Foxp3 Post-translational Modifications and Treg Suppressive Activity.

Regulatory T cells (Tregs) are engaged in maintaining immune homeostasis and preventing autoimmunity. Treg cells include thymic Treg cells and peripheral Treg cells, both of which can suppress the immune response via multiple distinct mechanisms. The differentiation, proliferation, suppressive function and survival of Treg cells are affected by distinct energy metabolic programs. Tissue-resident Treg cells hold unique features in comparison with the lymphoid organ Treg cells. Foxp3 transcription factor is a lineage master regulator for Treg cell development and suppressive activity. Accumulating evidence indicates that the activity of Foxp3 protein is modulated by various post-translational modifications (PTMs), including phosphorylation, O-GlcNAcylation, acetylation, ubiquitylation and methylation. These modifications affect multiple aspects of Foxp3 function. In this review, we define features of Treg cells and roles of Foxp3 in Treg biology, and summarize current research in PTMs of Foxp3 protein involved in modulating Treg function. This review also attempts to define Foxp3 dimer modifications relevant to mediating Foxp3 activity and Treg suppression. Understanding Foxp3 protein features and modulation mechanisms may help in the design of rational therapies for immune diseases and cancer.

PRMT5 Associates With the FOXP3 Homomer and When Disabled Enhances Targeted p185erbB2/neu Tumor Immunotherapy.

Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner.

Potential role of NK cells in the induction of immune responses: implications for NK cell-based immunotherapy for cancers and viral infections.

Natural killer (NK) cells recognize tumor cells and virus-infected cells and attack without being sensitized to antigens. The development of the antitumor/antivirus activities of NK cells is controlled by multiple mechanisms such as direct cytotoxic activity against target cells, antibody-dependent cell-mediated cytotoxicity, secretion of Th1-type cytokines, and interactions with dendritic cells. The development of these activities plays a significant role in both innate and adaptive immunities. Considering the recent progress made in elucidating the molecular and cellular biology of NK cells, we summarize the current situation and discuss future possibilities with regard to NK cell-based adoptive immunotherapy.

[Anti tumor activities of lentinan and micellapist in tumor-bearing mice].

Although Lentinan (LNT) is sold as a medicine, and Micellapist (MME) sold as a food supplement, both LNT and MME are beta-glucans isolated from the Shiitake mushroom (Lentinula edodes). These two substances have been thought to be the same component of Shiitake. In the present study, we evaluated anti tumor activities of LNT and MME in tumor-bearing mice (B10.D2 mice implanted with S908D2 tumor cells) and examined the mechanism of immunopotentiation of these substances. The tumor growth was significantly suppressed in the LNT-treated group. In ex vivo evaluation, the tumor cytotoxicity was significantly reduced by a treatment of splenocytes with anti-CD8 antibody in the LNT-treated group. Furthermore, the tumor cytotoxicity of the LNT-treated group was also significantly reduced by a treatment of splenocytes with anti-CD8 antibody and its complement and with an anti-CD4 and its complement in the effector phase and the induction phase, respectively. A significant prolongation of the survival of tumor-bearing mice as compared to the untreated control group was noted in the LNT-treated group. In the mice treated intraperitoneally with LNT, CD8-positive cells appeared to have suppressed tumor cell proliferation. CD4-positive cells appeared to be involved in this activity of CD8-positive cells. On the other hand, orally administered MME has exerted no clear cytotoxic effects.

Role of alpha4 integrin and its ligand VCAM-1 in the specific extravasation of a tumor-specific TH2 clone into tumor tissue that initiates its rejection.

