Mouse Model Of Skin Transplantation Research Articles

Early graft-infiltrating lymphocytes are not associated with graft rejection in a mouse model of skin transplantation.

Graft-infiltrating lymphocytes (GILs) play an important role in promoting rejection after organ transplantation. We recently reported that GILs that accumulated up to 3 days post-transplantation did not promote rejection, whereas GILs present 3-5 days post-transplantation promoted rejection in a mouse heart transplantation model. However, the immunological behaviour of GILs in murine skin transplantation remains unclear. GILs were isolated on days 3, 5 or 7 post-transplantation from C57BL/6 (B6) allogeneic skin grafts transplanted onto BALB/c mice. BALB/c Rag2-/- γc-/- mice (BRGs) underwent B6 skin graft transplantation 10 weeks after adoptive transfer of day 3, 5, or 7 GILs. BRGs reconstituted with day 5 or 7 GILs completely rejected B6 grafts. However, when B6 grafts harvested from recipient BALB/c mice on day 5 or 7 were re-transplanted into BRGs, half of the re-transplanted day 5 grafts established long-term survival, although all re-transplanted day 7 grafts were rejected. BRGs reconstituted with day 3 GILs did not reject B6 grafts. Consistently, re-transplantation using day 3 skin grafts resulted in no rejection. Administration of anti-CD25 antibodies did not prevent the phenomenon observed for the day 3 skin grafts. Furthermore, BRGs reconstituted with splenocytes from naïve BALB/c mice immediately rejected the naïve B6 skin grafts and the re-transplanted day 3 B6 grafts, suggesting that day 3 GILs were unable to induce allograft rejection during the rejection process. In conclusion, the immunological role of GILs depends on the time since transplantation. Day 3 GILs had neither protective nor alloreactive effects in the skin transplant model.

Triptolide promotes differentiation of human monocytes into immunosuppressive MDSCs

BackgroundMyeloid-derived suppressor cells (MDSCs) negatively modulate immune activity. Prior investigations have shown much promise in using MDSCs-assisted immunotherapy for organ transplantation patients. Additionally, owing to its immunosuppressive activity, MDSCs can also be used to manage immune-associated disorders. MethodsGranulocyte-macrophage colony-stimulating factor (GM-CSF) was employed to stimulate myeloid progenitor cell differentiation. Triptolide (PG490) was introduced toward the later phases of in vitro MDSCs induction. Lastly, real-time PCR (RT-PCR) and flow cytometry were used to assess transcript expression and cell phenotype, and a mouse skin transplantation model was established to evaluate the MDSCs-mediated immune suppression in vivo. ResultsCo-stimulation with PG490 and GM-CSF potently induced myeloid-derived monocytes to form MDSCs, with remarkable immune-suppressive activity. The underlying mechanism involved downregulation of T cell proliferation, activation, enhancement of inflammatory cytokine release, as well as T cell conversion to Treg cells. PG490 strongly enhanced iNOS expression in MDSCs, and iNOS inhibition successfully reversed the immune-suppression. The PG490- and GM-CSF-induced MDSCs substantially extended survival duration of murine skin grafts, thereby validating their strong immune-suppressive activity in vivo. ConclusionsHerein, we presented a new approach involving MDSCs-based immunosuppression in vitro. PG490 and GM-CSF co-treatment strongly induced immuno-suppressive activity in MDSCs both in vitro and in vivo. Our findings highlight the promise of applying MDSCs-based therapy in clinical organ transplantation treatment.

2-D-gal Targets Terminal Fucosylation to Inhibit T-cell Response in a Mouse Skin Transplant Model.

