Ramireddy Bommireddy

TGF-β1 Regulates Lymphocyte Homeostasis by Preventing Activation and Subsequent Apoptosis of Peripheral Lymphocytes

TGF-β1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-β1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-β1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1−/− mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-γ and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1−/− T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1−/− T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4+ T cells have elevated intracellular Ca2+ levels. However, upon subsequent stimulation in vitro, increases in Ca2+ levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1−/− splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca2+ levels.

TGFβ1 Inhibits Ca2+-Calcineurin-Mediated Activation in Thymocytes

TGFβ1 is a polypeptide growth modulatory and differentiation factor involved in many biological processes including immune homeostasis and self-tolerance. Tgfb1 knockout mice die around weaning age due to severe inflammation in most major organ systems, but the mechanism underlying this disease is not understood. In this study we demonstrate that Tgfb1−/− CD4+CD8+ and CD4+CD8− thymocytes are hyperresponsive to receptor-mediated and receptor-independent mitogenic stimulation. A suboptimal concentration of ionomycin in the presence of PMA fully activates Tgfb1−/− thymocytes, whereas the inhibitors of Ca2+ influx and calcineurin, EGTA and FK506, eliminate the hyperresponsiveness. Hence, the hypersensitivity of Tgfb1−/− thymocytes is due to a lowered threshold for Ca2+-dependent activation. Further, we demonstrate that the hypersensitivity of thymocytes results from the absence of TGFβ1 and not from the inflammatory environment because the thymocytes are hyperresponsive in preinflammatory-stage Tgfb1−/− mice. Our results suggest for the first time that TGFβ1 functions to inhibit aberrant T cell expansion by maintaining intracellular calcium concentration levels low enough to prevent a mitogenic response by Ca2+-independent stimulatory pathways alone. Consequently, TGFβ1 prevents autoimmune disease through a Ca2+ regulatory pathway that maintains the activation threshold above that inducible by self-MHC-TCR interactions.

Self-antigen recognition by TGFβ1-deficient T cells causes their activation and systemic inflammation

To investigate whether the multifocal inflammatory disease in TGFβ1-deficient mice is caused by self-antigen (self-Ag)-specific autoreactive T cells, or whether it is caused by antigen independent, spontaneous hyperactivation of T cells, we have generated Tgfb1−/− and Tgfb1−/− Rag1−/− mice expressing the chicken OVA-specific TCR transgene (DO11.10). On a Rag1-sufficient background, Tgfb1−/− DO11.10 mice develop a milder inflammation than do Tgfb1−/− mice, and their T cells display a less activated phenotype. The lower level of activation correlates with the expression of hybrid TCR (transgenic TCRβ and endogenous TCRα), which could recognize self-Ag and undergo activation. In the complete absence of self-Ag recognition (Tgfb1−/− DO11.10 Rag1−/− mice) inflammation and T-cell activation are eliminated, demonstrating that self-Ag recognition is required for the hyper-responsiveness of TGFβ1-deficient T cells. Thus, TGFβ1 is required for the prevention of autoimmune disease through its ability to control the activation of autoreactive T cells to self-Ag.

TGFβ1 deficiency does not affect the generation and maintenance of CD4+CD25+FOXP3+ putative Treg cells, but causes their numerical inadequacy and loss of regulatory function

TGFbeta1 is considered to be required for peripheral maintenance of CD4(+)CD25(+)FOXP3(+) T(reg) cells. However, we demonstrate no reduction in the percentage of such T cells in the spleens and thymi of Tgfb1(-/-) mice. Although putative T(reg) cells, characterized as CD4(+)CD25(+)FOXP3(+)CD62L(+) T cells, are increased in Tgfb1(-/-) mice, they may be inadequate to control activated T cells since the ratio of activated T cells:putative T(reg) cells is several-fold higher in Tgfb1(-/-) mice than in control mice. We further show that whereas Tgfb1(-/-) mice that express a chicken OVA-specific TCR transgene (DO11.10) have an increase in putative T(reg) cells, there are no detectable CD4(+)CD25(+) T cells in the spleens of DO11.10 Rag1(-/-) mice suggesting that T(reg)-cell generation is self-antigen dependent regardless of whether they express Tgfb1. Finally, we demonstrate that Tgfb1(-/-) T cells remain responsive to the suppressive effect of TGFbeta1 in vitro. These data suggest that TGFbeta1 is required for the regulatory function of T(reg) cells to prevent activation of T cells and autoimmunity.

