Wenhao Chen

Generation and regulation of human CD4+ IL-17-producing T cells in ovarian cancer

Despite the important role of Th17 cells in the pathogenesis of many autoimmune diseases, their prevalence and the mechanisms by which they are generated and regulated in cancer remain unclear. Here, we report the presence of a high percentage of CD4+ Th17 cells at sites of ovarian cancer, compared with a low percentage of Th17 cells in peripheral blood mononuclear cells from healthy donors and cancer patients. Analysis of cytokine production profiles revealed that ovarian tumor cells, tumor-derived fibroblasts, and antigen-presenting cells (APCs) secreted several key cytokines including IL-1β, IL-6, TNF-α and TGF-β, which formed a cytokine milieu that regulated and expanded human IL-17-producing T-helper (Th17) cells. We further show that IL-1β was critically required for the differentiation and expansion of human Th17 cells, whereas IL-6 and IL-23 may also play a role in the expansion of memory Th17 cells, even though IL-23 levels are low or undetectable in ovarian cancer. Further experiments demonstrated that coculture of naïve or memory CD4+ T cells with tumor cells, APCs, or both could generate high percentages of Th17 cells. Treatment with anti-IL-1 alone or a combination of anti-IL-1 and anti-IL-6 reduced the ability of tumor cells to expand memory Th17 cells. Thus, we have identified a set of key cytokines secreted by ovarian tumor cells and tumor-associated APCs that favor the generation and expansion of human Th17 cells. These findings should accelerate efforts to define the function of this important subset of CD4+ T cells in the human immune response to cancer.

Characterization of Distinct Conventional and Plasmacytoid Dendritic Cell-Committed Precursors in Murine Bone Marrow

The developmental pathways and differentiation relationship of dendritic cell (DC) subsets remain unclear. We report that murine CD11c+MHC II− bone marrow cells, which are immediate DC precursors of CD8α+, CD8α−, and B220+ DC in vivo, can be separated into B220+ and B220− DC precursor subpopulations. Purified B220− DC precursors expand, and generate exclusively mature CD11c+CD11b+B220− DC in vitro and after adoptive transfer. B220+ DC precursors, which resemble plasmacytoid pre-DC, have a lower proliferative potential than B220− DC precursors and generate both CD11b− B220+ and CD11b+B220− DC populations. Both DC precursor populations can give rise to CD8α+ and CD8α− DC subtypes. Our findings indicate that CD11c+MHC II−B220+ and CD11c+MHC II−B220− bone marrow cells are distinct DC lineage-restricted precursors.

In Situ Replication of Immediate Dendritic Cell (DC) Precursors Contributes to Conventional DC Homeostasis in Lymphoid Tissue

The developmental biology of dendritic cells (DC) under physiological conditions remains unclear. In this study, we show that mouse CD11c+ MHC class II−lineage− cells are immediate precursors of conventional DC and are widely distributed in both bone marrow and lymphoid tissues. These precursors have a high clonal efficiency, and when cocultured on a supportive stromal monolayer or adoptively transferred in vivo, generate a population CD11c+MHC class II+ DC that retain limited proliferation capacity. During steady state conditions, a small proportion of immediate DC precursors (DCp) and DCs are dividing actively in bone marrow and spleen. Cytokines that initiate and support proliferation of immediate DCp were defined. Collectively, our findings provide evidence of a distinct development pathway for conventional DC in both bone marrow and lymphoid tissues and highlight the importance of in situ replication of immediate DCp and DC in maintaining conventional DC populations.

