ABSTRACT
CD4+CD25+ regulatory T cells (Tregs) are critical for peripheral tolerance and prevention of autoimmunity. In vitro coculture studies have revealed that increased costimulation breaks Treg-mediated suppression in response to anti-CD3 or antigen. However, it was unclear whether loss of suppression arose from inactivation of Tregs or whether increased stimulation caused Th cells to escape suppression. We have investigated conditions that allow or override Treg-mediated suppression using DO11.10 TCR-transgenic T cells and chicken ovalbumin peptide 323-339-pulsed antigen-presenting cells. Treg suppression of Th proliferation is broken with potent stimulation, using activated spleen cells and high antigen dose, but is intact at low antigen dose. Costimulation with CD80 and CD86 expressed on activated dendritic cells was essential for Th cell escape from suppression at a high antigen dose. Potently stimulated Tregs were functional since they reduced levels of IL-2, IFN-gamma, IL-4 and Th CD25 expression in cocultures. Furthermore, Tregs responding to high antigen dose and activated splenocytes retained the ability to suppress proliferation, but only of Th cells responding to a sub-optimal dose of independent antigen. Together, our results demonstrate that under conditions of strong antigen-specific stimulation, Tregs remain functional, but Th cells escape Treg-mediated suppression.
Subject(s)
Antigen Presentation/immunology , Antigens/immunology , Dendritic Cells/immunology , Immune Tolerance/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes/immunology , Animals , Antigens, CD/physiology , Antigens, T-Independent/immunology , B7-1 Antigen/physiology , B7-2 Antigen , Cell Division , Coculture Techniques , Dose-Response Relationship, Immunologic , Humans , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-2/metabolism , Interleukin-4/metabolism , Lymphocyte Activation , Membrane Glycoproteins/physiology , Membrane Proteins/pharmacology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Ovalbumin/immunology , Peptide Fragments/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Interleukin-2/analysis , Recombinant Proteins/pharmacology , Specific Pathogen-Free Organisms , Spleen/cytology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes/metabolismABSTRACT
RasGRP1 is a guanine nucleotide exchange factor for Ras that is required for the efficient production of both CD4 and CD8 single-positive thymocytes. We found that RasGRP1 expression is rapidly up-regulated in double-negative thymocytes following pre-TCR ligation. Transgenic overexpression of RasGRP1 compensated for deficient pre-TCR signaling in vivo, enabling recombinase-activating gene 2(-/-) double-negative thymocytes to mature to the double-positive stage. RasGRP1 transgenic mice had a 4-fold increase in CD8 single-positive thymocytes, most of which had atypically low levels of CD3. The RasGRP1 transgene lowered the threshold of TCR signaling needed to initiate proliferation of single-positive thymocytes, with this effect being particularly evident among CD8 single-positive cells. In 3-day cultures, TCR stimulation via anti-CD3 caused a 10-fold increase in the ratio of CD8 to CD4 thymocytes among RasGRP1 transgenic vs nontransgenic thymocytes. These results demonstrate that in addition to driving the double-negative to double-positive transition, increased expression of RasGRP1 selectively increases CD8 single-positive thymocyte numbers and enhances their responsiveness to TCR signaling.
Subject(s)
Adjuvants, Immunologic/physiology , CD8 Antigens/biosynthesis , CD8-Positive T-Lymphocytes/cytology , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Guanine Nucleotide Exchange Factors , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thymus Gland/cytology , Adjuvants, Immunologic/biosynthesis , Adjuvants, Immunologic/genetics , Amino Acid Sequence , Animals , CD8 Antigens/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Division/genetics , Cell Division/immunology , Crosses, Genetic , DNA-Binding Proteins/physiology , Humans , Lymphocyte Count , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Nuclear Proteins , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/physiology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes/metabolism , Thymus Gland/immunology , Thymus Gland/metabolism , Transgenes/immunology , Up-Regulation/genetics , Up-Regulation/immunology , ras Proteins/physiologyABSTRACT
Programmed death-1 ligand 2 (PD-L2) is a ligand for programmed death-1 (PD-1), a receptor that plays an inhibitory role in T cell activation. Since previous studies have shown up-regulation of PD-L2 expression by Th2 cytokines, and asthma is driven by a Th2 response, we hypothesized that PD-L2 might be involved in regulation of the immune response in this disease. We have found that lungs from asthmatic mice had sustained up-regulation of PD-1 and PD-L2, with PD-L2 primarily on dendritic cells. Although addition of PD-L2-Fc in vitro led to decreased T cell proliferation and cytokine production, administration of PD-L2-Fc in vivo in a mouse asthma model resulted in elevated serum IgE levels, increased eosinophilic and lymphocytic infiltration into bronchoalveolar lavage fluid, higher number of cells in the draining lymph nodes, and production of IL-5 and IL-13 from these cells. Although PD-1 was expressed on regulatory T cells, PD-L2-Fc did not affect regulatory T cell activity in vitro. This study provides in vivo evidence of an exacerbated inflammatory response following PD-L2-Fc administration and indicates a potential role for this molecule in Th2-mediated diseases such as asthma.