The effectiveness of anticancer immunotherapeutic strategies involving the transfer of tumor-specific T cells depends on appropriate lymphocyte-endothelial cell interactions that facilitate the migration of lymphocytes into tumor. Here, we investigated the molecular mechanisms underlying the migration of the antigen-specific Th2 CD4(+) T-cell clone YS1093 into S1509a tumor tissue. YS1093 is specific for the S1509a tumor but does not recognize the S713a tumor. Transfer of YS1093 cells into mice bearing both S1509a and S713a tumors caused only the S1509a tumor to regress. This regression was markedly inhibited by pretreating YS1093 cells with an anti-alpha4 integrin MAb and administering an anti-VCAM-1 MAb at T-cell transfer. Since vascular endothelial cells in S1509a tumor tissues express VCAM-1 and the MHC class II (I-E(k)) molecule restricting YS1093 activity, labeled YS1093 cells migrated specifically into the S1509a tumor, and this migration was also blocked by the anti-TCRbeta F(ab')(2) and anti-I-E(k) MAbs. Furthermore, in vitro assays revealed that anti-CD3 MAb-mediated TCR cross-linkage initiated the binding of alpha4 integrin on YS1093 cells to VCAM-1. This adhesive activity was completely blocked by the anti-alpha4 integrin MAb. These results strongly suggest that i.v.-transferred YS1093 cells act in tumor regression by specifically recognizing their tumor antigen peptide in the context of I-E(k) on vascular endothelial cells in the S1509a tumor, which activates the binding of alpha4 integrin to VCAM-1 on the endothelial cells, facilitating YS1093 extravasation into the tumor. It is likely that this initial migration of specific CD4(+) T cells into tumor tissues promotes the subsequent infiltration into the tumor of other immunocytes that effect tumor destruction.

Monomethoxypolyethylene glycol-modified cardiac myosin treatment blocks the active and passive induction of experimental autoimmune myocarditis.

BACKGROUND: Injecting various protein antigens conjugated to monomethoxypolyethylene glycol (mPEG) results in antigen-specific tolerance to subsequent immunization. In the present study the ability of mPEG-modified cardiac myosin (CM) to block the development of experimental autoimmune myocarditis (EAM) induced by CM immunization or by the transfer of lymphocytes from CM-immunized donors was studied. METHODS AND RESULTS: A/J mice were injected with mPEG-CM before active or passive EAM induction. We examined the suppressive mechanism by the transfer of lymphocytes from mPEG-CM-treated mice into naïve mice. To ascertain the cells responsible for suppressing EAM induction, in vivo or in vitro depletion of CD4(+) or CD8(+) T cells was performed. mPEG-CM administered before active or passive EAM induction markedly suppressed the incidence and severity of EAM and reduced CM-specific antibody responses. When lymphocytes from mPEG-CM treated mice were transferred into naïve mice that were then immunized with CM, the suppressive effect was recapitulated. CONCLUSIONS: mPEG-CM treatment blocked the active and passive induction of EAM.

Experimental protection of mice against lethal Staphylococcus aureus infection by novel bacteriophage phi MR11.

The protective effects of bacteriophages were assessed against experimental Staphylococcus aureus infection in mice. Of the S. aureus phages isolated in the study, phi MR11 was representatively used for all testing, because its host range was the most broad and it carries no genes for known toxins or antibiotic resistance. Intraperitoneal injections (8 x 10(8) cells) of S. aureus, including methicillin-resistant bacteria, caused bacteremia and eventual death in mice. In contrast, subsequent intraperitoneal administration of purified phi MR11 (MOI > or = 0.1) suppressed S. aureus-induced lethality. This lifesaving effect coincided with the rapid appearance of phi MR11 in the circulation, which remained at substantial levels until the bacteria were eradicated. Inoculation with high-dose phi MR11 alone produced no adverse effects attributable to the phage. These results uphold the efficacy of phage therapy against pernicious S. aureus infections in humans and suggest that phi MR11 may be a potential prototype for gene-modified, advanced therapeutic S. aureus phages.

Molecular cloning and cytochemical analysis of exopolygalacturonase from carrot.

Exopolygalacturonase (exo-PGase, EC 3.2.1.67) attacks the non-reducing terminus of the polygalacturonic acid in pectic molecules, releasing galacturonic acid. We cloned the cDNA of exo-PGase purified from cell homogenates of suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells. The nucleotide sequence of the cDNA (1.4 kb) contains an open reading frame that encodes a 391-amino-acid polypeptide. Sequence homology research showed 97.9% identity to the glycoprotein EP4 obtained from cultured carrot cells and 49.3% identity to the ENOD8 gene product of alfalfa ( Medicago sativa). However, no significant similarity was found to known PGases. The Southern hybridization pattern indicated that this exo-PGase protein is a member of a small-sized gene family. Predominant expression of the exo-PGase gene was detected by in situ hybridization and immunohistochemistry in the root apical meristem and in the elongation region, but not in the root cap. A cross-immunoresponse with anti-exo-PGase also occurred in the root nodule meristem of alfalfa. These results suggest that this exo-PGase plays a role in the degradation of pectic molecules during root development.