CD4+CD25+ T cells have been proposed as the principal regulators of both self-tolerance and transplantation tolerance. Although CD4+CD25+ T cells do have a suppressive role in transplantation tolerance, so do CD4+CD25− T cells, although 10-fold less potent. Abs to CTLA-4, CD25, IL-10, and IL-4 were unable to abrogate suppression mediated by tolerant spleen cells so excluding any of these molecules as critical agents of suppression. CD4+CD25+ T cells from naive mice can also prevent rejection despite the lack of any previous experience of donor alloantigens. However, this requires many more naive than tolerized cells to provide the same degree of suppression. This suggests that a capacity to regulate transplant rejection pre-exists in naive mice, and may be amplified in “tolerized” mice. Serial analysis of gene expression confirmed that cells sorted into CD4+CD25+ and CD4+CD25− populations were distinct in that they responded to TCR ligation with very different programs of gene expression. Further characterization of the differentially expressed genes may lead to the development of diagnostic tests to monitor the tolerant state.

Alloreactive CD8+ T cells may persist in animals made tolerant of transplanted tissues; their function is controlled through continuous censorship by regulatory CD4+ T cells. We sought to establish the stage at which such censorship operates. We found that monospecific CD8+ T cells introduced into tolerant animals responded to the tolerated tissue antigen as if they had received CD4+ T cell "help": they proliferated and accumulated normally. However, they did show compromised graft rejection, interferon-gamma production and cell-mediated cytotoxicity. These findings suggest that tolerance mediated by regulatory T cells acts by censoring immune effector functions rather than by limiting the induction of T cell responses.

While immunotherapy holds great promise for combating cancer, the limited efficacy due to an immunosuppressive tumor microenvironment and systemic toxicity hinder the broader application of cancer immunotherapy. Here, we report a combinatorial immunotherapy approach that uses a highly efficient and tumor-selective gene carrier to improve anticancer efficacy and circumvent the systemic toxicity. In this study, we engineered tumor-targeted lipid-dendrimer-calcium-phosphate (TT-LDCP) nanoparticles (NPs) with thymine-functionalized dendrimers that exhibit not only enhanced gene delivery capacity but also immune adjuvant properties by activating the stimulator of interferon genes (STING)–cGAS pathway. TT-LDCP NPs delivered siRNA against immune checkpoint ligand PD-L1 and immunostimulatory IL-2–encoding plasmid DNA to hepatocellular carcinoma (HCC), increased tumoral infiltration and activation of CD8+ T cells, augmented the efficacy of cancer vaccine immunotherapy, and suppressed HCC progression. Our work presents nanotechnology-enabled dual delivery of siRNA and plasmid DNA that selectively targets and reprograms the immunosuppressive tumor microenvironment to improve cancer immunotherapy.

