Dorothee Wernet

HLA Antibody Specification Using Single-Antigen Beads—A Technical Solution for the Prozone Effect

Background. Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only. This led us to investigate complement component 1 (C1) as the cause of the prozone in SAB testing. We also found an easy way to overcome the prozone effect. Methods. Sera with a prozone effect were tested in the SAB assay, applying different methods of serum pretreatment to explore the parameters leading to the prozone. Results. The prozone was not observed when EDTA plasma or serum with EDTA added were tested. Further, addition of dithiothreitol, addition of C1 inhibitor, or heat inactivation of the sera abolished the prozone effect. Adding fresh nonimmune serum to heat-inactivated sera restored the prozone effect. Only beads showing a prozone were found to be covered with C1q. Conclusion. Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.

Distorted Relation between mRNA Copy Number and Corresponding Major Histocompatibility Complex Ligand Density on the Cell Surface

The major histocompatibility complex (MHC) presents peptides derived from degraded cellular proteins to T-cells and is thus crucial for triggering specific immune responses against viral infections or cancer. Up to now, there has been no evidence for a correlation between levels of mRNA (the “transcriptome”) and the density of MHC-peptide complexes (the “MHC ligandome”) on cells. Because such dependences are of intrinsic importance for the detailed understanding of translation efficiency and protein turnover and thus for systems biology in general and for tumor immunotherapy in practical application, we quantitatively analyzed the levels of mRNA and corresponding MHC ligand densities in samples of renal cell carcinomas and their autologous normal kidney tissues. Relative quantification was carried out by gene chip analysis and by stable isotope peptide labeling, respectively. In comparing more than 270 pairs of gene expression and corresponding peptide presentation ratios, we demonstrate that there is no clear correlation (r = 0.32) between mRNA levels and corresponding MHC peptide levels in renal cell carcinoma. A significant number of peptides presented predominantly on tumor or normal tissue showed no or only minor changes in mRNA expression levels. In several cases, peptides could even be identified despite the virtual absence of the respective mRNA. Thus we conclude that a majority of epitopes from tumor-associated antigens will not be found in approaches based mainly on mRNA expression studies as mRNA expression reflects a distorted picture of the situation on the cell surface as visible for T-cells.

Novel Multi-Peptide Vaccination In Hla-A2+ Hormone Sensitive Patients With Biochemical Relapse of Prostate Cancer

A phase I/II trial was conducted to assess feasibility and tolerability of tumor associated antigen peptide vaccination in hormone sensitive prostate carcinoma (PC) patients with biochemical recurrence after primary surgical treatment.

Unexpected Abundance of HLA Class II Presented Peptides in Primary Renal Cell Carcinomas

Purpose: To elicit a long-lasting antitumor immune response, CD8+ and CD4+ T cells should be activated. We attempted to isolate HLA-DR–presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumor-associated antigens (TAA) for their use in peptide-based immunotherapy. Experimental Design: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression. HLA class II peptides were subsequently isolated and identified by mass spectrometry. Gene expression analysis was done to detect genes overexpressed in tumor tissue. Peptides from identified TAAs were used to induce peptide-specific CD4+ T-cell responses in healthy donors and in tumor patients. Results: In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system. To our surprise, we were able to isolate and characterize hundreds of class II peptides directly from primary dissected solid tumors, especially from renal cell carcinomas, and from colorectal carcinomas and transitional cell carcinomas. Infiltrating leukocytes expressed MHC class II molecules and tumor cells, very likely under the influence of IFNγ. Our list of identified peptides contains ligands from several TAAs, including insulin-like growth factor binding protein 3 and matrix metalloproteinase 7. The latter bound promiscuously to HLA-DR molecules and were able to elicit CD4+ T-cell responses. Conclusions: Thus, our direct approach will rapidly expand the limited number of T-helper epitopes from TAAs for their use in clinical vaccination protocols.

Simultaneous Infiltration of Polyfunctional Effector and Suppressor T Cells into Renal Cell Carcinomas

Renal cell carcinoma is frequently infiltrated by cells of the immune system. This makes it important to understand interactions between cancer cells and immune cells so they can be manipulated to bring clinical benefit. Here, we analyze subsets and functions of T lymphocytes infiltrating renal cell tumors directly ex vivo following mechanical disaggregation and without any culture step. Subpopulations of memory and effector CD4(+) Th1, Th2, and Th17 and CD8(+) Tc1 cells were identified based on surface phenotype, activation potential, and multicytokine production. Compared with the same patients peripheral blood, T lymphocytes present inside tumors were found to be enriched in functional CD4(+) cells of the Th1 lineage and in effector memory CD8(+) cells. Additionally, several populations of CD4(+) and CD8(+) regulatory T cells were identified that may synergize to locally dampen antitumor T-cell responses.

