcoma model over-expressing these mutated protein variants and resulted in accelerated tumor outgrowth. In contrast, tumor outgrowth was delayed when vaccination was directed against tumor-intrinsic Kras/Tp53 mutations of lower immunogenicity. Conclusively, we show that LP vaccination targeting multiple mutated TSAs elicits polyvalent, multifunctional, and mutation-specific effector T cells capable of targeting tumors. However, the success of this therapeutic approach can be hampered by vaccination-induced, TSA-specific Tregs.

Strong T­cell costimulation can reactivate tumor antigen­specific T cells in late­stage metastasized colorectal carcinoma patients: results from a phase â clinical study.

T­cell costimulation is necessary to induce a response of naïve T cells. Whether T­cell costimulation can also cause reactivation of unreactive, possibly anergized memory T cells (MTCs) from late­stage cancer patients is unknown. To investigate this question, we developed a bispecific anti­CD28 fusion protein (bsHN­CD28) which can easily be attached to the vaccine ATV­NDV. This virus­modified autologous tumor cell vaccine has already shown effectivity in colon cancer patients following resection of liver metastases. In this phase â…  clinical study, 14 colorectal carcinoma (CRC) patients with late­stage disease which could not be operated anymore with curative intent were treated with the vaccine ATV­NDV to which bsHN­CD28 was attached. No severe adverse events were recorded. All patients showed an immunological response of tumor­reactive T cells, at least once during the course of five vaccinations. Also, we demonstrate a dose­response relationship with the costimulatory molecule added to the vaccine. A partial response of metastases was documented in four patients. The study suggests that the three­component vaccine is safe and can reactivate possibly anergized T cells from a chronic disease like advanced­stage cancer.

Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma.

Spontaneous antitumor T cell responses in cancer patients are strongly controlled by Tregs, and increased numbers of tumor-infiltrating Tregs correlate with reduced survival. However, the tumor antigens recognized by Tregs in cancer patients and the impact of these cells on tumor-specific T cell responses have not been systematically characterized. Here we used a broad panel of long synthetic peptides of defined tumor antigens and normal tissue antigens to exploit a newly developed method to identify and compare ex vivo the antigen specificities of Tregs with those of effector/memory T cells in peripheral blood of colorectal cancer patients and healthy subjects. Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens, suggesting that Tregs exert T cell suppression in an antigen-selective manner. Tumor-specific effector T cells were detectable in the majority of colorectal cancer patients but not in healthy individuals. We detected differences in the repertoires of antigens recognized by Tregs and effector/memory T cells in the majority of colorectal cancer patients. In addition, only effector/memory T cell responses against antigens recognized by Tregs strongly increased after Treg depletion. The selection of antigens according to preexisting T cell responses may improve the efficacy of future immunotherapies for cancer and autoimmune disease.