To investigate the antigen specificity of regulatory T cells capable of preventing transplant rejection, we have developed two different strategies to achieve tolerance to fully mismatched skin grafts in euthymic mice. A combination of nondepleting Abs targeting CD4, CD8, and CD154 (CD40 ligand) induces dominant transplantation tolerance to fully mismatched skin allografts. Such tolerance is antigen-specific, mediated by regulatory T cells, and can be extended through linked suppression to naïve lymphocytes. The same protocol, when combined with allogeneic bone marrow, enables the development of mixed hematopoietic chimerism and deletional tolerance. Although we cannot exclude that some regulatory T cells may persist in chimeric mice, these cells are insufficient to mediate linked suppression. CD4 ؉ CD25 ؉ T cells, whether taken from naïve mice or from mice tolerized through either treatment protocol, were always able to prevent rejection of skin grafts by naïve CD4 ؉ T cells, and did so with no demonstrable specificity for the tolerizing donor antigens. Such data question whether CD4 ؉ CD25 ؉ regulatory T cells alone can account for the antigen specificity of dominant transplantation tolerance. N atural CD4 ϩ CD25 ϩ regulatory T cells are known to prevent autoimmunity and gut immunopathology (1-3). They are presumed to interact with endogenous antigen, but such antigens are not yet characterized. Adoptive transfer studies have also shown that CD4 ϩ CD25 ϩ T cells can prevent graft rejection (4). This finding has led to the claim that these same cells are responsible for dominant transplantation tolerance after therapeutic intervention. If so, we would expect CD4 ϩ CD25 ϩ T cells from animals demonstrating dominant tolerance to show evidence of priming and specificity for the tolerated antigen. By the same token, such cells should not be detectable in animals tolerized through deletional mechanisms, where dominant tolerance has been excluded. We have used an Ab mixture of nondepleting CD4, CD8, and CD154 (CD40-ligand) Abs to produce both forms of tolerance to MHC-mismatched transplants. Tolerance induced to skin grafts as the tolerogen exhibited features of dominant tolerance. Tolerance induced to the combination of skin and donor bone marrow (BM), was associated with mixed hematopoietic chimerism and showed no linked suppression, indicating deletion as the likely mechanism. CD4 ϩ CD25 ϩ T cells from any of the two groups of tolerized animals behaved no differently from CD4 ϩ CD25 ϩ T cells obtained from naïve animals in being able to prevent graft rejection by naïve CD4 ϩ T cells, yet lacking specificity for the donor alloantigens. We cannot therefore implicate CD4 ϩ CD25 ϩ T cells alone as responsible for the exquisite donor antigen specificity of dominant transplantation tolerance. It is possible that interactions with other regulatory T cells are required. MethodsMice. CBA͞Ca (CBA, H-2 k ), RAG1 Ϫ/Ϫ -CBA͞Ca (RAG1 Ϫ/Ϫ , H-2 k ), BALB͞c (H-2 d ), and C57BL͞10 (B10, H-2 b ) mice were bred and maintained in spec...

Modulation of the cellular response by the administration of probiotic bacteria may be an effective strategy for preventing or inhibiting tumour growth. We orally pre-inoculated mice with probiotics Lactobacillus acidophilus NCFM (La) for 14 d. Subcutaneous dorsal-flank tumours and segmental orthotopic colon cancers were implanted into mice using CT-26 murine colon adenocarcinoma cells. On day 28 after tumour initiation, the lamina propria of the colon, mesenteric lymph nodes (MLN) and spleen were harvested and purified for flow cytometry and mRNA analyses. We demonstrated that La pre-inoculation reduced tumour volume growth by 50·3 %, compared with untreated mice at 28 d after tumour implants (2465·5 (SEM 1290·4) v. 4950·9 (SEM 1689·3) mm 3 , P, 0·001). Inoculation with La reduced the severity of colonic carcinogenesis caused by CT-26 cells, such as level of colonic involvement and structural abnormality of epithelial/ crypt damage. Moreover, La enhanced apoptosis of CT-26 cells both in dorsal-flank tumour and segmental orthotopic colon cancer, and the mean counts of apoptotic body were higher in mice pre-inoculated with La (P,0·05) compared with untreated mice. La pre-inoculation down-regulated the CXCR4 mRNA expressions in the colon, MLN and extra-intestinal tissue, compared with untreated mice (P,0·05). In addition, La pre-inoculation reduced the mean fluorescence index of MHC class I (H-2Dd, -Kd and -Ld) in flow cytometry analysis. Taken together, these findings suggest that probiotics La may play a role in attenuating tumour growth during CT-26 cell carcinogenesis. The down-regulated expression of CXCR4 mRNA and MHC class I, as well as increasing apoptosis in tumour tissue, indicated that La may be associated with modulating the cellular response triggered by colon carcinogenesis. Key words: Probiotics: Lactobacillus acidophilus NCFM: CT-26 cells: Colon carcinogenesis: ApoptosisColon carcinoma is a leading cause of digestive system neoplasm. The colon cancer mortality rate is second only to that of lung cancer in men and breast cancer in women, and colon cancer rates have increased over the past 20-30 years (1) . Diet contributes to colon cancer risk. In fact, up to 75 % of cases are thought to be associated with diet (2) , indicating that a person can reduce his or her colon cancer risk simply via diet modification.Probiotics consist of a preparation of viable micro-organisms that alter the existing microflora of the intestine, thereby