Organ allograft rejection is mainly driven by T-cell response. Studies have shown that fucosylation plays essential roles in the immune cell development and function. Terminal fucosylation inhibitor, 2-deoxy-D-galactose (2-D-gal), has been reported to suppress immunoresponse of macrophages, but its effects on T-cell-mediated immune response and transplant rejection have not been fully explored. The terminal fucosylation level in T cells was detected through ulex europaeus agglutinin-I staining. The consequences of 2-D-gal on murine T-cell proliferation, activation, cytokine secretion, and cell cycle were investigated in vitro. T-cell receptor signaling cascades were examined. Last, mouse skin transplant model was utilized to evaluate the regulatory effects of 2-D-gal on T-cell response in vivo. The expression of fucosyltransferase1 was upregulated in CD3/CD28-activated T cells along with an elevation of α(1,2)-fucosylation level as seen by ulex europaeus agglutinin-I staining. Furthermore, 2-D-gal suppressed T-cell activation and proliferation, decrease cytokines production, arrest cell cycle, and prevent the activation of T-cell receptor signaling cascades. In vivo experiments showed that 2-D-gal limited T-cell proliferation to prolong skin allograft in mice. This was accompanied by lower level of inflammatory cytokines, and were comparable to those treated with Cyclosporin A. Terminal fucosylation appears to play a role in T-cell activation and proliferation, and its inhibitor, 2-D-gal, can suppress T-cell activation and proliferation both in vitro and in vivo. In a therapeutic context, inhibiting terminal fucosylation may be a potential strategy to prevent allogeneic transplant rejection.

Induction of allograft tolerance by adoptive transfer of donor B cells: an immune regulatory strategy for transplantation using MHC-matched iPS cells.

For cellular or tissue transplantation using induced pluripotent stem cells (iPSCs), from the viewpoint of time and economic cost, the use of allogeneic ones is being considered. Immune regulation is one of the key issues in successful allogeneic transplantation. To reduce the risk of rejection, several attempts have been reported to eliminate effects of the major histocompatibility complex (MHC) on the iPSC-derived grafts. On the other hand, we have shown that minor antigen-induced rejection is not negligible even when the MHC's impact is mitigated. In organ transplantation, it is known that donor-specific transfusion (DST) can specifically control immune responses to the donor. However, whether DST could control the immune response in iPSC-based transplantation was not clarified. In this study, using a mouse skin transplantation model, we demonstrate that infusion of donor splenocytes can promote allograft tolerance in the MHC-matched but minor antigen-mismatched conditions. When narrowing down the cell types, we found that infusion of isolated splenic B cells was sufficient to control rejection. As a mechanism, the administration of donor B cells induced unresponsiveness but not deletion in recipient T cells, suggesting that the tolerance was induced in the periphery. The donor B cell transfusion induced allogeneic iPSC engraftment. These results suggest for the first time a possibility that DST using donor B cells could induce tolerance against allogeneic iPSC-derived grafts.

Ex vivo-expanded human CD19+TIM-1+ regulatory B cells suppress immune responses in vivo and are dependent upon the TIM-1/STAT3 axis

Regulatory B cells (Breg) are a heterogenous population with immune-modulating functions. The rarity of human IL-10+ Breg makes translational studies difficult. Here we report ex vivo expansion of human B cells with in vivo regulatory function (expBreg). CD154-stimulation of human CD19+ B cells drives >900-fold expansion of IL-10+ B cells that is maintained in culture for 14 days. Whilst expBreg-mediated suppressive function is partially dependent on IL-10 expression, CRISPR-mediated gene deletions demonstrate predominant roles for TIM-1 and CD154. TIM-1 regulates STAT3 signalling and modulates downstream suppressive function. In a clinically relevant humanised mouse model of skin transplantation, expBreg prolongs human allograft survival. Meanwhile, CD19+CD73-CD25+CD71+TIM-1+CD154+ Breg cells are enriched in the peripheral blood of human donors with cutaneous squamous cell carcinoma (SCC). TIM-1+ and pSTAT3+ B cells are also identified in B cell clusters within histological sections of human cutaneous SCC tumours. Our findings thus provide insights on Breg homoeostasis and present possible targets for Breg-related therapies.

Evaluation of immunosuppression protocols for MHC-matched allogeneic iPS cell-based transplantation using a mouse skin transplantation model