Cutting Edge: Predetermined Avidity of Human CD8 T Cells Expanded on Calibrated MHC/Anti-CD28-Coated Microspheres

Cytotoxic CD8 T cells are key effectors in the immunotherapy of malignant and viral diseases. However, the lack of efficient methods for their in vitro priming and expansion has become a bottleneck to the development of vaccines and adoptive transfer strategies. Synthetic artificial APCs (aAPCs) are now emerging as an attractive tool for eliciting and expanding CTL responses. We show that, by controlling the MHC density on aAPCs, high- or low-avidity tumor-directed human CTL lines can be raised effectively in vitro if costimulation via CD28 and IL-12 is provided. Compared with low-avidity CTL lines, high-avidity CTLs need 100- to 1000-fold less peptide for activation, bind more MHC tetramers, and, as expected, are superior in recognizing tumor cell lines expressing Ag. We believe that the possibility to raise Ag-specific T cells with predetermined avidity will be crucial for the future use of aAPCs in immunotherapeutical settings.

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells.

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ Tu2004cell responses by cross‐presentation of exogenous antigen via MHC classu2004I is well known. In contrast, their role in the activation of CD4+ Tu2004cells is less clearly defined, although several recent studies in mice and Tu2004cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC classu2004II. In this study we have investigated the potential of antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to the human stress‐inducible Hsp70 to enhance activation and proliferation of human memory CD4+ Tu2004cells. Hsp70:peptide complexes were found to amplify the proliferation of antigen‐specific CD4+ Tu2004cells as confirmed by HLA‐DR tetramer staining. Complex formation of the antigenic peptide with Hsp70 was absolutely required to elicit an antigen‐specific amplification. This effect was most pronounced at low doses of antigen and decreasing APC/CD4+ Tu2004cell ratios. Taken together, we show the potential of Hsp70 to enhance antigen‐specific CD4+ Tu2004cell proliferation and to increase the immunogenicity of presented peptides in human CD4+ Tu2004cells.

Promiscuous survivin peptide induces robust CD4+ T‐cell responses in the majority of vaccinated cancer patients

CD4+ T cells have been shown to be crucial for the induction and maintenance of cytotoxic T cell responses and to be also capable of mediating direct tumor rejection. Therefore, the anticancer therapeutic efficacy of peptide‐based vaccines may be improved by addition of HLA class II epitopes to stimulate T helper cells. Survivin is an apoptosis inhibiting protein frequently overexpressed in tumors. Here we describe the first immunological evaluation of a survivin‐derived CD4+ T cell epitope in a multipeptide immunotherapy trial for prostate carcinoma patients. The survivin peptide is promiscuously presented by several human HLA‐DRB1 molecules and, most importantly, is naturally processed by dendritic cells. In vaccinated patients, it was able to induce frequent, robust and multifunctional CD4+ T cell responses, as monitored by IFN‐γ ELISPOT and intracellular cytokine staining. Thus, this HLA‐DR restricted epitope is broadly immunogenic and should be valuable for stimulating T helper cells in patients suffering from a wide range of tumors.

“Electronic nose” detects major histocompatibility complex-dependent prerenal and postrenal odor components

Mice prefer to mate with individuals expressing different MHC genes from their own. Volatile components presenting MHC-dependent odor types are present in urine and can be detected by mice, as shown by extensive behavioral studies. Similar odor types are suspected to influence human behavior as well. Although a recent report indicates that MHC expression influences the ratio of volatile compounds such as phenylacetic acid, so far no other means than studying the behavior of mice or rats has been available to assess odor types. Here, we report the ability of a gas sensor array (referred to as “electronic nose”) to detect MHC-dependent odor types. The electronic nose consists of an array of chemophysical detectors, in our case quartz crystal microbalances and semiconducting metal-oxide sensors that change frequency or conductivity upon binding of very small numbers of individual molecules present in the gas phase of odorous fluids. The pattern of changes is characteristic for a particular smell. Our electronic nose distinguishes the urine odor types of MHC congenic mouse strains, MHC class I mutant mice, and HLA-A2 transgenic mice. In addition, MHC-dependent odor types can be detected in serum. The device also clearly differentiates between individual odor types of human sera from HLA homozygous individuals; however, HLA expression seems to have only a secondary influence. Thus, odor-type research can now be carried out with an objective and fast through-put system independent of behavioral studies.

Essential differences in ligand presentation and T cell epitope recognition among HLA molecules of the HLA-B44 supertype

Human leukocyte antigens (HLA) have long been grouped into supertypes to facilitate peptide‐based immunotherapy. Analysis of several hundreds of peptides presented by all nine antigens of the HLA‐B44 supertype (HLA‐B*18, B*37, B*40, B*41, B*44, B*45, B*47, B*49 and B*50) revealed unique peptide motifs for each of them. Taking all supertype members into consideration only 25 out of 670 natural ligands were found on more than one HLA molecule. Further direct comparisons by two mass spectrometric methods – isotope labeling as well as a label‐free approach – consistently demonstrated only minute overlaps of below 3% between the ligandomes of different HLA antigens. In addition, T cell reactions of healthy donors against immunodominant HLA‐B*44 and HLA‐B*40 epitopes from EBV lacked promiscuous T‐cell recognition within the HLA‐B44 supertype. Taken together, these results challenge the common paradigm of broadly presented epitopes within this supertype.