Peripheral tolerance to allogeneic organ grafts can be induced in rodents by treating with non-depleting CD4 and CD8 monoclonal antibodies. This tolerance is maintained by CD4+ T cells with a potent capacity to induce tolerance in further cohorts of T cells (i.e. infectious tolerance). We have cloned CD4+ T-cell subsets against the male transplantation antigen in vitro and find, in contrast to Th1 or Th2 clones that elicit rejection, that there is a distinct population of CD4+ T cells that suppress rejection by adoptive transfer (here called Treg). In order to identify molecular markers associated with tolerance and gain insights into the mechanisms of action of Treg cells, we carried out serial analysis of gene expression. We identified genes overexpressed in Treg compared to Th1 and Th2 cultures and found that some of these correlated in vivo with CD4-induced transplantation tolerance rather than rejection. The genes overexpressed in Treg cultures and within tolerated skin grafts were primarily expressed by mast cells (e.g. tryptophan hydroxylase and FcepsilonR1alpha), suggesting that regulatory cell activity and this form of tolerance may be associated with a localised but non-destructive form of Th2-like activation and a recruitment of mast cells.

BackgroundInterleukin-12 (IL-12) has long been considered to be effective in triggering an anticancer immune response, however, the dosage has been limited by potential systemic immunotoxicity. Since focused ultrasound (FUS) has been confirmed to temporally and locally open the blood–brain barrier (BBB), the purpose of this study was to elucidate the possibility of combining FUS-induced BBB opening with IL-12 delivery to enhance the anticancer immunological response for glioma treatment.MethodsFUS energy combined with microbubble administration was delivered transcranially to open BBB, and C-6 glioma rats were used in this study. The efficacy in inducing BBB opening and the corresponding immunological response were primarily evaluated in normal animals. The anticancer immune-triggering chemokine, IL-12, was intraperitoneally administered during the treatment phase to evaluate the effect of immunological response on tumor progression. Glioma animals were sub-grouped to evaluate the effect of the immune response in suppressing glioma when IL-12 was combined with FUS-induced BBB opening. We performed flow cytometry to verify consequent immune cell population changes of peripheral/tissue lymphocytes as well as macrophages from the animals. Brain sections of sacrificed animals were also used for histological and immunohistochemical analysis. IL-12 level among experimental groups were measured via ELISA analysis. We also analyzed survival and followed tumor progression in vivo via T2-weighted magnetic resonance imaging.ResultsFUS-induced BBB opening had no obvious effect on the T lymphocytes population in normal animals, either in the brain or systemically. Yet, it triggered mild changes in the tumor-infiltrating lymphocyte (TIL) population, particularly in numbers of CD3+CD8+ cytotoxic T lymphocytes (CTLs) in the tumor region. IL-12 administration triggered a profound increase in all TIL populations, including CD3+CD4+ T helper cells (Th), CTL, and CD4+CD25+ regulatory T cells (Treg), but combined FUS-BBB opening with IL-12 administration produced the most significant IL-12 increase, CTL increase and CTL/Treg ratio increase, thus contributing to the most significant suppression of tumor progression and increased animal survival.ConclusionThis study provides evidence that FUS-BBB opening can enhance immune-modulating agent delivery to the brain, which improve the anticancer immune response in brain tumor treatment.

BackgroundHigh-intensity focused-ultrasound (HIFU) has been successfully employed for thermal ablation of tumors in clinical settings. Continuous- or pulsed-mode HIFU may also induce a host antitumor immune response, mainly through expansion of antigen-presenting cells in response to increased cellular debris and through increased macrophage activation/infiltration. Here we demonstrated that another form of focused ultrasound delivery, using low-pressure, pulsed-mode exposure in the presence of microbubbles (MBs), may also trigger an antitumor immunological response and inhibit tumor growth.MethodsA total of 280 tumor-bearing animals were subjected to sonographically-guided FUS. Implanted tumors were exposed to low-pressure FUS (0.6 to 1.4 MPa) with MBs to increase the permeability of tumor microvasculature.ResultsTumor progression was suppressed by both 0.6 and 1.4-MPa MB-enhanced FUS exposures. We observed a transient increase in infiltration of non-T regulatory (non-Treg) tumor infiltrating lymphocytes (TILs) and continual infiltration of CD8+ cytotoxic T-lymphocytes (CTL). The ratio of CD8+/Treg increased significantly and tumor growth was inhibited.ConclusionsOur findings suggest that low-pressure FUS exposure with MBs may constitute a useful tool for triggering an anticancer immune response, for potential cancer immunotherapy.