Shuang Fu

TGF-β induces Foxp3 + T-regulatory cells from CD4 + CD25 - precursors

CD4 + CD25 + regulatory T cells (Tregs) are potent suppressors, playing important roles in autoimmunity and transplantation tolerance. Understanding the signals necessary for the generation and expansion of Tregs is important for clinical cellular therapy, but only limited progress has been made. Recent reports suggest a role for TGF‐β in the generation of Tregs from CD4 + CD25 − precursors, but the mechanism remains unknown. Here, we demonstrate that TGF‐β2 triggers Foxp3 expression in CD4 + CD25 − precursors, and these Foxp3 + cells act like conventional Tregs. The generation of Foxp3 + Tregs requires stimulation of the T‐cell receptor, the IL‐2R and the TGF‐β receptor. More importantly, strong costimulation through CD28 prevents Foxp3 expression and suppressive function in an IL‐4‐dependent manner. Furthermore, TGF‐β‐driven Tregs inhibit innate inflammatory responses to syngeneic transplanted pancreatic islets and enhance islet transplant survival. Thus, TGF‐β is a key regulator of the signaling pathways that initiate and maintain Foxp3 expression and suppressive function in CD4 + CD25 − precursors. TGF‐β and signaling through TGF‐β receptor, CD28 costimulation and IL‐4 may be key components for the manipulation of Treg. The de novo generation of Foxp3 + cells from CD4 + cells has the potential to be used for treatment of autoimmune diseases and induction of transplant tolerance.

CD4+ CD25+ CD62+ T-Regulatory Cell Subset Has Optimal Suppressive and Proliferative Potential

CD4+ CD25+ regulatory T cells (Treg) are potent suppressors, and play important roles in autoimmunity and transplantation. Recent reports suggest that CD4+ CD25+ Treg are not a homogeneous cell population, but the differences in phenotype, function, and mechanisms among different subsets are unknown. Here, we demonstrate CD4+ CD25+ Treg cells can be divided into subsets according to cell‐surface expression of CD62L. While both subsets express foxp3 and are anergic, the CD62L+ population is more potent on a per cell basis, and proliferates and maintains suppressive function far better than the CD62L– population and unseparated CD4+ CD25+ Treg. The CD62L+ population preferentially migrates to CCL19, MCP‐1 and FTY720. Both CD62L+ and CD62L– subsets prevent the development of autoimmune gastritis and colitis induced by CD4+ CD25–CD45RBhigh cells in severe combined immunodeficiency (SCID) mice. Overall, these results suggest CD4+ CD25+ Treg are not a homogenous cell population, but can be divided into at least two subsets according to CD62L expression. The CD62L+ subset is a more potent suppressor than the CD62L– population or unfractionated CD4+ CD25+ Treg cells, can be expanded far more easily in culture, and is more responsive to chemokine‐driven migration to secondary lymphoid organs. These properties may have significant implications for the clinical manipulation of the CD4+ CD25+ CD62L+ cells.

FTY720-Enhanced T Cell Homing Is Dependent on CCR2, CCR5, CCR7, and CXCR4: Evidence for Distinct Chemokine Compartments

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.

Adenovirus transduction induces expression of multiple chemokines and chemokine receptors in murine beta cells and pancreatic islets.

Adenoviral vectors are highly efficient for transferring genes to islets. However, the inflammatory and immune responses stimulated by adenovirus may be detrimental to islet survival. Given the role of chemokines and their receptors in inflammation, we analyzed their expression in isolated murine islets, in a murine β cell line and in syngeneic islet grafts after adenovirus transduction (AdRSVLacZ). AdRSVLacZ transduction enhanced and induced the expression of a variety of chemokines. Transduced syngeneic transplanted islets showed significantly enhanced expression of multiple chemokines and receptors, including monocyte chemoattractant protein‐1 (MCP‐1), CC chemokine receptor 2 (CCR2) and regulated upon activation, normal T cell expressed and secreted (RANTES), compared with untransduced islet grafts. AdRSVLacZ‐transduced islet grafts had significant mononuclear infiltrates, and in situ hybridization demonstrated intragraft expression of MCP‐1, CCR2 and RANTES. Although adenovirus transduction did not impair in vitro insulin secretion, diabetes was reversed in only one of six recipients of a marginal mass of AdRSVLacZ‐transduced islets, compared with six of six control recipients. In conclusion, multiple chemokines and chemokine receptors are expressed by murine islets constitutively and in response to adenovirus transduction. Adenovirus transduction impairs engraftment of marginal mass of transplanted islets. This is not because of direct vector toxicity of islet secretory capacity, but may be related to host innate immunity in response to adenovirus vector.