Divergent effects of calcineurin Aβ on regulatory and conventional T-cell homeostasis.

Calcineurin (CN) is a phosphatase that activates nuclear factor of activated T cells (NFAT). While the CN inhibitors cyclosporine A (CsA) and tacrolimus (FK506) can prevent graft rejection, they also cause inflammatory diseases. We investigated the role of calcineurin using mice deficient in the CN catalytic subunit Aβ (CNAβ). Cnab(-/-) mice exhibit defective thymocyte maturation, splenomegaly and hepatomegaly. Further, as Cnab(-/-) mice age, they exhibit spontaneous T-cell activation and enhanced production of proinflammatory cytokines (IL-4, IL-6, and IFNγ). FOXP3(+) T(reg) cells were significantly decreased in Cnab(-/-) mice likely contributing to increased T-cell activation. Interestingly, we found that CNAβ is critical for promotion of BCL-2 expression in FOXP3(+) T(reg) and for permitting TGFβ signaling, as TGFβ induces FOXP3 in control but not in Cnab(-/-) T-cells. Together, these data suggest that CNAβ is important for the production and maintenance of T(reg) cells and to ensure mature T-cell quiescence.

Calcineurin deficiency decreases inflammatory lesions in transforming growth factor β1‐deficient mice

Transforming growth factor (TGF) β1) is an immunoregulatory cytokine involved in self‐tolerance and lymphocyte homeostasis. Tgfb1 knock‐out (KO) mice develop severe multi‐focal autoimmune inflammatory lesions due to [Ca2+]i deregulation in T cells, and die within 3 weeks after birth. Because the calcineurin inhibitor FK506 inhibits the hyperresponsiveness of Tgfb1−/− thymocytes, and because calcineurin Aβ (CNAβ)‐deficient mice do not reject allogenic tumours, we have generated Tgfb1−/−Cnab−/− mice to address whether CNAβ deficiency prevents T cell activation and inflammation in Tgfb1−/− mice. Here we show that in Tgfb1−/−Cnab−/− mice inflammation is reduced significantly relative to that in Tgfb1−/− mice. However, both CD4+ and CD8+ T cells in double knock‐out (DKO) mice are activated, as revealed by up‐regulation of CD11a lymphocyte function‐associated antigen‐1 (LFA‐1), CD44 and CD69 and down‐regulation of CD62L. These data suggest that deficiency of CNAβ decreases inflammatory lesions but does not prevent activation of autoreactive T cells. Also Tgfb1−/− T cells can undergo activation in the absence of CNAβ, probably by using the other isoform of calcineurin (CNAα) in a compensatory manner. CNAβ‐deficient T cells undergo spontaneous activation in vivo and are activated upon anti‐T cell receptor stimulation in vitro. Understanding the role of calcineurin in T cell regulation should open up new therapeutic opportunities for inflammation and cancer.

Time-serial Assessment of Drug Combination Interventions in a Mouse Model of Colorectal Carcinogenesis Using Optical Coherence Tomography

