IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response

Immunization with serological identification of Ags by recombinant expression cloning (SEREX)-defined self-Ags leads to generation/activation of CD4+ CD25+ regulatory T cells with suppressive activities and enhanced expression of Foxp3. This is associated with increased susceptibility to pulmonary metastasis following challenge with syngeneic tumor cells and enhanced development of 3-methylcholanthrene-induced primary tumors. In contrast, coimmunization with the same SEREX-defined self-Ags mixed with a CTL epitope results in augmented CTL activity and heightened resistance to pulmonary metastasis, both of which depend on CD4+ Th cells. These active regulatory T cells and Th cells were derived from two distinct CD4+ T cell subsets, CD4+ CD25+ T cells and CD4+ CD25- T cells, respectively. In the present study, IFN-gamma was found to abrogate the generation/activation of CD4+ CD25+ regulatory T cells by immunization with SEREX-defined self-Ag. CD4+ CD25+ T cells from these IFN-gamma-treated mice failed to exhibit immunosuppressive activity as measured by 1) increased number of pulmonary metastasis, 2) enhanced development of 3-methylcholanthrene-induced primary tumors, 3) suppression of peptide-specific T cell proliferation, and 4) enhanced expression of Foxp3. The important role of IFN-gamma produced by CD8+ T cells was shown in experiments demonstrating that CD4+ CD25+ T cells cotransferred with CD8+ T cells from IFN-gamma(-/-) mice, but not from wild-type BALB/c mice, became immunosuppressive and enhanced pulmonary metastasis when recipient animals were subsequently immunized with a SEREX-defined self-Ag and a CTL epitope. These findings support the idea that IFN-gamma regulates the generation/activation of CD4+ CD25+ regulatory T cells.     

Cutting edge: Lipopolysaccharide induces IL-10-producing regulatory CD4+ T cells that suppress the CD8+ T cell response

TLR ligands are potent activators of dendritic cells and therefore function as adjuvants for the induction of immune responses. We analyzed the capacity of TLR ligands to enhance CD8+ T cell responses toward soluble protein Ag. Immunization with OVA together with LPS or poly(I:C) elicited weak CD8+ T cell responses in wild-type C57BL/6 mice. Surprisingly, these responses were greatly increased in mice lacking CD4+ T cells indicating the induction of regulatory CD4+ T cells. In vivo, neutralization of IL-10 completely restored CD8+ T cell responses in wild-type mice and OVA-specific IL-10 producing CD4+ T cells were detected after immunization with OVA plus LPS. Our study shows that TLR ligands not only activate the immune system but simultaneously induce Ag specific, IL-10-producing regulatory Tr1 cells that strongly suppress CD8+ T cell responses. In this way, excessive activation of the immune system may be prevented.     

CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127^lowPD-1^highTIM-3^high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.     

Naturally arising CD25+CD4+ regulatory T cells in maintaining immunologic self-tolerance and preventing autoimmune disease

A large body of evidence indicates that T cell-mediated dominant suppression of self-reactive T cells is indispensable for maintaining immunologic unresponsiveness to self-constituents (i.e., self-tolerance) and preventing autoimmune disease. CD25+CD4+ regulatory T cells naturally present in normal animals, in particular, engage in this function, as their reduction or functional abnormality leads to the development of autoimmune disease in otherwise normal animals. They are at least in part produced by the normal thymus as a functionally mature and distinct subpopulation of T cells. Recent studies have demonstrated that CD25+CD4+ regulatory T cells control not only autoimmune reactions but also other immune responses, including tumor immunity, transplantation tolerance and microbial infection. Thus, this unique population of regulatory T cells can be exploited to control pathological as well as physiological immune responses.     