“Default” Generation of Neonatal Regulatory T Cells

CD4+Foxp3+ regulatory T (Treg) cells were shown to control all aspects of immune responses. How these Treg cells develop is not fully defined, especially in neonates during development of the immune system. We studied the induction of Treg cells from neonatal T cells with various TCR stimulatory conditions, because TCR stimulation is required for Treg cell generation. Independent of the types of TCR stimulus and without the addition of exogenous TGF-β, up to 70% of neonatal CD4+Foxp3− T cells became CD4+Foxp3+ Treg cells, whereas generally <10% of adult CD4+Foxp3− T cells became CD4+Foxp3+ Treg cells under the same conditions. These neonatal Treg cells exert suppressive function and display relatively stable Foxp3 expression. Importantly, this ability of Treg cell generation gradually diminishes within 2 wk of birth. Consistent with in vitro findings, the in vivo i.p. injection of anti-CD3 mAb to stimulate T cells also resulted in a >3-fold increase in Treg cells in neonates but not in adults. Furthermore, neonatal or adult Foxp3− T cells were adoptively transferred into Rag1−/− mice. Twelve days later, the frequency of CD4+Foxp3+ T cells converted from neonatal cells was 6-fold higher than that converted from adult cells. Taken together, neonatal CD4+ T cells have an intrinsic “default” mechanism to become Treg cells in response to TCR stimulations. This finding provides intriguing implications about neonatal immunity, Treg cell generation, and tolerance establishment early in life.

Protection against allograft rejection with intercellular adhesion molecule-1 antisense oligodeoxynucleotides

BACKGROUND We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125). METHODS IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting. RESULTS IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days). CONCLUSIONS Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.

Role of Double-Negative Regulatory T Cells in Long-Term Cardiac Xenograft Survival

A novel subset of CD3+CD4−CD8− (double negative; DN) regulatory T cells has recently been shown to induce donor-specific skin allograft acceptance following donor lymphocyte infusion (DLI). In this study, we investigated the effect of DLI on rat to mouse cardiac xenotransplant survival and the ability of DN T cells to regulate xenoreactive T cells. B6 mice were given either DLI from Lewis rats, a short course of depleting anti-CD4 mAb, both DLI and anti-CD4 treatment together, or left untreated. DLI alone did not prolong graft survival when compared with untreated controls. Although anti-CD4-depleting mAb alone significantly prolonged graft survival, grafts were eventually rejected by all recipients. However, the combination of DLI and anti-CD4 treatment induced permanent cardiac xenograft survival. We demonstrate that recipients given both DLI and anti-CD4 treatment had a significant increase in the total number of DN T cells in their spleens when compared with all other treatment groups. Furthermore, DN T cells harvested from the spleens of DLI plus anti-CD4-treated mice could dose-dependently inhibit the proliferation of syngeneic antidonor T cells. Suppression mediated by these DN T cells was specific for antidonor T cells as T cells stimulated by third-party Ags were not suppressed. These results demonstrate for the first time that a combination of pretransplant DLI and anti-CD4-depleting mAb can induce permanent survival of rat to mouse cardiac xenografts and that DN T regulatory cells play an important role in preventing long-term concordant xenograft rejection through the specific suppression of antidonor T cells.

Targeted Deletion of Fgl-2/Fibroleukin in the Donor Modulates Immunologic Response and Acute Vascular Rejection in Cardiac Xenografts

Background—Xenografts ultimately fail as a result of acute vascular rejection (AVR), a process characterized by intravascular thrombosis, fibrin deposition, and endothelial cell activation. Methods and Results—We studied whether targeted deletion of Fgl-2, an inducible endothelial cell procoagulant, (Fgl-2−/−) in the donor prevents AVR in a mouse-to-rat cardiac xenotransplantation model. By 3 days after transplant, Fgl-2+/+ grafts developed typical features of AVR associated with increased levels of donor Fgl-2 mRNA. Grafts from Fgl-2−/− mice had reduced fibrin deposition but developed cellular rejection. Treatment with a short course of cobra venom factor and maintenance cyclosporine resulted in long-term acceptance of both Fgl-2+/+ and Fgl-2−/− grafts. On withdrawal of cyclosporine, Fgl-2+/+ grafts developed features of AVR; in contrast, Fgl-2−/− grafts again developed acute cellular rejection. Rejecting Fgl-2+/+ hearts stained positively for IgG, IgM, C3, and C5b-9, whereas rejecting Fgl-2−/− hearts had minimal Ig and complement deposition despite xenoantibodies in the serum. Furthermore, serum containing xenoantibodies failed to stain Fgl-2−/− long-term treated hearts but did stain wild-type heart tissues. Treatment of Fgl-2−/− xenografts with mycophenolate mofetil and tacrolimus, a clinically relevant immune suppression protocol, led to long-term graft acceptance. Conclusions—Deletion of Fgl-2 ameliorates AVR by downregulation of xenoantigens and may facilitate successful clinical heart xenotransplantation.