BackgroundOff-the-shelf major histocompatibility complex (MHC)-matched iPS cells (iPSC) can potentially initiate host immune responses because of the existence of numerous minor antigens. To suppress allo-immune responses, combination of immunosuppressants is usually used, but its efficacy to the allogeneic iPSC-based transplantation has not been precisely evaluated.MethodsThree transplantation models were used in this study; MHC-matched, minor antigen-mismatched mouse skin or iPSC-graft transplantation, and fully allogeneic human iPSC-derived liver organoid transplantation in immune-humanized mice. The recipients were treated with triple drugs combination (TDC; tacrolimus, methylprednisolone, and mycophenolate mofetil) or co-stimulatory molecule blockade (CB) therapy with some modifications. Graft survival as well as anti-donor T and B cell responses was analyzed.ResultsIn the mouse skin transplantation model, immunological rejection caused by the minor antigen-mismatch ranged from mild to severe according to the donor-recipient combination. The TDC treatment could apparently control the mild skin graft rejection when combined with a transient T cell depletion, but unexpected anti-donor T or B cell response was observed. On the other hand, CB therapy, particularly when combined with rapamycin treatment, was capable of attenuating both mild and severe skin graft rejection and allowing them to survive long-term without any unfavorable anti-donor immune responses. The efficacy of the CB therapy was confirmed in both mouse and human iPSC-derived graft transplantation.ConclusionsThe findings suggest that the CB-based treatment seems suitable to well manage the MHC-matched allogeneic iPSC-based transplantation. The TDC-based treatment may be also used to suppress the rejection, but screening of its severity prior to the transplantation seems to be needed.

PMA induces the differentiation of monocytes into immunosuppressive MDSCs.

The induction of immune tolerance without the use of immunosuppressive drugs is a crucial problem in organ transplantation. The use of myeloid-derived suppressor cells (MDSCs) as a cell-based adjuvant immunosuppressive therapy is a bright clinical prospect in organ transplantation. MDSCs with stable immunosuppressive activities can be used to treat immune-related diseases. In this study, macrophage colony-stimulating factor (M-CSF) was used to promote myeloid progenitor cell differentiation, and phorbol 12-myristate 13-acetate (PMA) was added to induce MDSCs at the later stage of induction in vitro. Cell phenotypes were detected by flow cytometry and mRNA was detected by real-time-polymerase chain reaction (RT-PCR). A mouse skin transplantation model was used to investigate the cell inhibitory function. The combination of PMA and M-CSF induced the differentiation of myeloid-derived monocytes into MDSCs. MDSCs were found to induce immune tolerance by inhibiting the proliferation and activation of T cells, promoting cytokine secretion and inducing T cell transformation to regulatory T cells (Treg ). PMA significantly up-regulated the expression of Arg-1 and the Arg-1 protein expression in MDSCs and arginase 1 (Arg-1) inhibitor nor-NOHA reversed the MDSC immunosuppressive activity, indicating the involvement of the Arg-1 pathway in MDSC-mediated immunosuppression. M-CSF+PMA-induced MDSCs also significantly prolonged the survival time of skin grafts in mice, showing that MDSCs exert immunosuppressive effects in vivo. We describe a novel scheme to induce immunosuppressive MDSCs in vitro. MDSCs induced by M-CSF with PMA showed stable immunosuppression. MDSCs induced by this protocol may benefit patients with organ transplantation through immune regulation.

Blockade of the IL-21 Pathway: A New Perspective for the Treatment of T and B Cell Mediated Allogeneic Responses after Transplantation

Purpose IL-21 is a T cell growth factor for and secreted by Th17 and T follicular helper (Tfh)-cells. This cytokine regulates CD8+ T cell expansion and their effector functions, and is crucial for T cell-dependent B cell differentiation of antigen-activated B cells into antibody-producing plasma cells. Little is known about IL-21 mediated T and B cell responses in alloreactivity after transplantation. Methods First, we explored the actions of IL-21 in alloreactive T cell proliferation and cytotoxicity in mixed lymphocyte reactions. Second, to test the role of IL-21 producing Tfh cells in the regulation of B cells in an alloantigen-driven setting, we performed co-culture experiments of transplant recipient Tfh cells and B cells stimulated ith alloantigen. Third, the effect of an αIL-21R blocking antibody on allograft rejection was studied in a humanized skin transplantation model in mice reconstituted with human T and B cells. Results Alloactivated T and B cells highly express IL-21R. In the presence of IL-21, a marked increased proliferative response of alloactivated T cells was found. Also, the expanded CD8+ T cells had significant cytolytic functions, while blockade of the IL-21 route by an αIL-21R antagonist inhibited the proliferation of alloactivated T-cells. Next, we determined the role of IL-21 in T cell dependent B cell responses. Donor antigen stimulation of the co-cultured Tfh - B cells initiated expression of the activation markers ICOS and PD-1 on Tfh cells with a shift toward a mixed Tfh2 and Tfh17 phenotype. The alloantigen activated memory B cells underwent class switch recombination and differentiated toward IgM- and IgG-producing plasmablasts. Anti-IL-21R antagonists significantly inhibited B cell differentiation. Finally, in the humanized mouse skin transplant model, in mice treated with the αIL-21R antagonist reduced signs of alloreactivity were measured including significantly less CD4+ and CD8+ T and B cell infiltration and less expression of inflammatory markers Keratin 17 and Ki67. Conclusion These data show that comparing the effects of new IL-21-targeting therapeutics against the conventional immunosuppressants used in transplantation would be of considerable interest.

