TAS0314, a novel multi-epitope long peptide vaccine, showed synergistic antitumor immunity with PD-1/PD-L1 blockade in HLA-A*2402 mice.

Cancer peptide vaccines are a promising cancer immunotherapy that can induce cancer-specific cytotoxic T lymphocytes (CTLs) in tumors. However, recent clinical trials of cancer vaccines have revealed that the efficacy of the vaccines is limited. Targeting single antigens and vaccination with short peptides are partly the cause of the poor clinical outcomes. We synthesized a novel multi-epitope long peptide, TAS0314, which induced multiple epitope-specific CTLs in HLA knock-in mice. It also showed superior epitope-specific CTL induction and antitumor activity. We also established a combination treatment model of vaccination with PD-1/PD-L1 blockade in HLA-A*2402 knock-in mice, and it showed a synergistic antitumor effect with TAS0314. Thus, our data indicated that TAS0314 treatment, especially in combination with PD-1/PD-L1 blockade, is a promising therapeutic candidate for cancer immunotherapy.

Generation of a Novel HLA Class I Transgenic Mouse Model Carrying a Knock-in Mutation at the ß2-Microglobulin Locus.

We generated a series of monochain HLA class I knock-in (KI) mouse strains, in which a chimeric HLA class I molecule (α1/α2 domain of HLA-A*0201, HLA-A*0301, HLA-A*2402, or HLA-A*3101 and α3 domain of H-2Db) was covalently linked with 15 aa to human ß2-microglobulin (ß2m) and introduced into the endogenous mouse ß2m locus. In homozygous KI mice, mouse ß2m gene disruption resulted in loss of the endogenous H-2 class I molecules and reduction in the peripheral CD8+ T cell population that was partially restored by monochain HLA class I expression. A gene dosage-dependent expression of HLA, similar to that in human PBMCs, was detected in heterozygous and homozygous HLA KI mice. Upon vaccination with various virus epitopes, HLA-restricted, epitope-specific CTLs were induced in HLA KI mice, similar to the response in the commonly used HLA transgenic mice. Importantly, the CTL responses induced in heterozygous KI mice were similar to those in homozygous KI mice. These results suggest that coexpression of H-2 class I does not affect HLA-restricted CTL responses in HLA KI mice, which differs from the situation reported for monochain HLA Tg × ß2m-/- mice. Furthermore, we generated double KI mice harboring two different HLA (HLA-A*2402 and HLA-A*0301) KI alleles, which showed a CTL response against both HLA-A24 and HLA-A3 epitopes when immunized with a mixture of both peptides. These results indicated that this HLA class I KI mouse model provides powerful research tools not only for the study of HLA class I-restricted CTL responses, but also for preclinical vaccine evaluation.

Regulation of activated CD4+ T cells by NK cells via the Qa-1-NKG2A inhibitory pathway.

The ability of natural-killer cells to regulate adaptive immunity is not well understood. Here we define an interaction between the class Ib major histocompatibility complex (MHC) molecule Qa-1-Qdm on activated T cells responsible for adaptive immunity and CD94-NKG2A inhibitory receptors expressed by natural-killer cells by using Qa-1-deficient and Qa-1 knockin mice containing a point mutation that selectively abolishes Qa-1-Qdm binding to CD94-NKG2A receptors. The Qa-1-NKG2A interaction protected activated CD4+ T cells from lysis by a subset of NKG2A+ NK cells and was essential for T cell expansion and development of immunologic memory. Antibody-dependent blockade of this Qa-1-NKG2A interaction resulted in potent NK-dependent elimination of activated autoreactive T cells and amelioration of experimental autoimmune encephalomyelitis. These findings extend the functional reach of the NK system to include regulation of adaptive T cell responses and suggest a new clinical strategy for elimination of antigen-activated T cells in the context of autoimmune disease and transplantation.

Induction and activation of the aryl hydrocarbon receptor by IL-4 in B cells.

