Shared αß TCR Usage in Lungs of Sarcoidosis Patients with Löfgren's Syndrome.

Sarcoidosis is a granulomatous disease that primarily affects the lungs and is characterized by an accumulation of CD4+ T cells in the bronchoalveolar lavage (BAL). Previous work has indicated that HLA-DRB1*03:01+ (DR3+) patients diagnosed with the acute form of the disease, Löfgren's syndrome (LS), have an accumulation of CD4+ T cells bearing TCRs using TRAV12-1 (formerly AV2S3). However, the importance of these α-chains in disease pathogenesis and the paired TCRß-chain remains unknown. This study aimed to identify expanded αßTCR pairs expressed on CD4+ T cells derived from the BAL of DR3+ LS patients. Using a deep-sequencing approach, we determined TCRα- and TCRß-chain usage, as well as αßTCR pairs expressed on BAL CD4+ T cells from LS patients. TRAV12-1 and TRBV2 (formerly BV22) were the most expanded V region gene segments in DR3+ LS patients relative to control subjects, and TRAV12-1 and TRBV2 CDR3 motifs were shared among multiple DR3+ LS patients. When assessing αßTCR pairing, TRAV12-1 preferentially paired with TRBV2, and these TRAV12-1/TRBV2 TCRs displayed CDR3 homology. These findings suggest that public CD4+ TCR repertoires exist among LS patients and that these T cells are recognizing the putative sarcoidosis-associated Ag(s) in the context of DR3.

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR.

Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.

Improving antigenic peptide vaccines for cancer immunotherapy using a dominant tumor-specific T cell receptor.

Vaccines that incorporate peptide mimics of tumor antigens, or mimotope vaccines, are commonly used in cancer immunotherapy and function by eliciting increased numbers of T cells that cross-react with the native tumor antigen. Unfortunately, they often elicit T cells that do not cross-react with or that have low affinity for the tumor antigen. Using a high affinity tumor-specific T cell clone, we identified a panel of mimotope vaccines for the dominant peptide antigen from a mouse colon tumor that elicits a range of tumor protection following vaccination. The TCR from this high affinity T cell clone was rarely identified in ex vivo evaluation of tumor-specific T cells elicited by mimotope vaccination. Conversely, a low affinity clone found in the tumor and following immunization was frequently identified. Using peptide libraries, we determined if this frequently identified TCR improved the discovery of efficacious mimotopes. We demonstrated that the representative TCR identified more protective mimotopes than the high affinity TCR. These results suggest that targeting a dominant fraction of tumor-specific T cells generates potent immunity and that consideration of the available T cell repertoire is necessary for targeted T cell therapy. These results have important implications when optimizing mimotope vaccines for cancer immunotherapy.

A novel miRNA produced during lytic HSV-1 infection is important for efficient replication in tissue culture.

The influence of miRNAs on the host-pathogen environment is largely unknown and under intensive investigation. Whether produced by the pathogen or by the host cell, these miRNAs will sculpt the intracellular landscape, as their activity will ultimately affect levels of target proteins. Using a high-throughput sequencing approach, we identified 19 novel small RNAs produced during the early hours of herpes simplex virus type 1 (HSV-1) infection in epithelial cells. Six of the novel RNAs had predicted folds characteristic of miRNAs. One of the six, miR-92944, which resides in the 5' UTR of the ul42 gene in the sense orientation, was confirmed as a bona fide miRNA by RT-PCR and stem-loop PCR analysis. Northern blot analysis was used to observe the precursor forms of miR-92944. Viral mutants that do not produce miR-92944 exhibited significant reductions in viral titers in both single and multi-step growth analysis and a fourfold reduction in plaque size. The miR-92944 mutants produce wild-type levels of ICP4, UL42, VP5, and gC proteins contain no additional changes in the DNA sequence surrounding the site of mutagenesis. The defective phenotype of miR-92944 mutants was complemented in V42.3 cells, which contain the 5'UTR of ul42. We also found that miR-H1 expression was diminished in cells infected with the miR-92944 mutant virus. This study provides new information on the miRNA landscape during the early stages of HSV-1 infection and reveals novel targets for antagonistic molecules that may curtail the establishment of lytic or latent virus infection.

MHC class I loaded ligands from breast cancer cell lines: A potential HLA-I-typed antigen collection.