SINOMENINE ENHANCES IMDC DERIVED FROM IPS CELL-DIRECTED DIFFERENTIATION TO INDUCE TRANSPLANT IMMUNE HYPORESPONSIVENESS IN MOUSE

Introduction: Induced pluripotent stem cells (iPS) have a wide source and is not restricted by ethics, so that it is expected to become a new source of immature dendritic cells (imDCs). The Chinese medicine monomer sinomenine (SN) can reduce the expression of co-stimulatory molecules on the surface of imDCs, thereby inhibiting the maturation of imDCs. Therefore, in this study, we intend to explore the process of SN acting on induced pluripotent stem (iPS) to differentiate into imDCs, and in order to obtain imDCs (SN-iPS-imDCs) that can remain immature for a long time. Methods: IPS cells were differentiated into imDCs (SN-iPS-imDCs and iPS-imDCs) with three-steps differentiation method with SN. A mouse skin transplantation model was established; SN-iPS-imDCs (1×106 cells) were infused into the mouse skin transplantation model, and 5 groups of controls were established; the skin rejection score, graft survival rate and body weight were observed in recipient mouse; the serum levels of cytokines, the proportion of lymphocyte subsets CD4+CD25+Foxp3+Tregs in the spleen, and the relative expression of Foxp3 mRNA were detected in the recipient mouse; unrelated third-party lymphocytes were added to the lymphocyte mixed culture, and the specificity of induced transplant immunoreactivity by SN-iPS-imDCs and iPS-imDCs were observed; the tissue destruction and inflammatory cell infiltration in the skin graft were detected by histopathology. Results: A mouse skin transplantation model was successfully established. The scores of skin rejection in the SN-iPS-imDCs group were significantly lower than those in the PBS, SN and iPS-imDCs groups at each time point after surgery; on day 28 of transplantation, the body weight of the SN-iPS-imDCs group was increased significantly compared with day 0 (P<0.05); survival analysis showed that the survival time of skin grafts in the SN-iPS-imDCs group was significantly longer than that in the SN, iPS-imDCs and PBS groups (P<0.05);from the 3rd day to the 7th day of the transplantation, the expression levels of IL-12, IL-2 and IFN-γ in the serum of each group were increased except the CsA group, and the elevation level in the SN-iPS-imDCs group was significantly lower than that in the SN, iPS-imDCs and PBS groups (P<0.05); the serum IL-10 levels in the SN-iPS-imDCs group were second only to the CsA group but significantly higher than those in the PBS, iPS-imDCs and SN groups (P<0.05); in the SN-iPS-imDCs group, the spleen CD4+CD25+Tregs and CD4+CD25+Foxp3+Tregs accounted for the highest proportion of CD4+T cells, and there were significant differences compared with other groups (P<0.05); the Foxp3 mRNA expression in SN-iPS-imDCs group was significantly higher than that in iPS-imDCs group (P <0.05); on day 7 of transplantation, spleen T cells in SN-iPS-imDCs group and iPS-imDCs group were significantly less reactive to donor lymphocytes than unrelated third-party C3H mouse. The SN-iPS-imDCs group had lower response to donor lymphocytes than the iPS-imDC group (P<0.05). HE staining of skin grafts showed that the inflammatory response of the grafts in the SN-iPS-imDCs group was significantly lower than that in the iPS-imDCs and PBS groups. Conclusions: Infusion of donor-derived SN-iPS-imDCs could induce donor-specific transplantation immunoreactivity, could reduce the incidence of skin graft rejection, and could prolong the survival time of transplanted skin grafts. National Nature Science Foundation of China (No. 81900686). National Nature Science Foundation of China (No. 81100179).

