Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.

Constitutive activation of the KRAS oncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes, KRAS acts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutant KRAS(G12V)gene in wild-type KRAS tumor cells led to regulatory T-cell (Treg) induction. We also demonstrate that mutant KRAS induces the secretion of IL10 and transforming growth factor-ß1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutant KRAS oncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.

CD8+ regulatory T cells are responsible for GAD-IgG gene-transferred tolerance induction in NOD mice.

Our previous studies demonstrated that lipopolysaccharide (LPS)-stimulated splenocytes, retrovirally transduced with a glutamate decarboxylate 65 (GAD) and immunoglobulin G (IgG) fusion construct, can protect non-obese diabetic (NOD) mice from diabetes by inducing GAD-specific tolerance, and also that there are increased numbers of CD4(+) regulatory T cells (Tregs) in GAD-IgG-treated NOD mice. However, little is known about the role of CD8(+) Tregs in GAD-IgG gene-transferred tolerance induction in NOD mice. Here, we found that GAD-IgG-transduced splenocytes induced an increase in the number of CD8(+) Foxp3(+) Tregs in vitro. Using a T-cell depletion assay, we found that, compared with undepleted groups, NOD recipients transfused with CD8(-) or CD8(-) CD25(-) GAD-IgG-transduced splenocytes showed a decrease in the percentage of CD8(+) Foxp3(+) T cells, a high incidence of diabetes, serious insulitis, GAD-specific hyperresponsiveness at both the cellular and humoral levels, and changes in cytokine expression. These results indicate that CD8(+) Tregs, which were induced in vitro by GAD-IgG-transduced splenocytes, were also responsible for GAD-IgG gene-transferred tolerance induction in NOD mice.

Gene delivery GAD 500 autoantigen by AAV serotype 1 prevented diabetes in NOD mice: transduction efficiency do not play important roles.

We previously found that adeno-associated viral vector serotype 2 (AAV-2) muscle gene delivery of GAD 500-585 autoantigen efficiently prevented autoimmune diabetes in NOD mice. Recent reports suggest that AAV vectors based on serotype 1 (AAV-1) transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2 to 1000-fold. To determine whether this increased efficacy of AAV-1 could result in increased therapeutic effects in mice, we constructed rAAV1/GAD 500-585 vectors and compared their effects in preventing autoimmune diabetes in NOD mice with those of rAAV2/GAD 500-585 after muscle injection. rAAV(1)/GAD(500-585) gene therapy prevented diabetes in NOD mice. However, although much higher level of GAD 500-585 expression was found in mice using AAV-1 as gene delivery vector than those using AAV-2, no increased efficiency of AAV-1 vectors were found in their capability to prevent autoimmune diabetes, as higher titers of rAAV1/GAD 500-585 virus (3x10(11)v.g./mouse) were needed to obtain therapeutic effects in NOD mice, a titer not different from that of AAV-2. Protection resulted from rAAV1/GAD 500-585 gene therapy were marked by enhanced Th2 immune response and up-regulated CD4+ Foxp3+T regulatory cells, which might actively suppress effector T cells in NOD mice. As here we found that the therapeutic effects of AAV1 were not positively correlated to it's transduction efficiency, our data suggested that the safety and other factors besides efficiency should be considered when use different AAV serotype to treat autoimmune disease.

The role of STAT3 in antigen-IgG inducing regulatory CD4(+)Foxp3(+)T cells.

Unraveling the events that control the suppressive function of regulatory T (Treg) cells is extremely important because it will enable investigators to manipulate these cells to inhibit or enhance their functions as necessary. One of the members of the Signal Transducer and Activators of Transcription (STATs) family, STAT3, has emerged as a negative regulator of inflammatory responses. Here, we study the role of STAT3 in Treg cell induction. We found that GAD-IgG-transduced splenocytes induce a CD4(+)Foxp3(+)Treg cell increase in NOD mice. In parallel with the Treg cell increase, an IL-6-STAT3 signal pathway is activated. When STAT3 activation is blocked, GAD-specific tolerance disappears, the percentage of Treg cells decreases and IL-10 secretion is reduced in the splenocytes of NOD mice recipients of GAD-IgG-transduced splenocytes. Our findings indicate that transcription factor STAT3 plays an important role in immune tolerance.

The combination of GM-CSF and IL-2 as local adjuvant shows synergy in enhancing peptide vaccines and provides long term tumor protection.