Differential Chemokine and Chemokine Receptor Gene Induction by Ischemia, Alloantigen, and Gene Transfer in Cardiac Grafts

Transplantation of allogeneic grafts presents several challenges to the innate and adaptive immune systems including chemokine leukocyte recruitment, activation, and effector function. We defined the chemokines and receptors induced by the transplant procedure/ischemia injury, alloantigen and gene transfer vector administration in murine cardiac grafts. E1, E3 deleted AdRSVβgal was transferred into grafts at the time of transplantation, grafts were harvested after 1–14 days, and a pathway‐specific cDNA array was used to evaluate the levels of 67 chemokine and chemokine receptor genes. Transplantation resulted in ischemic injury and induction of a number of similar genes in both the syngeneic and allogeneic grafts, such as CXCL1 and CXCL5, which increased dramatically on day 1 and returned rapidly to baseline in the syngeneic grafts. Alloantigen stimulated the adaptive immune response and induced the presence of more inflammatory genes within the grafts, particularly at later time points. The adenovirus vector induced a broader panel of genes, among them potent inflammatory chemokines CXCL9 and CXCL10, that are induced earlier or more strongly compared with alloantigen stimulation alone. As alloantigen and adenovirus vectors both induce similar sets of genes, targeting these molecules may not only inhibit alloimmunity, but also enhance the utility of the gene transfer vector.

Viral IL‐10 Gene Transfer Inhibits the Expression of Multiple Chemokine and Chemokine Receptor Genes Induced by Inflammatory or Adaptive Immune Stimuli

Previously we have shown that adenovirus‐mediated gene transfer and expression of vIL‐10 are able to prolong cardiac allograft survival, through the inhibition of the immune response to both alloantigen and adenoviral antigens. In the current study, we have defined further mechanisms of Ad.vIL‐10‐mediated prolongation of cardiac allograft survival. E1‐ and E3‐deleted adenoviral vectors encoding β‐galactosidase or vIL‐10 were transferred into grafts at the time of transplantation, chemokine and chemokine receptor expression were evaluated by a pathway‐specific cDNA array, and the results were confirmed with real time RT‐PCR on selected genes. Ischemic injury, alloantigen and adenovirus vector induced the expression of multiple pro‐inflammatory chemokines in the grafts, which likely amplify allograft rejection. Most of these Th1‐related chemokine genes were inhibited or down‐regulated by Ad.vIL‐10 administration, which may help to decrease leukocytic infiltration and improve graft survival. Among the potent Th1 type chemokines inhibited were the CXCR3 ligands CXCL9 and CXCL10, which could directly inhibit vector‐mediated gene expression in myoblasts, although targeting CXCR3 or its ligands did not prolong allograft survival with vIL‐10 gene transfer. Ad.vIL‐10 administration also induced the expression of the Th2‐associated chemokines eotaxin‐2 and MIP‐1 γ, suggesting Th1 to Th2 immune deviation. These results demonstrated that the vIL‐10 gene transfer inhibits chemokine expression, preventing stimulation of innate and adaptive immunity.

Transduction of pancreatic islets with pseudotyped adeno-associated virus: effect of viral capsid and genome conversion.

Background. Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be packaged with other serotype capsids to assemble infectious virions. During transduction, the ssDNA to dsDNA conversion is one of the major rate-limiting steps that contribute to the slow onset of transgene expression. Methods. Using pseudotyping strategy, we produced double-stranded (dsAAV) and single-stranded (ssAAV) rAAV2 genomes carrying the GFP reporter gene packaged into AAV1, AAV2, and AAV5 capsids. The ability of cross-packaged AAV1, AAV2, and AAV5 at the same genome containing particle (gcp) concentration to transduce murine and human pancreatic islets was evaluated by GFP positive cell percentage. Transgenic expression was also determined by transplant transduced human islet into SCID mice. Results. Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islets transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. dsAAV, which do not require second strand synthesis and thus are potentially more efficient, evidenced 5 fold higher transduction ability than ssAAV vectors. Pseudotyped rAAV transduced islet grafts maintained normal function, expressed transgenic product persistently in vivo, and reversed diabetes. Conclusions. The transduction efficiency of rAAV vectors was dependent on the cross-packaged capsid. The vector capsids permit species-specific transduction. For human islets, dsAAV2/2 vectors may be the most efficient vector for clinical development.