Monitoring Early-Stage Acute Rejection by Imaging CXCR3-Positive Cell Infiltration: Evaluation of ¹²⁵Iodine-Labeled CXCL10.

It has been reported that CXCR3 is related to inflammatory cell infiltration. The purpose of this study was to investigate iodine-125-labeled CXCL10, a ligand of CXCR3, as a tracer targeting CXCR3 to detect acute rejection in a mouse skin transplant model. The isograft and allograft skin models were established with BALB/c and C57BL/6 mouse skin, respectively, as donors and BALB/c mice as recipients. We used reverse transcriptase-polymerase chain reaction and immunochemistry staining to test CXCR3 expression. ¹²⁵I-labeled CXCL10 was produced with the iodogenic method. Allograft/isograft mice were examined with whole body autoradiography and ex vivo biodistribution after tail vein injection of ¹²⁵I-labeled CXCL10 on day 8 posttransplant. CXCR3 expression was higher in allograft tissue than in isograft control. ¹²⁵I-labeled CXCL10 was prepared with high specificity and affinity. Biodistribution results showed higher ¹²⁵I-labeled CXCL10 uptake in allograft tissue. The target-to-nontarget ratio was 3.01 ± 0.25 at 24 hours, a result higher than that shown in the isograft group. Pharmacokinetic analyses of ¹²⁵I-labeled CXCL10 showed that distribution half-life was 0.34 hour and the elimination half-life was 9.83 hours. Dynamic whole body autoradiography images of ¹²⁵I-labeled CXCL10 showed excellent graft visualization in the allograft compared with the isograft group at all checking points, with visualization much more obvious at 12 and 24 hours. These data suggest that CXCR3 is a promising imaging target for immune cell infiltration in early-stage acute rejection and ¹²⁵I-labeled CXCL10 can successfully image acute rejection with good pharmacokinetics.

Regulatory T Cell Extracellular Vesicles Modify T-Effector Cell Cytokine Production and Protect Against Human Skin Allograft Damage.

Regulatory T cells (Tregs) are a subpopulation of CD4+ T cells with a fundamental role in maintaining immune homeostasis and inhibiting unwanted immune responses using several different mechanisms. Recently, the intercellular transfer of molecules between Tregs and their target cells has been shown via trogocytosis and the release of small extracellular vesicles (sEVs). In this study, CD4+CD25+CD127lo human Tregs were found to produce sEVs capable of inhibiting the proliferation of effector T cells (Teffs) in a dose dependent manner. These vesicles also modified the cytokine profile of Teffs leading to an increase in the production of IL-4 and IL-10 whilst simultaneously decreasing the levels of IL-6, IL-2, and IFNγ. MicroRNAs found enriched in the Treg EVs were indirectly linked to the changes in the cytokine profile observed. In a humanized mouse skin transplant model, human Treg derived EVs inhibited alloimmune-mediated skin tissue damage by limiting immune cell infiltration. Taken together, Treg sEVs may represent an exciting cell-free therapy to promote transplant survival.

The Effects of an IL-21 Receptor Antagonist on the Alloimmune Response in a Humanized Mouse Skin Transplant Model.

Interleukin 21 (IL-21) is involved in regulating the expansion and effector function of a broad range of leukocytes, including T cells and B cells. In transplantation, the exact role of IL-21 in the process of allograft rejection is unknown. To further explore this, the aim of this study is to test the effect of an IL-21 receptor (IL-21R) blocking antibody on the early phase of allograft rejection in a humanized skin transplantation model in mice reconstituted with human T and B cells. Immunodeficient Balb/c IL2rγRag2 mice were transplanted with human skin followed by adoptive transfer of human allogeneic splenocytes. Control animals were treated with a phosphate buffered saline vehicle while the other group was treated with a humanized anti-IL-21R antibody (αIL-21R). In the phosphate buffered saline-treated animals, human skin allografts were infiltrated with lymphocytes and developed a thickened epidermis with increased expression of the inflammatory markers Keratin 17 (Ker17) and Ki67. In mice treated with αIL-21R, these signs of allograft reactivity were significantly reduced. Concordantly, STAT3 phosphorylation was inhibited in this group. Of note, treatment with αIL-21R attenuated the process of T and B cell reconstitution after adoptive cellular transfer. These findings demonstrate that blockade of IL-21 signaling can delay allograft rejection in a humanized skin transplantation model.