It is widely known that IL-4 and IL-13 act on various kinds of cells, including B cells, resulting in enhancement of proliferation, class switching to IgE and expression of several surface proteins. These functions are important for the recognition of the various antigens in B cells and are known to be involved in the pathogenesis of allergic diseases. However, it has not been known whether IL-4/IL-13 is involved in the metabolism of various kinds of xenobiotics including 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), and it remains undetermined whether TCDD, an environmental pollutant, influences IgE production in B cells, exaggerating allergic reactions. We identified IL-4- or IL-13-inducible genes in a human Burkitt lymphoma cell line, DND-39, using microarray technology, in which the AHR gene was included. The AHR gene product, the aryl hydrocarbon receptor (AhR), was induced by IL-4 in both mouse and human B cells in a STAT6-dependent manner. IL-4 alone had the ability to translocate the induced AhR to the nuclei. TCDD, a ligand for AhR, rapidly degraded the induced AhR by the proteasomal pathway, although IL-4-activated AhR sustained its expression. AhR activated by IL-4 caused expression of a xenobiotic-metabolizing gene, CYP1A1, and TCDD synergistically acted on the induction of this gene by IL-4. However, the induction of AhR had no effect on IgE synthesis or CD23 expression. These results indicate that the metabolism of xenobiotics would be a novel biological function of IL-4 and IL-13 in B cells, whereas TCDD is not involved in IgE synthesis in B cells.

Signaling by the kinase MINK is essential in the negative selection of autoreactive thymocytes.

Signaling through the T cell antigen receptor leading to elimination (negative selection) or differentiation (positive selection) of developing thymocytes generates a self-tolerant T cell repertoire. Here we report that the serine-threonine kinase MINK selectively connects the T cell receptor to a signaling pathway that mediates negative but not positive selection. Analysis of this pathway suggested that the essential function of MINK in the elimination of self-reactive thymocytes may be associated with 'downstream' activation of Jun kinase and enhancement of expression of the proapoptotic molecule Bim.

Analysis of regulatory CD8 T cells in Qa-1-deficient mice.

The mouse protein Qa-1 (HLA-E in humans) is essential for immunological protection and immune regulation. Although Qa-1 has been linked to CD8 T cell-dependent suppression, the physiological relevance of this observation is unclear. We generated mice deficient in Qa-1 to develop an understanding of this process. Qa-1-deficient mice develop exaggerated secondary CD4 responses to foreign and self peptides. Enhanced responses to proteolipid protein self peptide were associated with resistance of Qa-1-deficient CD4 T cells to Qa-1-restricted CD8 T suppressor activity and increased susceptibility to experimental autoimmune encephalomyelitis. These findings delineate a Qa-1-dependent T cell-T cell inhibitory interaction that prevents the pathogenic expansion of autoreactive CD4 T cell populations and consequent autoimmune disease.

Cell type-dependent divergence of transactivation by glucocorticoid receptor ligand.

The glucocorticoid receptor regulates gene expression mainly by two mechanisms; transactivation and trans-repression. A ligand with strong transrepression and weak transactivation activity is predicted to be a beneficial agent with potent anti-inflammatory activity and minor adverse effects. Recently, the profile of a synthetic steroid, RU24858, has been reported to fulfill this condition in vitro, but others have reported no dissociation between the anti-inflammatory activity and side effects in vivo. To gain further information on the profile of this compound, we evaluated its transactivation ability using a reporter gene analysis both in vitro and in vivo. In the in vitro analysis, RU24858 demonstrated only a weak transactivation activity in HeLa cells, when compared with prednisolone. In CV-1 cells, however, these two glucocorticoids exhibited equivalent transactivation activities. This behavior is independent of whether the reporter gene is regulated by mouse mammary tumor virus promoter or multiple copies of glucocorticoid response element. When the reporter plasmid was inoculated into mouse abdominal skin using a gene gun, followed by orally administration of glucocorticoids, RU24858 induced significantly higher reporter enzyme activity than prednisolone. These results suggest that the profile of RU24858 is divergent and its transactivation ability is comparable to prednisolone depending on the cell-type both in vitro and in vivo.

