Activation of the TLR signaling pathway in CD8+ T cells counteracts liver endothelial cell-induced T cell tolerance

Abstract Acquired multidrug resistance (MDR) remains a major challenge in the treatment of cancer with chemotherapeutic drugs. It can be mediated by the upregulated expression of different proteins within the tumor cell membrane. Here we used murine MDR-1 as a target-antigen for the immunotherapy of cancer. We successfully demonstrated that peripheral T cell-tolerance can be broken by oral administration of a DNA vaccine encoding MDR-1 and carried by attenuated Salmonella typhimurium to secondary lymphoid organs. Thus, mice, immunized orally three times at 2-week intervals and challenged two weeks thereafter with either MDR-1 expressing CT-26 colon carcinoma cells or MDR-1 expressing Lewis lung carcinoma cells, revealed a significant increase in lifespan. This was evident, when compared to animals vaccinated with the empty control vaccine or to animals challenged with the maternal cell lines lacking overexpression of MDR-1. The immune response induced was antigen specific and CD8+ T cell-mediated. The presence of the target antigen led to upregulation of activation markers on CD8+ T cells and resulted in a strong cytotoxic T cell response as well as lysis of tumor target cells in vitro. We furthermore established the vaccine to be an effective treatment for established multidrug resistant tumor metastases resulting in a significantly increased lifespan of experimental animals. Absence of CD8+ T cells due to in vivo depletion led to abrogation of effectiveness. Taken together our results demonstrate, that T cell tolerance against the MDR-1 self antigen can be broken. It is anticipated that the combination of such an approach with chemotherapy could lead to more effective treatments of cancer.

Download Full-text

CD8+ T Cell Tolerance to a Tumor-associated Antigen Is Maintained at the Level of Expansion Rather than Effector Function

Journal of Experimental Medicine ◽

10.1084/jem.20011063 ◽

2002 ◽

Vol 195 (11) ◽

pp. 1407-1418 ◽

Cited By ~ 81

Author(s):

Claes Öhlén ◽

Michael Kalos ◽

Laurence E. Cheng ◽

Aaron C. Shur ◽

Doley J. Hong ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Tumor Antigen ◽

Interleukin 2 ◽

Cd8 T Cell ◽

High Avidity ◽

T Cell Tolerance ◽

Cell Tolerance

CD8+ T cell tolerance to self-proteins prevents autoimmunity but represents an obstacle to generating T cell responses to tumor-associated antigens. We have made a T cell receptor (TCR) transgenic mouse specific for a tumor antigen and crossed TCR-TG mice to transgenic mice expressing the tumor antigen in hepatocytes (gag-TG). TCRxgag mice showed no signs of autoimmunity despite persistence of high avidity transgenic CD8+ T cells in the periphery. Peripheral CD8+ T cells expressed phenotypic markers consistent with antigen encounter in vivo and had upregulated the antiapoptotic molecule Bcl-2. TCRxgag cells failed to proliferate in response to antigen but demonstrated cytolytic activity and the ability to produce interferon γ. This split tolerance was accompanied by inhibition of Ca2+ flux, ERK1/2, and Jun kinasephosphorylation, and a block in both interleukin 2 production and response to exogenous interleukin 2. The data suggest that proliferation and expression of specific effector functions characteristic of reactive cells are not necessarily linked in CD8+ T cell tolerance.

Download Full-text

Analysis of the Fate and Function of Antigen-Specific CD8 T Cells during B Cell Lymphoma Progression.