Human CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells

Germline encoded pattern recognition receptors, such as TLRs, provide a critical link between the innate and adaptive immune systems. There is also evidence to suggest that pathogen-associated molecular patterns may have the capacity to modulate immune responses via direct effects on CD4+ T cells. Given the key role of both CD4+CD25+ T regulatory (Treg) cells and the TLR5 ligand flagellin in regulating mucosal immune responses, we investigated whether TLR5 may directly influence T cell function. We found that both human CD4+CD25+ Treg and CD4+CD25- T cells express TLR5 at levels comparable to those on monocytes and dendritic cells. Costimulation of effector T cells with anti-CD3 and flagellin resulted in enhanced proliferation and production of IL-2, at levels equivalent to those achieved by costimulation with CD28. In contrast, costimulation with flagellin did not break the hyporesponsiveness of CD4+CD25+ Treg cells, but rather, potently increased their suppressive capacity and enhanced expression of FOXP3. These observations suggest that, in addition to their APC-mediated indirect effects, TLR ligands have the capacity to directly regulate T cell responses and modulate the suppressive activity of Treg cells.     

High accumulation of T regulatory cells prevents the activation of immune responses in aged animals

In our previous in vivo study we demonstrated that young BALB/c mice effectively rejected the BM-185 tumor cells expressing enhanced GFP (EGFP) as a surrogate tumor Ag. In contrast, old BALB/c mice succumbed to the BM-185-EGFP tumors, indicating that there is a deficiency in old animals preventing the rejection of immunogenic tumors. There is cumulative evidence indicating that regulatory T (T(reg)) cells control the activation of primary and memory T cell responses. However, very little is known about whether there is a relation between T(regs) and the lack of immune responses in the aged. We evaluated young and aged animals, and our results demonstrated that there are significantly more CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ T(regs) in the spleen and lymph nodes of old animals when compared with the young. Depletion of CD25+ cells with anti-CD25 mAb induces the rejection of BM-185-EGFP cells, restores antitumor T cell cytotoxic activity, and results in the generation of a protective memory response against the BM-185 wild-type tumors in old mice. Furthermore, vaccination with CpG-oligodeoxynucleotide decreases the number of T(reg) cells in old animals to the same levels as young mice, restoring the primary and memory antitumor immune responses against BM-185-EGFP tumors. Taken together, these results indicate that there is a direct correlation between the expansion of T(reg) cells and immune deficiency in the old, and that depletion of these cells might be critical for restoring immune responses in aged animals.     

Single administration of low dose cyclophosphamide augments the antitumor effect of dendritic cell vaccine

Single administration of low dose cyclophosphamide (CTX) was previously reported to enhance the antitumor efficacy of immunotherapies. To investigate the possible mechanisms for this effect, we examined whether a single administration of low dose CTX could augment the immunogenicity of dendritic cell (DC) vaccines. Fifty milligrams per kilogram body weight dose of CTX was administrated intraperitoneally to mice after B16 melanoma or C26 colon carcinoma tumor models were established, DC vaccine generated from mouse bone marrow and pulsed with B16 or C26 tumor cells lysates were vaccinated 4 days later. CTX treatment potentiated the antitumor effects of the DC vaccine, and increased the proportion of IFN-γ secreting lymphocytes in spleens. Furthermore, a significantly reduced proportion of CD4+CD25+FoxP3+ regulatory T (T_reg) cells was detected by flow cytometry in spleen lymphocytes from tumor-bearing mice treated with CTX. Thus, a single administration of low dose CTX could augment antitumor immune responses of DC vaccine by reducing the proportion of CD4+CD25+FoxP3+ T_reg cells in tumor-bearing mice. Our results suggested a possible mechanism of CTX-induced immunopotentiation and provided a strategy of immunotherapy combining a low dose CTX with DC vaccine. J.-Y. Liu and Y. Wu contributed equally to this work.     

Cutting Edge: Anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells

CD4+CD25+ T regulatory (T(R)) cells are an important regulatory component of the adaptive immune system that limit autoreactive T cell responses in various models of autoimmunity. This knowledge was generated by previous studies from our lab and others using T(R) cell supplementation and depletion. Contrary to dogma, we report here that injection of anti-CD25 mAb results in the functional inactivation, not depletion, of T(R) cells, resulting in exacerbated autoimmune disease. Supporting this, mice receiving anti-CD25 mAb treatment display significantly lower numbers of CD4+CD25+ T cells but no change in the number of CD4+FoxP3+ T(R) cells. In addition, anti-CD25 mAb treatment fails to both reduce the number of Thy1.1+ congenic CD4+CD25+ T(R) cells or alter levels of CD25 mRNA expression in treatment recipients. Taken together, these findings have far-reaching implications for the interpretation of all previous studies forming conclusions about CD4+CD25+ T(R) cell depletion in vivo.     