In situ repurposing of dendritic cells with CRISPR/Cas9-based nanomedicine to induce transplant tolerance

Organ transplantation is the only effective method to treat end-stage organ failure. However, it is continuously plagued by immune rejection, which is mostly caused by T cell-mediated reactions. Dendritic cells (DCs) are professional antigen-presenting cells, and blocking the costimulatory signaling molecule CD40 in DCs inhibits T cell activation and induces transplant tolerance. In this study, to relieve graft rejection, Cas9 mRNA (mCas9) and a guide RNA targeting the costimulatory molecule CD40 (gCD40) were prepared and encapsulated into poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLAN), denoted CLANmCas9/gCD40. CLAN effectively delivered mCas9/gCD40 into DCs and disrupted CD40 in DCs at the genomic level both in vitro and in vivo. After intravenous injection into an acute mouse skin transplant model, CLANmCas9/gCD40-mediated CD40 disruption significantly inhibited T cell activation, which reduced graft damage and prolonged graft survival. This work provides a promising strategy for reprogramming DCs with nanoparticles carrying the CRISPR/Cas9 system to abate transplant rejection.

Transiently Activated Human Regulatory T Cells Upregulate BCL-XL Expression and Acquire a Functional Advantage in vivo.

Regulatory T cells (Tregs) can control excessive or undesirable immune responses toward autoantigens, alloantigens, and pathogens. In transplantation, host immune responses against the allograft are suppressed through the use of immunosuppressive drugs, however this often results in life-threatening side effects including nephrotoxicity and an increased incidence of cancer and opportunistic infections. Tregs can control graft-vs.-host disease and transplant rejection in experimental models, providing impetus for the use of Tregs as a cellular therapy in clinical transplantation. One of the major barriers to the widespread use of Treg cellular therapy is the requirement to expand cells ex vivo to large numbers in order to alter the overall balance between regulatory and effector cells. Methods that enhance suppressive capacity thereby reducing the need for expansion are therefore of interest. Here, we have compared the function of freshly-isolated and ex vivo-manipulated human Tregs in a pre-clinical humanized mouse model of skin transplantation. Sorted human CD127loCD25+CD4+ Tregs were assessed in three different conditions: freshly-isolated, following transient in vitro activation with antiCD3/antiCD28 beads or after ex vivo-expansion for 2 weeks in the presence of antiCD3/antiCD28 beads and recombinant human IL2. While ex vivo-expansion of human Tregs increased their suppressive function moderately, transient in vitro-activation of freshly isolated Tregs resulted in a powerful enhancement of Treg activity sufficient to promote long-term graft survival of all transplants in vivo. In order to investigate the mechanisms responsible for these effects, we measured the expression of Treg-associated markers and susceptibility to apoptosis in activated Tregs. Transiently activated Tregs displayed enhanced survival and proliferation in vitro and in vivo. On a molecular level, Treg activation resulted in an increased expression of anti-apoptotic BCL2L1 (encoding BCL-XL) which may be at least partially responsible for the observed enhancement in function. Our results suggest that in vitro activation of human Tregs arms them with superior proliferative and survival abilities, enabling them to more effectively control alloresponses. Importantly, this transient activation results in a rapid functional enhancement of freshly-isolated Tregs, thereby providing an opportunity to eliminate the need for in vitro expansion in select circumstances. A protocol employing this technique would therefore benefit from a reduced requirement for large cell numbers for effective therapy.

Peptide Tk-PQ induces immunosuppression in skin allogeneic transplantation via increasing Foxp3+ Treg and impeding nuclear translocation of NF-κB

Solid organ transplantation is used as the last resort for patients with end-stage disease, but allograft rejection is an unsolved problem. Here, we showed that Tk-PQ, a peptide derived from trichosanthin, had an immune-suppressive effect without obvious cytotoxicity in vitro and in a mouse skin allo-transplantation model. In vitro, treatment of Tk-PQ administrated type 2 T helper cell (Th2)/regulatory T-cell (Treg) cytokines, and increased the ratio of CD4+CD25+Foxp3+ Treg by repressing the PI3K/mTOR pathway. In addition, Tk-PQ decreased NF-κB activation to downregulate pro-inflammatory cytokines. Tk-PQ treatment in the mouse skin transplantation model also caused the similar molecular and cellular phenotypes. Furthermore, Tk-PQ enhanced the suppressive function of Treg by increasing Foxp3 expression, and substantially improved allograft survival. These finding demonstrate that Tk-PQ has the potential to be used in clinical allogeneic transplantation.

