Downregulation of TCF7 and LEF1 is a key determinant of tumor-infiltrating regulatory T-cell function.

Forkhead box P3 (Foxp3)-expressing regulatory T (Treg) cells play essential roles in immune homeostasis but also contribute to establish a favorable environment for tumor growth by suppressing anti-tumor immune responses. It is thus necessary to specifically target tumor-infiltrating Treg cells to minimize effects on immune homeostasis in cancer immunotherapy. However, molecular features that distinguish tumor-infiltrating Treg cells from those in secondary lymphoid organs remain unknown. Here we characterize distinct features of tumor-infiltrating Treg cells by global analyses of the transcriptome and chromatin landscape. They exhibited activated phenotypes with enhanced Foxp3-dependent transcriptional regulation, yet being distinct from activated Treg cells in secondary lymphoid organs. Such differences may be attributed to the extensive clonal expansion of tumor-infiltrating Treg cells. Moreover, we found that TCF7 and LEF1 were specifically downregulated in tumor-infiltrating Treg cells both in mice and humans. These factors and Foxp3 co-occupied Treg suppressive function-related gene loci in secondary lymphoid organ Treg cells, whereas the absence of TCF7 and LEF1 accompanied altered gene expression and chromatin status at these gene loci in tumor-infiltrating Treg cells. Functionally, overexpression of TCF7 and LEF1 in Treg cells inhibited the enhancement of Treg suppressive function upon activation. Our results thus show the downregulation of TCF7 and LEF1 as markers of highly suppressive Treg cells in tumors and suggest that their absence controls the augmentation of Treg suppressive function in tumors. These molecules may be potential targets for novel cancer immunotherapy with minimum effects on immune homeostasis.

Immunotherapy Targeting CCR8+ Regulatory T Cells Induces Antitumor Effects via Dramatic Changes to the Intratumor CD8+ T Cell Profile.

Regulatory T cells (Tregs) contribute to the formation of a tumor-immunosuppressive microenvironment. CCR8 is reportedly selectively expressed in tumor Tregs, and an anti-CCR8 Ab can exert potent antitumor effects by eliminating intratumor Tregs in murine tumor models. In this study, we analyzed changes to intratumor immunity after anti-CCR8 Ab administration, especially in CD8+ T cells, which are involved in cancer cell killing, using the CT26 colorectal carcinoma mouse model. Immunophenotyping of tumor-infiltrating cells by mass cytometry after Ab administration on day 5 of tumor inoculation revealed that CD8+ T cell subsets were dramatically altered in the CCR8 Ab-treated group, with an increase in naive cells and nonexhausted effector cells and a decrease in exhausted cells with high expression levels of TOX. These results were corroborated with flow cytometry analysis. Delayed administration of the anti-CCR8 Ab on day 9 or 12, when the amount of CCR8+ Tregs and CD8+ T cell exhaustion were more progressed, also resulted in a decrease in exhausted CD8+ T cells, leading to tumor regression. Finally, we confirmed that high CCR8+ Treg infiltration was associated with high TOX expression in CD8+ T cells in human cancer patients. In conclusion, administration of an anti-CCR8 Ab can dramatically alter the activation and exhaustion state of intratumor CD8+ T cells, resulting in strong antitumor effects. In cancer patients with an advanced tumor-immunosuppressive environment, CD8+ T cell exhaustion has progressed along with CCR8+ Treg induction. Therefore, targeted depletion of CCR8+ Tregs is expected to be effective in these patients.

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

Neural-net-based cell deconvolution from DNA methylation reveals tumor microenvironment associated with cancer prognosis.

DNA methylation is a pivotal epigenetic modification that defines cellular identity. While cell deconvolution utilizing this information is considered useful for clinical practice, current methods for deconvolution are limited in their accuracy and resolution. In this study, we collected DNA methylation data from 945 human samples derived from various tissues and tumor-infiltrating immune cells and trained a neural network model with them. The model, termed MEnet, predicted abundance of cell population together with the detailed immune cell status from bulk DNA methylation data, and showed consistency to those of flow cytometry and histochemistry. MEnet was superior to the existing methods in the accuracy, speed, and detectable cell diversity, and could be applicable for peripheral blood, tumors, cell-free DNA, and formalin-fixed paraffin-embedded sections. Furthermore, by applying MEnet to 72 intrahepatic cholangiocarcinoma samples, we identified immune cell profiles associated with cancer prognosis. We believe that cell deconvolution by MEnet has the potential for use in clinical settings.

Novel Adjuvant S-540956 Targets Lymph Nodes and Reduces Genital Recurrences and Vaginal Shedding of HSV-2 DNA When Administered with HSV-2 Glycoprotein D as a Therapeutic Vaccine in Guinea Pigs.

Herpes simplex virus type 2 (HSV-2) is a leading cause of genital ulcer disease and a major risk factor for acquisition and transmission of HIV. Frequent recurrent genital lesions and concerns about transmitting infection to intimate partners affect the quality of life of infected individuals. Therapeutic vaccines are urgently needed to reduce the frequency of genital lesions and transmission. S-540956 is a novel vaccine adjuvant that contains CpG oligonucleotide ODN2006 annealed to its complementary sequence and conjugated to a lipid that targets the adjuvant to lymph nodes. Our primary goal was to compare S-540956 administered with HSV-2 glycoprotein D (gD2) with no treatment in a guinea pig model of recurrent genital herpes (studies 1 and 2). Our secondary goals were to compare S-540956 with oligonucleotide ODN2006 (study1) or glucopyranosyl lipid A in a stable oil-in-water nano-emulsion (GLA-SE) (study 2). gD2/S-540956 reduced the number of days with recurrent genital lesions by 56%, vaginal shedding of HSV-2 DNA by 49%, and both combined by 54% compared to PBS, and was more efficacious than the two other adjuvants. Our results indicate that S-540956 has great potential as an adjuvant for a therapeutic vaccine for genital herpes, and merits further evaluation with the addition of potent T cell immunogens.