Suppression of antitumor immunity by IL-10 and TGF-beta-producing T cells infiltrating the growing tumor: influence of tumor environment on the induction of CD4+ and CD8+ regulatory T cells

We examined the hypothesis that a failure of the immune system to eradicate tumors is due to the immunosuppressive environment created by the growing tumor, which is influenced by the site of tumor growth. We demonstrated that T cell responses to a bystander Ag in mice were suppressed by a growing CT26 tumor. T cells purified from the growing tumor expressed mRNA for IL-10, TGF-beta, and Foxp3. Intracellular cytokine staining revealed a high frequency of IL-10-secreting macrophages, dendritic cells, and CD4+ and CD8+ T cells infiltrating the tumor. In contrast, T cell IFN-gamma production was weak and CD8+ CTL responses were undetectable in mice with CT26 lung metastases and weak and transient following s.c. injection of CT26 cells, but were enhanced in the presence of anti-IL-10 and anti-TGF-beta. Consistent with this, removal of CD8+ T cells abrogated CTL responses and promoted progression of the s.c. tumor. However, in the lung model, depletion of CD8+ T cells significantly reduced the tumor burden. Furthermore, depletion of CD4+ or CD25+ T cells in vivo reduced tumor burden in s.c. and lung models, and this was associated with significantly enhanced IFN-gamma production by CD8+ T cells. These findings suggest that tumor growth facilitates the induction or recruitment of CD4+ regulatory T cells that secrete IL-10 and TGF-beta and suppress effector CD8+ T cell responses. However, CD8+ T regulatory cells expressing IL-10 and TGF-beta are also recruited or activated by the immunosuppressive environment of the lung, where they may suppress the induction of antitumor immunity.     

Cancer Immunotherapy Employing an Innovative Strategy to Enhance CD4+ T Cell Help in the Tumor Microenvironment

Chemotherapy and/or radiation therapy are widely used as cancer treatments, but the antitumor effects they produce can be enhanced when combined with immunotherapies. Chemotherapy kills tumor cells, but it also releases tumor antigen and allows the cross-presentation of the tumor antigen to trigger antigen-specific cell-mediated immune responses. Promoting CD4+ T helper cell immune responses can be used to enhance the cross-presentation of the tumor antigen following chemotherapy. The pan HLA-DR binding epitope (PADRE peptide) is capable of generating antigen-specific CD4+ T cells that bind various MHC class II molecules with high affinity and has been widely used in conjunction with vaccines to improve their potency by enhancing CD4+ T cell responses. Here, we investigated whether intratumoral injection of PADRE and the adjuvant CpG into HPV16 E7-expressing TC-1 tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and PADRE was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and PADRE also enhanced the generation of PADRE-specific CD4+ T cells and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci. The treatment regimen presented here represents a universal approach to cancer control.     

Suppression of CD4+ T lymphocyte effector functions by CD4+CD25+ cells in vivo

CD4+CD25+ regulatory T cells have been extensively studied during the last decade, but how these cells exert their regulatory function on pathogenic effector T cells remains to be elucidated. Naive CD4+ T cells transferred into T cell-deficient mice strongly expand and rapidly induce inflammatory bowel disease (IBD). Onset of this inflammatory disorder depends on IFN-gamma production by expanding CD4+ T cells. Coinjection of CD4+CD25+ regulatory T cells protects recipient mice from IBD. In this study, we show that CD4+CD25+ regulatory T cells do not affect the initial activation/proliferation of injected naive T cells as well as their differentiation into Th1 effectors. Moreover, naive T cells injected together with CD4+CD25+ regulatory T cells into lymphopenic hosts are still able to respond to stimuli in vitro when regulatory T cells are removed. In these conditions, they produce as much IFN-gamma as before injection or when injected alone. Finally, when purified, they are able to induce IBD upon reinjection into lymphopenic hosts. Thus, prevention of IBD by CD4+CD25+ regulatory T cells is not due to deletion of pathogenic T cells, induction of a non reactive state (anergy) among pathogenic effector T cells, or preferential induction of Th2 effectors rather than Th1 effectors; rather, it results from suppression of T lymphocyte effector functions, leading to regulated responses to self.     

CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4(+) TCRBV8(+) T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-gamma secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4(+) T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4(+)CD25(+) splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4(+)CD25(-) T regulatory cells prevented SEB-induced death independently of CD4(+)CD25(+) T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25(+) cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4(+)CD25(+) T regulatory cells are required for the induction of SAg-induced tolerance, CD4(+)CD25(-) T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.     

Three different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection

Low-frequency CTL and low-titer IgM responses against tumor-associated Ag MUC1 are present in cancer patients but do not prevent cancer growth. Boosting MUC1-specific immunity with vaccines, especially effector mechanisms responsible for tumor rejection, is an important goal. We studied immunogenicity, tumor rejection potential, and safety of three vaccines: 1) MUC1 peptide admixed with murine GM-CSF as an adjuvant; 2) MUC1 peptide admixed with adjuvant SB-AS2; and 3) MUC1 peptide-pulsed dendritic cells (DC). We examined the qualitative and quantitative differences in humoral and T cell-mediated MUC1-specific immunity elicited in human MUC1-transgenic (Tg) mice compared with wild-type (WT) mice. Adjuvant-based vaccines induced MUC1-specific Abs but failed to stimulate MUC1-specific T cells. MUC1 peptide with GM-CSF induced IgG1 and IgG2b in WT mice but only IgM in MUC1-Tg mice. MUC1 peptide with SB-AS2 induced high-titer IgG1, IgG2b, and IgG3 Abs in both WT and MUC1-Tg mice. Induction of IgG responses was T cell independent and did not have any effect on tumor growth. MUC1 peptide-loaded DC induced only T cell immunity. If injected together with soluble peptide, the DC vaccine also triggered Ab production. Importantly, the DC vaccine elicited tumor rejection responses in both WT and MUC1-Tg mice. These responses correlated with the induction of MUC1-specific CD4+ and CD8+ T cells in WT mice, but only CD8(+) T cells in MUC1-Tg mice. Even though MUC1-specific CD4+ T cell tolerance was not broken, the capacity of MUC1-Tg mice to reject tumor was not compromised.     

Co-administration of GP96 and Her2/neu DNA vaccine in a Her2 breast cancer model

Heat-shock proteins have biochemical and immunological roles in chaperoning/signaling and activation of innate and adaptive immune responses, respectively. Their effect on the immune response is due to a phenomenon known as cross-priming of antigen, in which exogenous antigens are presented via MHC class I by antigen presenting cells. GP96 exerts adjuvant activity with some viral and bacterial antigens when applied in the form of a DNA vaccine. In this study, animals with Her2-expressing tumors were vaccinated by co-administration of GP96+ Her2/neu DNA vaccines. Analyses of the immune response, 2 weeks after the last immunization revealed decreased CD4+ CD25+ Foxp3+ naturally occurring regulatory T cells (Tregs) at the tumor site and increased IFN-γ/IL-4 level. Nevertheless, the graph of tumor size demonstrated a bi-phasic pattern in which partial control of tumor progression initially occurred, but finally its effectiveness was inversely affected by tumor size.     

In vitro and in vivo down-regulation of regulatory T cell activity with a peptide inhibitor of TGF-beta1

Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-beta1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-beta1 and TGF-beta2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo. In vitro studies demonstrate that P17 inhibits murine and human Treg-induced unresponsiveness of effector T cells to anti-CD3 stimulation, in an MLR or to a specific Ag. Moreover, administration of P17 to mice immunized with peptide vaccines containing tumor or viral Ags enhanced anti-vaccine immune responses and improved protective immunogenicity against tumor growth or viral infection or replication. When CD4+ T cells purified from OT-II transgenic mice were transferred into C57BL/6 mice bearing s.c. EG.7-OVA tumors, administration of P17 improved their proliferation, reduced the number of CD4+Foxp3+ T cells, and inhibited tumor growth. Also, P17 prevented development of immunotolerance induced by oral administration of OVA by genetically modified Lactococcus lactis in DO11.10 transgenic mice sensitized by s.c. injection of OVA. These findings demonstrate that peptide inhibitors of TGF-beta may be a valuable tool to enhance vaccination efficacy and to break tolerance against pathogens or tumor Ags.