Blood ◽

10.1182/blood.v104.11.80.80 ◽

2004 ◽

Vol 104 (11) ◽

pp. 80-80 ◽

Cited By ~ 3

Author(s):

Jason Brayer ◽

Fengdong Cheng ◽

Pedro Horna ◽

Ildefonso Suarez ◽

Hongwei Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

B Cell Lymphoma ◽

Cd8 T Cell ◽

T Cell Tolerance ◽

Cell Tolerance ◽

Stat3 Signaling ◽

And Function

Abstract There is now overwhelming evidence that tumor-induced antigen (Ag)-specific T cell tolerance represents a critical problem in tumor immunology. Early studies of CD8 T cell tolerance equated peripheral tolerance with either ignorance or clonal deletion, although more recent evidence has suggested that this may be only partly accurate. While murine modeling outwardly supports the contention that high-affinity tumor-specific CD8 T cell responses are centrally deleted, cognate CD8 T cells displaying an Ag-experienced phenotype can nonetheless be detected in regional draining lymph nodes (dLN) or in non-lymphoid sites where the Ag is present. However, these CD8 T cells are typically deficient in one or more effector functions, including cytokine production, cytotoxicity, or proliferative capacity. To better define the state of Ag-specific CD8 T cell responsiveness in the face of progressive tumor, we adoptively transferred hemagglutinin (HA) Ag-specific Clone 4 (CLN4) CD8 T cells into animals bearing a genetically modified B cell lymphoma expressing HA as a model tumor antigen (A20HA). Analysis of the fate and function of these transferred antigen-specific CD8 T cells revealed that they encountered antigen in vivo, were capable of mounting an initial response to A20HA but this response was not sustained. Indeed, while a prominent CTL activation was observed in the spleen and draining lymph nodes of tumor bearing mice within 14 days of T cell transfer, responses (HA-specific proliferation, IFN-γ production and cytotoxicity) began to wane by day 21 after T cell transfer, and in particular their ability to produce IFN-γ. A similar pattern of transient activation followed by loss of CD8 T cell function has been also observed in an in vivo model of high-dose peptide induced antigen-specific CD8 T cell tolerance. Given our recent demonstration that the disruption of Stat3 signaling in APCs overcomes CD4 T cell tolerance we determined next whether Stat3 deficient APCs may be inherently better at cross-presenting tumor-Ags and elicit therefore a more productive and sustained CD8 T cell response. In an in vitro system in which tumor cells expressing a model tumor antigen (EL4mOVA) were cultured with APCs genetically devoid of Stat3 signaling and anti-OVA CD8 T-cells (OT-I), we found that these T cells displayed an enhanced function relative to antigen-specific CD8 T-cells that encountered antigen on APCs with an intact Stat3 signaling. Currently, we are investigating whether CD8 T-cell tolerance to tumor antigens occurred -or not- in tumor bearing mice with a genetic disruption of Stat3 signaling in APCs. Furthermore, given the emerging role of other members of the STAT family in regulation of APC function, we are exploring whether targeted disruption of Stat1, 4 and 6 can alter the ability of the CD8 T-cell to sustain a protective response or, more importantly to recover function once tolerance is induced.

Download Full-text

Disruption of STAT3 Signaling in Antigen Presenting Cells Represents a Novel Strategy To Overcome Tumor-Induced Antigen-Specific CD4+ T-Cell Tolerance.

Blood ◽

10.1182/blood.v104.11.109.109 ◽

2004 ◽

Vol 104 (11) ◽

pp. 109-109

Author(s):

Fengdong Cheng ◽

Hongwei Wang ◽

Alex G. Cuenca ◽

Pedro Horna ◽

Lldefonso Suarez ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Signaling Pathway ◽