Optical coherence tomography (OCT) is a high-resolution, nondestructive imaging modality that enables time-serial assessment of adenoma development in the mouse model of colorectal cancer. In this study, OCT was utilized to evaluate the effectiveness of interventions with the experimental antitumor agent α-difluoromethylornithine (DFMO) and a nonsteroidal anti-inflammatory drug sulindac during early [chemoprevention (CP)] and late stages [chemotherapy (CT)] of colon tumorigenesis. Biological endpoints for drug interventions included OCT-generated tumor number and tumor burden. Immunochistochemistry was used to evaluate biochemical endpoints [Ki-67, cleaved caspase-3, cyclooxygenase (COX)-2, β-catenin]. K-Ras codon 12 mutations were studied with polymerase chain reaction-based technique. We demonstrated that OCT imaging significantly correlated with histological analysis of both tumor number and tumor burden for all experimental groups (P < 0.0001), but allows more accurate and full characterization of tumor number and burden growth rate because of its time-serial, nondestructive nature. DFMO alone or in combination with sulindac suppressed both the tumor number and tumor burden growth rate in the CP setting because of DFMO-mediated decrease in cell proliferation (Ki-67, P < 0.001) and K-RAS mutations frequency (P = 0.04). In the CT setting, sulindac alone and DFMO/sulindac combination were effective in reducing tumor number, but not tumor burden growth rate. A decrease in COX-2 staining in DFMO/sulindac CT groups (COX-2, P < 0.01) confirmed the treatment effect. Use of nondestructive OCT enabled repeated, quantitative evaluation of tumor number and burden, allowing changes in these parameters to be measured during CP and as a result of CT. In conclusion, OCT is a robust minimally invasive method for monitoring colorectal cancer disease and effectiveness of therapies in mouse models.

Imaging colon cancer development in mice: IL-6 deficiency prevents adenoma in azoxymethane-treated Smad3 knockouts

The development of colorectal cancer in the azoxymethane-induced mouse model can be observed by using a miniaturized optical coherence tomography (OCT) imaging system. This system is uniquely capable of tracking disease development over time, allowing for the monitoring of morphological changes in the distal colon due to tumor development and the presence of lymphoid aggregates. By using genetically engineered mouse models deficient in Interleukin 6 (IL-6) and Smad family member 3 (Smad3), the role of inflammation on tumor development and the immune system can be elucidated. Smad3 knockout mice develop inflammatory response, wasting, and colitis associated cancer while deficiency of proinflammatory cytokine IL-6 confers resistance to tumorigenesis. We present pilot data showing that the Smad3 knockout group had the highest tumor burden, highest spleen weight, and lowest thymus weight. The IL-6 deficiency in Smad3 knockout mice prevented tumor development, splenomegaly, and thymic atrophy. This finding suggests that agents that inhibit IL-6 (e.g. anti-IL-6 antibody, non-steroidal anti-inflammatory drugs [NSAIDs], etc.) could be used as novel therapeutic agents to prevent disease progression and increase the efficacy of anti-cancer agents. OCT can also be useful for initiating early therapy and assessing the benefit of combination therapy targeting inflammation.

Tumor Suppressor Functions of TGFβ1 in T Cells

Transforming growth factor β1 (TGFβ1) is an important pleiotropic immunoregulatory cytokine, which is involved in several cellular processes, such as inhibition of T-cell activation, proliferation, differentiation, apoptosis, cytokine production, and effector function. In contrast to its inhibitory effects on naive T cells, it is also involved in stimulation of suppressor/regulatory T-cell expansion and function. In order to achieve so many diverse effects TGFβ utilizes distinct signaling mechanisms in the same cell type. Mouse models with genetic ablation of functional TGFβ signaling mediators have revealed several interesting mechanisms that play an important role in T-cell homeostasis, Treg-cell function, and tumorigenesis. TGFβ1 primarily functions to prevent abnormal T-cell activation through modulation of a Ca2+-calcineurin signaling pathway in a SMAD3/4-independent manner. Additionally, in Treg cells TGFβ1 mediates its effects through a SMAD signaling cascade in a unique manner. TGFβ1 plays duel roles in tumor development and progression.

Transforming growth factor-β: From its effect in T cell activation to a role in dominant tolerance

Transforming growth factor (TGF)β1 is an important immunoregulatory cytokine involved in maintenance of self tolerance and T cell homeostasis [1]. It is produced by several immune and non-immune cell types and functions in both autocrine and paracrine manners [1]–[3]. Regulatory T cells (Treg) cells are the primary source of TGFβ1 for controlling autoimmune disease. Blockade of TGFβ signaling in T cells causes severe autoimmune disease in mice [4, 5]. However, TGFβ signaling in T cells under inflammatory conditions also causes the development of experimentally induced autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis (MS) disease [6]. This review focuses on recent findings concerning the function of TGFβ1 in T cells and immune tolerance, and it discusses implications of these findings for therapeutic intervention in autoimmune and inflammatory diseases.