Feline Immunodeficiency Virus‐Mediated Viral Interleukin‐10 Gene Transfer Prolongs Non‐Vascularized Cardiac Allograft Survival

Previous experiments demonstrated plasmid‐, retroviral‐, or adenoviral‐mediated vIL‐10 gene transfer could prolong allograft survival, but transgene expression was rapidly extinguished. Feline immunodeficiency virus (FIV) can integrate into genomic DNA of nondividing cells, resulting in indefinite transgene expression. We hypothesized FIV‐mediated gene transfer could provide long‐term gene expression, and improved allograft survival. FIV‐vIL‐10 and FIV‐β‐gal were produced using the FELIX vector system. With vector transfer to syngeneic cardiac grafts, β‐galactosidase reporter gene expression was noted as early as day 5, was strongly expressed at days 10 and 20, and persisted for 50 days after transplantation. For allografts, FIV‐vIL‐10 gene transfer more than doubled mean survival from 10 ± 1.6 to 22.3 ± 3 days. When combined with other immunosuppressants, such as anti‐CD40L mAb, FTY720, or anti‐CD3 mAb, the mean survival times were prolonged to 27 ± 4.6 days, 27.8 ± 4.6 days, and 45.5 ± 4.9 days, respectively. Multiple chemokine and chemokine receptor genes were induced by ischemia‐reperfusion injury in syngeneic grafts, and in allogeneic grafts more genes were induced and to a greater degree. In allogeneic grafts transduced with FIV‐IL‐10, a number of the chemokine genes were suppressed. Therefore, FIV virus‐mediated vIL‐10 gene transfer prolongs allograft survival and, in combination with other agents, produces an additive effect.

CD2 is a Dominant Target for Allogeneic Responses

CD2 and 2B4 (CD244) are members of the immunoglobulin gene superfamily and are both ligands for another family member, CD48. CD2 is widely distributed on T, NK, and B cells and some antigen‐presenting cells, while 2B4 is expressed on NK and some T cells and monocytes and is known to participate in NK cytotoxicity. Since indefinite allograft survival could be obtained by a combination of anti‐CD48 plus anti‐CD2 mAb administration, it was important to determine the role of 2B4 blockade in allograft rejection. MAbs directed against CD2, CD48, or 2B4 were administered singly or in pairs to cardiac allograft recipients. The experiments show that only anti‐CD2 plus anti‐CD48 mAbs result in indefinite allograft survival, while anti‐CD2 plus anti‐2B4 mAbs substantially prolong graft survival, and anti‐CD48 plus anti‐2B4 mAbs were no better than each mAb alone. The effect of these mAbs on anti‐CD3 mAb and alloantigen‐driven proliferation and IFN‐γ production were also assessed. In general, anti‐CD2 inhibited both anti‐CD3 mAb and alloantigen‐driven responses, while anti‐CD48 inhibited only anti‐CD3 mAb but not alloantigen‐driven proliferative and cytokine responses. Anti‐2B4 mAbs were generally ineffective alone. Combinations of mAbs were more effective than single mAbs only in alloantigen‐driven proliferation, commensurate with allograft survival results. Using CD2–/– and CD48–/– T cells and antigen‐presenting cells, we also demonstrate that these inhibitory mAbs act primarily by blocking intercellular interactions, rather than directly delivering negative signals to T cells. These results suggest that, unlike CD2, 2B4 is not a potent regulatory molecule or ligand for CD48 in the response to alloantigen. Blocking the 2B4–CD48 receptor–ligand pair does not inhibit T‐cell responses and alloreactivity to the same degree as CD2–CD48 blockade.

CHAPTER 25 – Interleukin-10

IL-10 is produced by many different cell types under a wide variety of stimuli. interleukin -10 (IL-10) is a key regulator of immune responses. Because of its ability to turn off cytokine production by T cells, IL-10 was originally described as a cytokine synthesis inhibitory factor (CSIF). IL-10 receptor complex is expressed by many different immune and parenchymal cell types, and signal transduction is regulated by multiple other signaling pathways, and it is responsible for many different pathophysiological events. The chapter discusses a major area that delineates the immunomodulatory roles of IL-10 in important disease states, particularly autoimmune disease, transplantation, infection, and ischemia-reperfusion. The study is directed toward separating out the immunosuppressive from the immunostimulatory activities of IL-10. The biological spectrum of activity of vIL-10 versus cIL-10 suggests that it may be possible to channel the activity of IL-10 in the desired direction. The agonist and antagonist ligands remain to be discovered and characterized. The demonstration that IL-10 and IL-22 share the IL-10R2 subunit suggests an additional level of regulation through alternative heterodimers on the cell surface.