Many strategies have been used to enhance the peptide vaccine immune response and to establish therapeutic benefits. This includes the utilization of cytokines to improve antigen presentation or enhance T cell response. Here, we have tested the combination of GM-CSF and IL-2 as locally administered adjuvant to enhance the immune response to the HPV16 E7 peptide. Female C57BL/6 mice were immunized intradermally with a 9-mer HPV16 E7 peptide (aa: 49-57) alone, or in combination with GM-CSF, IL-2, or both cytokines. Specific immune responses were measured by ELISA and Chromium-Release Assays. Furthermore, therapeutic effects of these vaccines and long term tumor protection were assessed in mice bearing established tumors. We showed that GM-CSF and IL-2, when co-administered locally in an emulsion with peptide, exert a synergistic effect in enhancing the immune response to the antigen. This combination induced higher CTL and cytokine release responses and did not increase the T(reg) population. Therapeutic intervention with this synergistic combination led to a complete response of established tumors. Furthermore, this combination induced a memory response which protected mice against subsequent additional tumor challenge. We identified a new vaccine adjuvant, a local combination of GM-CSF and IL-2, which is synergistic in enhancing peptide specific immune response through local effect without increasing T(reg) cells. This immune response was found to be long lasting and protective in tumor bearing mice.

Combined prophylactic and therapeutic cancer vaccine: enhancing CTL responses to HPV16 E2 using a chimeric VLP in HLA-A2 mice.

We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions. Since T cell response to E2 is less defined, we first evaluated the strategies to enhancing CD8(+) T cell responses to HPV E7, using different combinations of immune-modulators with VLP-E7E2. Data showed that the CTL response to E7 could be significantly enhanced by coinjection of GM-CSF and anti-CD40 antibodies with chimeric VLP-E7E2 without adjuvant. However, using the same combination, a low level of CD8(+) T cell response to E2 was detected. To enhance the CD8+ T cell response to E2, we analyzed T cell epitopes from E2 sequence. A heterogenous prime-boost with chimeric VLP-E7E2 and E2 peptides was performed. The data showed that the priming with chimeric VLP-E7E2, followed by boosting with E2 peptides, gave a better CTL response than 2 immunizations with E2 peptides. The enhanced immunity is due to the increase of CD11c(+) and CD11c(+) CD40(+) double positive dendritic cells in mice that received immune-modulators, GM-CSF and anti-CD40. Furthermore, the level of anti-L1 antibodies remains similar in mice immunized with chimeric VLP with/without immune-modulators. Thus, the data suggested that the chimeric VLP-E7E2 has a therapeutic potential for the treatment of HPV-associated CINs and cancer without diminishing VLPs potential as a prophylactic vaccine by inducing anti-L1 antibodies against free virus.

Identification of H-2Db-specific CD8+ T-cell epitopes from mouse VEGFR2 that can inhibit angiogenesis and tumor growth.

Vascular endothelial growth factor receptor 2 (VEGFR2/KDR) plays a crucial role in tumor-associated angiogenesis and vascularization. It has been established that monoclonal antibodies against VEGFR2 can inhibit angiogenesis. In this study, two naturally processed CD8 T-cell epitopes (VILTNPISM and FSNSTNDILI) were identified from murine KDR. Cytotoxic T lymphocytes targeting endothelial cells could be directly monitored by KDR2 and KDR3 Elispots or major histocompatibility complex class I tetramer staining. Immunization with these two peptides effectively reduced angiogenesis and inhibited tumor growth in mouse models. Thus, vaccination with KDR peptides alone or in combination with other anti-angiogenesis agents may afford a novel immunotherapy for inhibition of tumor growth.

Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD(500-585).

Tolerance induction of autoreactive T cells against pancreatic beta cell-specific autoantigens such as glutamic acid decarboxylase 65 (GAD65) and insulin has been attempted as a method to prevent autoimmune diabetes. In this study, we investigate whether adenoassociated virus (AAV) gene delivery of multiple immunodominant epitopes expressing GAD(500-585) could induce potent immune tolerance and persistently suppress autoimmune diabetes in NOD mice. A single muscle injection of 7-wk-old female NOD mice with rAAV/GAD(500-585) (3 x 10(11) IU/mouse) quantitatively reduced pancreatic insulitis and efficiently prevented the development of overt type I diabetes. This prevention was marked by the inactivation of GAD(500-585)-responsive T lymphocytes, the enhanced GAD(500-585)-specific Th2 response (characterized by increased IL-4, IL-10 production, and decreased IFN-gamma production; especially elevated anti-GAD(500-585) IgG1 titer; and relatively unchanged anti-GAD(500-585) IgG2b titer), the increased secretion of TGF-beta, and the production of protective regulatory cells. Our studies also revealed that peptides 509-528, 570-585, and 554-546 in the region of GAD(500-585) played important roles in rAAV/GAD(500-585) immunization-induced immune tolerance. These data indicate that using AAV, a vector with advantage for therapeutic gene delivery, to transfer autoantigen peptide GAD(500-585), can induce immunological tolerance through active suppression of effector T cells and prevent type I diabetes in NOD mice.

Gene transfer of Ig-fusion proteins into B cells prevents and treats autoimmune diseases.

Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PLxSJL F(1) mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7-10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9-23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.