Synthesis and structure-activity relationships of an orally available and long-acting analgesic peptide, N(alpha)-amidino-Tyr-D-Arg-Phe-MebetaAla-OH (ADAMB).

A novel dermorphin tetrapeptide N(alpha)-amidino-Tyr-D-Arg-Phe-MebetaAla-OH (ADAMB) was designed based on the structures of several dermorphin tetrapeptide analogues, including N(alpha)-amidino-Tyr-D-Arg-Phe-Gly-OH (ADA-DER), H-Tyr-D-Arg-Phe-betaAla-OH (TAPA), and H-Tyr-D-Arg-Phe-Sar-OH (DAS-DER). These parent compounds were known to show a weak oral analgesic activity in animals and/or to possess a different mechanism of analgesia from other mu-opioid peptides. Six analogues of ADAMB were also synthesized to investigate the effect on potency of N-terminal amidination and N-methyl-beta-alanine (MebetaAla) substitution at position 4. Compounds were assessed using the tail pressure test in mice after subcutaneous and oral administration. Among the peptides tested, ADAMB showed the strongest oral antinociceptive activity, with an ED(50) of 5.8 vs 22.2 mg/kg for morphine, as well as a 38-fold stronger activity after subcutaneous administration. ADAMB also showed long-lasting antinociceptive activity, with 50% of the maximum effect persisting in the tail pressure test at 10 h after oral administration (10 mg/kg). In contrast, orally administered morphine (80 mg/kg) showed a rapid decrease of activity in the same test and its antinociceptive effect disappeared within 4 h. When the antinociceptive effect of ADAMB was compared with that of analogues possessing betaAla(4) (1) or Sar(4) (2), as well as analogues with N-substitution (3-6), it was found that both the N(alpha)-amidino substitution and the MebetaAla(4) were synergistically involved in creating ADAMB's exceptionally high antinociceptive activity.

Plasmodium falciparum: the fungal metabolite gliotoxin inhibits proteasome proteolytic activity and exerts a plasmodicidal effect on P. falciparum.

The in vitro antimalarial activity of the fungal metabolite gliotoxin (GTX) was evaluated, and its mechanism of action was studied. GTX showed plasmodicidal activity against both Plasmodium falciparum chloroquine-resistant strain K-1 and chloroquine-susceptible strain FCR-3. GTX cytotoxicity was significantly lower against a normal liver cell line (Chang Liver cells). The intracellular reduced glutathione level of parasitized and of normal red blood cells was not affected by GTX treatment. However, GTX decreased the chymotrypsin-like activity of parasite proteasomes in a time-dependent manner. The results of this study indicate that GTX possesses plasmodicidal activity and that this effect is due to inhibition of parasite proteasome activity, suggesting that GTX may constitute a useful antimalarial therapy.

Structure-activity relationships (SAR) of [D-Arg(2)]dermorphin(1-4) analogues, N(alpha)-amidino-Tyr-D-Arg-Phe-X.

In investigating the development of compounds with potent analgesic effects after oral administration, 74 C-terminal analogues (N(alpha)-amidino-Tyr-D-Arg-Phe-X), based on the structure of N(alpha)-amidino-Tyr-D-Arg-Phe-Me beta Ala-OH (ADAMB), were synthesized. Their analgesic activity was evaluated using the mouse-tail pressure test after both subcutaneous and oral administration, and the structure-activity relationships (SAR) were examined in detail. The results clearly indicated that compounds containing beta-amino acid without a side chain at the X position are preferable for expression of potent analgesic activity, and that the free carboxyl group is superior in its analgesic activity to that of the esterified or amidated carboxy group at the C-terminal. In addition, N-methylation of the amide bond at the 4th position contributed to improved analgesic activity. These results indicated that the strong and long-lasting analgesic effect of ADAMB is expressed by the synergistic effects of N(alpha)-amidination, the N-methylation of the amide bond at the 4th position and the carbon chain length (beta-Ala) of the residue at the 4th position, and that this is the most suitable structure.

Synthesis and antinociceptive activity of orally active opioid peptides: improvement of oral bioavailability by esterification.

To improve the oral bioavailability of a dermorphin tetrapeptide analog, N(alpha)-1-iminoethyl-Tyr-D-MetO-Phe-MebetaAla-OH (III), which has a potent analgesic activity after oral administration, various derivatives were synthesized to increase lipophilicity by esterification of the C-terminal carboxyl group and/or acylation of the phenolic hydroxyl group on Tyr1. Antinociceptive activity was evaluated after subcutaneous or oral administration using the mouse tail pressure test. As a result, increased antinociceptive activity after oral administration as well as an improved ED50(p.o.)/ED50(s.c.) ratio, which is an indicator of oral bioavailability, were found for some compounds. With regard to the improvement of bioavailability, derivatives with acylation of the phenolic hydroxyl group on Tyr1 showed better results than derivatives with esterification of the C-terminal carboxyl group. In particular, an ED50(p.o.)/ED50(s.c.) ratio equivalent to that of morphine was found for an acetylated derivative, N(alpha)-1-iminoethyl-Tyr(COMe)-D-MetO-Phe-MebetaAla-OH (7a), as well as for a methoxycarbonylated derivative, N(alpha)-1-iminoethyl-Tyr(CO2Me)-D-MetO-Phe-MebetaAla-OH (7l).