Tumor Antigen ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

T Cell Tolerance ◽

Cell Tolerance ◽

Tumor Bearing ◽

Stat3 Signaling

Abstract Tumor antigen-specific T-cell tolerance imposes a significant barrier to the development of effective therapeutic cancer vaccines. Bone marrow-derived antigen presenting cells (APCs) are critical in the induction of this unresponsive state. Recently, we have identified STAT3 signaling in APCs as an important regulatory pathway that determines the functional outcome of antigen-specific CD4+ T-cells in response to cognate antigen. Indeed, while disruption of this signaling pathway in APCs led to effective T cell priming, enhanced STAT3 activity resulted in the induction of T cell unresponsiveness1. Given the above results, we explored in this study whether disruption of STAT3 signaling in APCs may preserve the responsiveness of antigen-specific CD4+ T-cells during the growth of a tumor that induces antigen specific T-cell tolerance. First, mice with a genetic disruption of Stat3 in macrophages, neutrophils and a sub-population of myeloid DCs (LysMcre/Stat3flox/− mice) or control C57BL/6 mice were given subcutaneously 1x106 B16 melanoma tumor cells engineered to express Ovalbumin as a model tumor antigen (B16-OVA). Four days later, naive CD4+ T-cells (1x106) specific for a MHC class II-restricted epitope of Ovalbumin (OT-II cells) were adoptively transferred intravenously into tumor bearing mice as well as into tumor-free controls. Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses to in vitro re-stimulation with OVA-peptide were evaluated. As expected, antigen-specific T cells re-isolated from tumor-bearing C57BL/6 mice were fully tolerant (lack of HA-specific proliferation and cytokine production). In sharp contrast, anti-OVA CD4+ T-cells isolated from tumor bearing LysMcre/Stat3flox/− mice remained fully functional as determined by their capacity to proliferate and produce IL-2 and IFN-gamma in response to cognate OVA-peptide. The demonstration that tumor-induced antigen-specific CD4+ T-cell tolerance occurs in mice with an intact STAT3 signaling in APCs, but not in mice with genetic disruption of this signaling pathway, led us next to evaluate the efficacy of pharmacologic inhibitors of STAT3 in preventing and or overcoming tumor-induced T-cell tolerance. In vivo treatment of tumor bearing mice with Tyrphostin AG490 (0.5 mg/ i.p. /twice a day x 5 days), a well-known inhibitor of STAT3 signaling, also resulted in preservation of the responsiveness of tumor-antigen specific CD4+ T-cells. Furthermore, in vitro treatment of APCs with this compound led to effective priming of naive antigen-specific T cells and breaking of antigen-specific T-cell anergy. More recently, we have evaluated the efficacy of a novel STAT3 inhibitor, compound 295558, which efficiently inhibits the DNA-binding activity of STAT3. Treatment of DCs or macrophages with this specific inhibitor led to the generation of inflammatory APCs capable of restoring the responsiveness of tolerized CD4+ T-cells isolated from tumor bearing mice. Taken together, our findings establish a critical role for STAT3 signaling in the induction of tolerance to tumor antigens in vivo. Inhibition of this signaling pathway in APCs provides a novel molecular target to overcome the remarkable barrier that tolerance to tumor antigens imposes to cancer vaccination strategies.

Download Full-text

FoxP3+ regulatory T cells essentially contribute to peripheral CD8+ T-cell tolerance induced by steady-state dendritic cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0910620107 ◽

2009 ◽

Vol 107 (1) ◽

pp. 199-203 ◽

Cited By ~ 58

Author(s):

A. Schildknecht ◽

S. Brauer ◽

C. Brenner ◽

K. Lahl ◽

H. Schild ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Steady State ◽

T Cell ◽

Regulatory T Cells ◽

Cd8 T Cell ◽

T Cell Tolerance ◽

Cell Tolerance

Download Full-text

Mechanisms of acquired tolerance induced by adoptive transfer of ALLOMHC peptide activated T cells: migration of effector T cells to the thymus to induce T cell tolerance

Journal of the American College of Surgeons ◽

10.1016/s1072-7515(00)00525-1 ◽

2000 ◽

Vol 191 (4) ◽

pp. S49-S50

Author(s):

R Gopinathan ◽

H.A DePaz ◽

O.O Oluwole ◽

A Ali ◽

R.A Fawwaz ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Transfer ◽

Effector T Cells ◽

T Cell Tolerance ◽

Cell Tolerance ◽

Activated T Cells

Download Full-text

Regulatory T Cells Are Not Required for Prevention of RBC-Specific Autoantibody Generation

Blood ◽

10.1182/blood.v124.21.568.568 ◽

2014 ◽

Vol 124 (21) ◽

pp. 568-568

Author(s):