Skin transplantation in mice as an animal model for functional analysis of T regulatory cells.

The role of T regulatory cells (Tregs) in transplantology has been widely studied over the years. The mouse model of skin transplantation is used as a quick and easy way to monitor the immune response of Tregs, vital for creation and maintenance of homeostasis and immunological tolerance. This model is extremely useful in the assessment of allograft rejection and immune tolerance induction dependent on Tregs. The procedure of skin transplantation is widely used not only in transplantology, but also in studies on the effectiveness of various immunosuppressive compounds and studies on autoimmune diseases, including diabetes, multiple sclerosis and albinism. The improved protocol of mouse tail skin grafting is easy to master, and its wide application gives many opportunities for new solutions in biomedical research..

Recognition of Intact Donor Class I by Recipient CD8+ T Cells is Required for Tolerance Induction Following Liver-Directed Transgene Expression

Introduction: AAV-mediated expression of donor MHC I (H-2Kb or H-2Kd) in recipient liver induces donor-specific tolerance in a mouse skin transplant model where Kb or Kd are mismatched between donor and host [1]. Recognition of both intact and processed allogeneic class I could contribute to tolerance induction. Aims and Methods: To determine the relative importance of intact and processed class I in tolerance induction, we generated rAAV-Kb-D227K and Kd-D227K vectors where a point mutation in the class I alpha 3 domain abrogates CD8 binding [2] and direct allorecognition but does not interfere with recognition of processed peptides. Expression of WT or mutant class I on hepatocytes was assessed by FACS and IHC. TCR-transgenic T cells recognizing intact (Des-RAG) or processed (TCR75) allogeneic class I were used as reporter cells in adoptive transfer experiments. Skin bearing the mismatched MHC I was grafted onto uninjected recipients or mice inoculated with rAAV encoding WT or D227K mutant Kb or Kd. In some recipients, Tregs were depleted using anti-FR4 or Diphtheria toxin (DT). Interferon gamma production by responding T cells was determined using ELISpot. Results: WT and D227K class I were expressed on hepatocytes at comparable levels. Adoptive transfer assays confirmed that the D227K mutation abrogated recognition of intact Kb by CD8+ Des-RAG T cells, whilst recognition of processed Kd peptides by TCR75-RAG cells was unimpaired. Uninjected B10.BR mice rejected Kb-mismatched 178.3 skin (MST = 16 d), while grafts onto rAAV-Kb-injected mice survived indefinitely (p < 0.001). rAAV-Kb-D227K only slightly prolonged graft survival (MST = 27d, p < 0.05). These results were recapitulated in the B6.Kd to C57BL/6 model. The modest survival prolongation of skin grafts in mice inoculated with Kb-D227K was abrogated when the recipients were treated with anti-FR4 (clone TH6) to deplete Tregs. Similarly, the survival prolongation of Kd-mismatched B6.Kd grafts onto DEREG recipients treated with rAAV-Kd-D227K (MST = 28d compared with 20d in mice receiving DT alone) was blocked when mice receiving Kd-D227K were also treated with DT (MST 18 days, p = 0.0046). Treg depletion did not shorten survival of skin grafts in mice inoculated with WT MHC class I vectors. Interferon-gamma production by unfractionated or CD8-enriched splenocytes from primed C57BL/6 mice in response to B6.Kd stimulators was over fourfold lower in mice inoculated with WT Kd than in those receiving Kd-D227K (p = 0.038). Conclusions: Recognition of intact MHC class I by alloreactive CD8+ T cells is required for tolerance induction via liver-directed expression of donor MHC class I. Indirect recognition of class I allopeptides and generation of Tregs can produce a modest prolongation of class I-mismatched skin graft survival but does not induce tolerance.FigureFigure

The Effect of Traditional Chinese Formula Danchaiheji on the Differentiation of Regulatory Dendritic Cells.