CD40-mediated tumor necrosis factor receptor-associated factor 3 signaling upregulates IL-4-induced germline Cepsilon transcription in a human B cell line.

Induction of germline C epsilon transcription in B cells by IL-4, which is a critical initiating step for IgE class switching, is enhanced by CD40 engagement. Although signaling by CD40 is initiated by the binding of tumor necrosis factor receptor-associated factor (TRAF) family members to its cytoplasmic domain, whether those TRAF family proteins mediate enhancement of germline Cepsilon transcription is not evident. We report here that CD40-induced TRAF3-dependent activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) is involved in the upregulation of IL-4-driven germline C epsilon transcription in a human Burkitt's lymphoma B cell line, DG75. Among the six known TRAF proteins, TRAF2, 3, 5, and 6 associated with CD40 in an unstimulated state, and the levels of these four proteins were unaffected by anti-CD40 stimulation. Antisense oligodeoxynucleotide (ODN) for TRAF3 inhibited CD40-induced activation of MEK1-ERK pathway by decreasing expression of TRAF3 protein, but antisense ODNs for TRAF2, 5, and 6 were ineffective. Furthermore, CD40-mediated enhancement of IL-4-driven germline C epsilon transcription was inhibited by antisense ODN for TRAF3 and by a MEK1 inhibitor, PD98059. These results suggest that in DG75 cells, TRAF3-induced MEK1 activation may be involved in CD40-mediated upregulation of IL-4-driven germline C epsilon transcription.

Species-specific differences in the glucocorticoid receptor transactivation function upon binding with betamethasone-esters.

Glucocorticoids (GCs) are the most effective drugs for anti-inflammatory diseases. A number of adverse side effects, however, limit chronic treatment with GCs. To improve their therapeutic usefulness, attempts have been made to dissociate the two main actions of the glucocorticoid receptor (GR), transactivation and transrepression, which are believed to be responsible for the side effects and anti-inflammatory effects, respectively. We report here species-specific differences in the transactivation response mediated by GR. Dexamethasone (DEX), betamethasone (BM), and their esterified-derivatives had full transrepression agonistic activity in a reporter assay using CV-1 cells transfected with either human or rat GR. These GCs also had full transactivation agonistic activity in CV-1 cells transfected with human GR. The esterified-BM, however, had only partial transactivation agonistic activity in cells transfected with rat GR, whereas BM and esterified-DEX had full transactivation agonistic activity. Moreover, in rat hepatoma H4-II-E cells, the esterified-BM failed to induce tyrosine aminotransferase, which is regulated by GR-mediated transactivation activity. There were no significant differences between the binding affinity of these GCs to human and rat GR. Consistent with the weak transactivation activity of esterified-BM mediated by rat GR, there were few side effects, evaluated by thymus involution and body weight loss, in an antigen-induced asthmatic model in rats. These results suggest that the potency of esterified-BM to induce transactivation activity is different between species and that this difference is not due to differences in receptor binding.

In vivo analysis of glucocorticoid-induced reporter gene expression using gene gun DNA delivery.

Glucocorticoid regulates various physiological processes via the activation and repression of gene expression. The anti-inflammatory effects and the adverse effects are believed to be dependent on the repression and the activation of genes, respectively. Reporter gene assay is a useful technique to separately evaluate these two functions and has been used for in vitro screening of novel ligands for the glucocorticoid receptor (GR). We report here the application of a reporter gene assay for the in vivo determination of the GR-mediated gene activation. A reporter plasmid containing glucocorticoid response elements was introduced to abdominal mouse skin using a gene gun. Administration of prednisolone induced the expression of the reporter gene, only when the GR expression plasmid was co-transfected with the reporter plasmid. Endogenous levels of corticosterone appeared to be negligible in this protocol. The dose response for this induction was comparable to those for the decreases in thymus weight and serum corticosterone. These results suggest that gene gun-mediated skin transfection enables the in vivo reporter gene assay and that this technique can be used to predict the potency of ligands for the GR-mediated gene activation.