Krystalyn E. Hudson ◽

James C. Zimring

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cd4 T Cells ◽

T Cell Tolerance ◽

Cell Tolerance ◽

Tolerance Mechanisms ◽

Antibody Treatment ◽

Isotype Control

Introduction: Loss of humoral tolerance to red blood cell (RBC) antigens may lead to the generation of pathogenic autoantibodies and result in autoimmune hemolytic anemia (AIHA), a severe and potentially fatal disease. Failure of tolerance to RBC antigens occurs with considerable frequency (1-3 cases/1,000 adults) and prevalence of AIHA is as high as 30% in persons with compromised B and/or T cell tolerance mechanisms. However, RBC-specific tolerance mechanisms are poorly understood. To elucidate the immune tolerances to RBC autoantigens, we utilized HOD mice. The HOD mouse expresses an RBC-specific transgene consisting of hen egg lysozyme (HEL), ovalbumin (OVA), and Duffy. Using the HOD model, we previously demonstrated B cell tolerance to RBC-specific HOD antigen is incomplete; however, T cell tolerance is stringent. HOD mice have similar detectable frequencies of HOD-specific CD4+ T cells compared to B6 mice. Although present, autoreactive HOD-specific CD4+ T cells are non-functional. Circumventing T cell tolerance by adoptive transfer, HOD mice make high titer anti-HOD autoantibodies in vivo. Thus, despite the presence of autoreactive B cells, no HOD-reactive antibodies are detectable unless CD4+ T cells are given, indicating T cell tolerance is a stopgap to autoimmunity. Methods: Leukocytes from C57BL/6 (B6) and HOD mice were harvested and OVA-specific CD4+ T cell responses were assessed by tetramer-pulldown assays with pooled tetramers I-Ab-OVA 329-337/326-334. Isolated cells were stained for surface and intracellular markers and analyzed via flow cytometry. For in vivo analysis, mice were treated with 300ug anti-CD25 (clone PC-61) depleting antibody or isotype control; a subset of antibody-treated mice was immunized with OVA/CFA. Antibodies bound to HOD RBCs were determined by direct antibody test. Anti-HOD antibodies were quantified by indirect immunofluorescence using HOD RBCs as targets. Results: Tetramer pull-down assays revealed similar numbers of OVA-reactive CD4+ T cells from HOD and B6 mice (mean 56 and 40, respectively, p = 0.3). However, cell surface and intracellular marker staining demonstrated that HOD mice had higher numbers of OVA-tetramer reactive CD4+ T cells that express regulatory markers CD25 and FoxP3, and exhaustion marker PD1 as compared to control B6 mice. Inhibitory CTLA4 expression was not detectable on OVA-reactive CD4+ T cells from HOD or B6 mice. To test whether regulatory T cells were required for RBC-specific immune tolerance, HOD and B6 mice were treated with CD25 depleting antibody or isotype control antibody. Anti-CD25 antibody treated mice had a significant reduction of CD25+ cells 4 days post treatment (p < 0.001, 2 independent experiments). Similarly, there was a significant reduction in FoxP3+CD25+CD4+ T cells (Tregs) in anti-CD25 treated mice (p < 0.001), compared to isotype. Mice received weekly injections of anti-CD25 or isotype antibody to maintain depletion for one month. A subset of mice received an OVA/CFA immunization. Sustained CD25+ depletion did not result in anti-HOD autoantibody generation. Further, there was no change in the endogenous frequency of OVA-reactive CD4+ T cells between HOD and B6 mice, regardless of antibody treatment. Similarly, HOD mice treated with depletion (or isotype) antibody and immunized with OVA/CFA did not make detectable anti-HOD autoantibodies. Consistent with lack of detectable autoantibodies, no expansion of OVA-tetramer reactive CD4+ T cells was observed in HOD mice. In contrast, B6 mice (treated with anti-CD25 or isotype antibody) had a detectable expansion of OVA-specific CD4+ T cells as a result of immunization. Conclusions: The data demonstrate a phenotypic difference between the OVA-reactive CD4+ T cells from HOD and B6 mice, with an increase in number of Tregs detectable in HOD mice. Administration of anti-CD25 antibody significantly reduced the number of overall CD25+ cells and Tregs. Prolonged depletion of these cellular subsets did not elicit autoantibodies in HOD mice. Further, immunization of CD25 depleted mice with a strong immune stimulus (OVA/CFA, known to expand OVA-reactive T cells in B6 mice), did not induce anti-HOD autoantibodies nor did it expand OVA-specific autoreactive CD4+ T cells in HOD mice. Together, these data demonstrate that CD25+ cells are not required for the maintenance of RBC-specific T cell tolerance and suggest a role for other regulatory mechanisms. Disclosures No relevant conflicts of interest to declare.