Recently, regulatory dendritic cells (DCregs), a newly described dendritic cell subset with potent immunomodulatory function, have attracted increased attention for their utility in treating immune response-related diseases, such as graft-versus-host disease, hypersensitivity, and autoimmune diseases. Danchaiheji (DCHJ) is a traditional Chinese formula that has been used for many years in the clinic. However, whether DCHJ can program dendritic cells towards a regulatory phenotype and the underlying mechanism behind this process remain unknown. Herein, we investigate the effects of traditional Chinese DCHJ on DCregs differentiation and a mouse model of skin transplantation. The current study demonstrates that DCHJ can induce dendritic cells to differentiate into DCregs, which are represented by high CD11b and low CD86 and HLA-DR expression as well as the secretion of IL-10 and TGF-β. In addition, DCHJ inhibited DC migration and T cell proliferation, which correlated with increased IDO expression. Furthermore, DCHJ significantly prolonged skin graft survival time in a mouse model of skin transplantation without any liver or kidney toxicity. The traditional Chinese formula DCHJ has the potential to be a potent immunosuppressive agent with high efficiency and nontoxicity.

Defective CD8 Signaling Pathways Delay Rejection in Older Recipients.

CD8+ T cells play a cardinal feature in response to alloantigens and are able to generate effector/memory T cells independently from CD4+ T cells. To investigate the impact of aging on CD8 T cells, we used a fully mismatched mouse skin transplant model. Our findings showed a prolonged allograft survival in older recipients associated with a significant increase of CD4+ and CD8+ CD44high CD62Llow effector/memory T cells and a reduced systemic IFNγ production. When reconstituting young CBA Rag-1 mice that lack mature T and B cells with old CD8+ T cells expressing clonal anti-H2K T cell receptor (TCR) alloreactive for MHC I, graft survival was significantly prolonged and comparable to those receiving young CD8+ T cells. Moreover, our data showed that reduced systemic IFNγ levels observed in old recipients had been linked to a compromised expression of the IL-2R β subunit (CD122) by old CD8+ T cells. In addition, we observed an impaired IFNγ production on IL-2 receptor activation. At the same time, gene profiling analysis of old CD8 T cells demonstrated reduced chemokine ligand-3 and CD40L expression that resulted in compromised CD8+ T cell/dendritic cell communication, leading to impaired migratory and phagocytic activity of CD11c cells.Collectively, our study demonstrated that aging delays allograft rejection. CD8 T cells play a critical role in this process linked to a compromised production of IFNγ, in addition to a defective IL-2 receptor signaling machinery and a defective communication between CD8 T cells and dendritic cells.

Hypercapnic acidosis prolongs survival of skin allografts

BackgroundEvidence reveals that hypercapnic acidosis (HCA) modulates immune responses. However, the effect of HCA on allogenic skin graft rejection is unknown. We examined whether HCA might improve skin graft survival in a mouse model of skin transplantation. MethodsA major histocompatibility-complex-incompatible BALB/c to C57BL/6 mouse skin transplantation model was used. Animals were divided into sham control, air, and HCA groups. Mice in the HCA group were exposed daily to 5% CO2 in air for 1 h. Skin grafts were harvested for histologic analyses. Nuclear factor (NF)-κB activation was determined in harvested draining lymph nodes. Spleen weights and serum levels of tumor necrosis factor-α and chemokine (C-X-C motif) ligand 2 were serially assessed after skin transplantation. ResultsSkin allografts survived significantly longer in the HCA group of mice than those in the air group. Allografted mice in the air group underwent a 2.1-fold increase in spleen weight compared with a 1.1-fold increase in the mice with HCA on day 3. There were increased inflammatory cell infiltration, folliculitis, focal dermal–epidermal separation, and areas of epidermal necrosis in the air group that were reduced with HCA treatment. In the HCA group, CD8+ T cell infiltration at day 7 decreased significantly but not CD4+ T cell infiltration. In addition, HCA significantly suppressed serum tumor necrosis factor-α on days 1 and 3 and chemokine (C-X-C motif) ligand 2 on days 1 and 10. Furthermore, the HCA group had remarkably suppressed NF-κB activity in draining lymph nodes. ConclusionsHCA significantly prolonged the survival of incompatible skin allografts in mice by reducing proinflammatory cytokine production, immune cell infiltration, and NF-κB activation.