To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2740. Of the unique peptides eluted, more than 1750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, half of these immunogenic peptides were shared between different breast cancer cell lines. MHC class I binding probability was used to plot the distribution of the eluted peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines. SIGNIFICANCE: By employing proteomic analyses of eluted peptides from breast cancer cells, the current study has built an initial HLA-I-typed antigen collection for breast cancer research. It was also determined that immunogenic epitopes can be identified using established cell lines and that shared immunogenic peptides can be found in different cancer types such as breast cancer and leukemia. Importantly, out of 3196 eluted peptides that included duplicate peptides in different cells 89 peptides either contained mutation in their sequence or were derived from aberrant translation suggesting that mutation-containing epitopes are on the order of 2-3% in breast cancer cells. Finally, our results suggest that interfering with MHC class I function is one of the mechanisms of how tumor cells escape immune system attack.

Androgen receptor expression in the rat prostate is down-regulated by dietary phytoestrogens.

BACKGROUND: It is well established that the growth of the prostate gland is a hormone-dependent phenomenon involving both androgenic and estrogenic control. Proliferation of prostate cells is, at least in part, under control of estrogen receptor beta (ER-beta). Phytoestrogens bind ER-beta with high affinity and therefore may have antiproliferative effects in the prostate. METHODS: The prostates of male Long-Evans rats fed a diet high in phytoestrogens (Phyto-600) or very low levels of phytoestrogens (Phyto-free) were analyzed to determine the impact of dietary phytoestrogens on prostate weight and androgen receptor (AR) expression in the prostate. RESULTS: Dietary phytoestrogens significantly decreased post-pubertal prostate weight gain in Phyto-600 vs Phyto-free fed males. Additionally, dietary phytoestrogens (Phyto-600) decreased AR expression in the prostate as determined by in situ hybridization. CONCLUSIONS: Soy phytoestrogens, present in diet, alter prostate growth presumably by binding ER-beta and subsequently reducing AR expression within the prostate.

Dihydrotestosterone may inhibit hypothalamo-pituitary-adrenal activity by acting through estrogen receptor in the male mouse.

The corticosterone (CORT) response to environmental perturbation has been shown to be enhanced by estrogen but inhibited by the androgen dihydrotestosterone (DHT). However, the mechanism of androgen's action has not been identified. This study examined the effects of estradiol benzoate (EB), the non-aromatizable androgen DHT, and the DHT metabolite 5alpha-androstan-3beta, 17beta-diol (3beta-diol) on the corticosterone response to stress. Adult male CBB6/F1 mice were gonadectomized and injected subcutaneously (once a day for 4 days) with the above compounds (controls received oil vehicle injections). Animals (within treatments) were randomly assigned to stress or non-stress conditions. The non-stress animals were taken directly from their home cages and killed. Animals were stressed by a 30 min restraint prior to being killed. Hormone levels were determined in plasma via radioimmunoassay. In agreement with previous studies, the CORT response to immobilization was enhanced by EB and inhibited by DHT. Surprisingly, 3beta-diol inhibited the CORT response similar to the effect of DHT. In a second study, concomitant injections of the androgen receptor antagonist flutamide only partially blocked DHT's, but had no effect on 3beta-diol's, inhibitory action. In contrast, injections with the estrogen receptor antagonist tamoxifen completely blocked the effects of 3beta-diol and partially blocked DHT's effect. Taken together these studies suggest that DHT's inhibitory effects may be, at least in part, via the estrogen receptor, through its conversion to 3beta-diol. These studies also suggest that the DHT metabolites may be functionally relevant when considering hormonal responses to stress.

Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback.

Equol (7-hydroxy-3[4'hydroxyphenyl]-chroman) is the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abundantly in soybeans and soy foods. Equol may be an important biologically active molecule based on recent studies demonstrating that equol can modulate reproductive function. In this study, we examined the effects of equol on prostate growth and LH secretion and determined some of the mechanisms by which it might act. Administration of equol to intact male rats for 4-7 days reduced ventral prostate and epididymal weight and increased circulating LH levels. Using binding assays, we determined that equol specifically binds 5alpha-dihydrotestosterone (DHT), but not testosterone, dehydroepiandrosterone, or estrogen with high affinity. Equol does not bind the prostatic androgen receptor, and has a modest affinity for recombinant estrogen receptor (ER) beta, and no affinity for ERalpha. In castrated male rats treated with DHT, concomitant treatment with equol blocked DHT's trophic effects on the ventral prostate gland growth and inhibitory feedback effects on plasma LH levels without changes in circulating DHT. Therefore, equol can bind circulating DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equol's biological properties. The significance of equol's ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment of androgen-mediated pathologies.