Download Full-text

T cell tolerance to non-H-2-encoded stimulatory alloantigens is induced intrathymically but not prethymically.

Journal of Experimental Medicine ◽

10.1084/jem.158.2.365 ◽

1983 ◽

Vol 158 (2) ◽

pp. 365-377 ◽

Cited By ~ 16

Author(s):

P J Morrissey ◽

D Bradley ◽

S O Sharrow ◽

A Singer

Keyword(s):

T Cells ◽

Major Histocompatibility Complex ◽

T Cell ◽

Present Report ◽

T Cell Tolerance ◽

Cell Tolerance ◽

Major Histocompatibility ◽

Histocompatibility Complex ◽

Specific Tolerance

The present report has evaluated the differentiation compartment in which T cells are tolerized to non-major histocompatibility complex (MHC)-encoded minor lymphocyte-stimulating locus (MLS) alloantigens. It was observed that T cell precursors are not tolerized prethymically to MLS alloantigens but are tolerized intrathymically and postthymically to MLS alloantigens. The failure of prethymic T cells to be tolerized indicates either that T cell precursors are unable to be tolerized to MLS alloantigens or that cells in the prethymic compartment are unable to induce MLS-specific tolerance. In either case, these results demonstrate that the thymus is the initial site in which T cell tolerance to MLS alloantigen is induced. The present results also demonstrate a striking disparity in the reactivity of thymocytes to MHC and MLS alloantigens expressed in the extrathymic host through which their precursors had migrated. In the experimental mice constructed for these studies, intrathymic T cells were tolerant to the MHC alloantigens but were reactive to the MLS alloantigens expressed by the extrathymic host. This observation is consistent with the concept that T cell precursors may be tolerized to MHC alloantigens at an earlier point in their differentiation than they are tolerized to non-MHC-encoded MLS alloantigens.

Download Full-text

Self-antigen presentation by mouse B cells results in regulatory T-cell induction rather than anergy or clonal deletion

Blood ◽

10.1182/blood-2011-02-336115 ◽

2011 ◽

Vol 118 (4) ◽

pp. 984-991 ◽

Cited By ~ 29

Author(s):

Sara Morlacchi ◽

Cristiana Soldani ◽

Antonella Viola ◽

Adelaida Sarukhan

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Antigen Expression ◽

T Cell Tolerance ◽

Cell Tolerance ◽

Clonal Deletion ◽

Thymic Epithelium ◽

Self Antigen ◽

Self Antigens

Abstract Multiple mechanisms operate to ensure T-cell tolerance toward self-antigens. Three main processes have been described: clonal deletion, anergy, and deviation to CD4+ regulatory T cells (Tregs) that suppress autoreactive T cells that have escaped the first 2 mechanisms. Although it is accepted that dendritic cells (DCs) and B cells contribute in maintaining T-cell tolerance to self-antigens, their relative contribution and the processes involved under physiologic conditions remain only partially characterized. In this study, we used different transgenic mouse models to obtain chimeras where a neo self-antigen is expressed by thymic epithelium and/or by DCs or B cells. We found that expression of cognate ligand in the thymus enhances antigen-specific FoxP3+ cells independently of whether the self-antigen is expressed on thymic epithelium or only on DCs, but not on B cells. On the contrary, self-antigen expression by B cells was very efficient in inducing FoxP3+ cells in the periphery, whereas self-antigen expression by DC led mainly to deletion and anergy of antigen-specific FoxP3− cells. The results presented in this study underline the role of B cells in Treg induction and may have important implications in clinical protocols aimed at the peripheral expansion of Tregs in patients.

Download Full-text