Donor Lymphocyte Infusion Induces Long-Term Donor-Specific Cardiac Xenograft Survival through Activation of Recipient Double-Negative Regulatory T Cells

Previous studies have shown that pretransplant donor lymphocyte infusion (DLI) can enhance xenograft survival. However, the mechanism by which DLI induces xenograft survival remains obscure. Using T cell subset-deficient mice as recipients we show that CD4+, but not CD8+, T cells are necessary to mediate the rejection of concordant cardiac xenografts. Adoptive transfer of naive CD4+ T cells induces rejection of accepted cardiac xenografts in CD4−/− mice. This rejection can be prevented by pretransplant DLI in the absence of any other treatment. Furthermore, we demonstrate that DLI activates αβ-TCR+CD3+CD4−CD8− double-negative (DN) regulatory T (Treg) cells in xenograft recipients, and that DLI-activated DN Treg cells can inhibit the proliferation of donor-specific xenoreactive CD4+ T cells in vitro. More importantly, adoptive transfer of DLI-activated DN Treg cells from xenograft recipients can suppress the proliferation of xenoreactive CD4+ T cells and their ability to produce IL-2 and IFN-γ in vivo. Adoptive transfer of DLI-activated DN Treg cells also prevents CD4+ T cell-mediated cardiac xenograft rejection in an Ag-specific fashion. These data provide direct evidence that DLI can activate recipient DN Treg cells, which can induce donor-specific long-term cardiac xenograft survival by suppressing the proliferation and function of donor-specific CD4+ T cells in vivo.

STAT3 : An Important Regulator of Multiple Cytokine Functions

Maintaining T cell homeostasis is critical for normal immune response. Three sequential signals activate T cells, with signal 3 delivered by multiple cytokines that regulate cell proliferation, differentiation, and survival/death. Cytokines binding to their receptors engages two key molecular families, namely, Janus tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats). Among Stats, Stat3 is involved in the generation of T helper 17 (Th17) cells, regulation of dendritic cells, and acute inflammatory response. These aspects of Stat3 function are important for transplantation. We discuss Stat3s role in innate and adaptive immunity as well as its potential for therapeutic intervention.

Peptide‐activated double‐negative T cells can prevent autoimmune type‐1 diabetes development

Autoimmune diseases may develop because of defective maturation, activation, differentiation and function of regulatory T cells. Previous studies have shown that exposure to donor antigen activates peripheral TCRαβ+CD3+CD4–CD8–NK1.1–, double‐negative (DN) T cells, which specifically suppress anti‐donor T cells and enhance survival of skin and heart grafts from allogeneic and xenogeneic donors. However, the role of DN T cells in preventing T cell‐mediated autoimmune disease is unknown. Here, we analyzed the ability of DN T cells to recognize peptides expressed on self MHC and to suppress peptide‐reactive CD8+ T cells, using the P14 mouse model that expresses a transgenic TCR specific for gp33 peptide presented on self MHC class I‐Db. We found that injection of gp33 peptide resulted in increased DN and decreased CD8+ T cell numbers in the lymph nodes when compared to untreated mice. Injection of gp33, but not TCR‐non‐specific AV peptide, increased expression of T cell activation markers on DN T cells. Moreover, gp33‐activated DN T cells suppressed proliferation of syngeneic CD8+ T cells via killing activated CD8+ T cells in an antigen‐specific fashion in vitro. Furthermore, transferring gp33‐activated DN T cells inhibited the development of autoimmune diabetes, suggesting that DN T cells may provide a novel therapy for T cell‐mediated autoimmune diseases.