T cell antigenicity and immunogenicity of allogeneic exosomes

Extracellular vesicles, including exosomes, are regularly released by allogeneic cells after transplantation. Recipient antigen-presenting cells (APCs) capture these vesicles and subsequently display donor MHC molecules on their surface. Recent evidence suggests that activation of alloreactive T cells by the so-called cross-dressed APCs plays an important role in initiating the alloresponse associated with allograft rejection. On the other hand, whether allogeneic exosomes can bind to T cells on their own and activate them remains unclear. In this study, we showed that allogeneic exosomes can bind to T cells but do not stimulate them in vitro unless they are cultured with APCs. On the other hand, allogeneic exosomes activate T cells in vivo and sensitize mice to alloantigens but only when delivered in an inflammatory environment.     

Successful semiallogeneic and allogeneic bone marrow reconstitution of lethally irradiated adult mice mediated by neonatal spleen cells

Spleens of fetal/newborn mice less than 3-4 days of age contain a naturally occurring cell population capable of suppressing T-dependent and T-independent immune responses of third-party adult cells both in vitro and in vivo. We have utilized newborn spleen cells to prevent acute graft-versus-host (GVH) disease in lethally irradiated adult hosts reconstituted with semiallogeneic or even allogeneic bone marrow cells. Pretreatment of reconstituting cell populations with newborn spleen cells reduced the incidence of GVH disease from 100% to 20% in semiallogeneic and from 100% to 40% in allogeneic combinations. Long-term-surviving reconstituted hosts proved immunologically unresponsive to both donor and host histocompatibility antigens, yet possessed a fully chimeric lymphoid system responsive to T and B cell mitogens, as well as unrelated third-party alloantigens.     

人CD8+CD28-抑制性T淋巴细胞的体外扩增及其抗原特异性调节作用

目的 探讨在体外大量扩增CD8+CD28-抑制性T淋巴细胞(Ts细胞)的方法,并检验其免疫调节作用.方法 分离健康志愿者全血中CD8+T淋巴细胞,在含不同细胞因子和异体抗原提呈细胞(APC)的培养条件下进行体外扩增.应用流式细胞术对扩增过程中CD28 -细胞亚群的比例进行监测.分为3组进行混合淋巴细胞培养,反应细胞均为CD4+T淋巴细胞:B-APc组以来源于原致敏供者的APC作为刺激细胞,I-APC组以HLA-A、B、DR全错配的无关供者的APC作为刺激细胞,Dynabeads组以包被有抗CD3和CD28单克隆抗体的免疫微球Dynabeads作为刺激细胞;扩增后的Ts细胞作为第三方调节细胞加入混合淋巴细胞培养中,测定其对CD4+T淋巴细胞增殖的抑制作用.结果 在含白细胞介素2(IL-2)+ IL-7+ IL-15的培养条件下,CD8+T淋巴细胞培养后CD8+CD28-T淋巴细胞亚群的比例最高(P＜0.05).B-APC组不加入Ts细胞时,增殖的CD4+T淋巴细胞比例为66.7％,当加入的Ts细胞与反应细胞的比例分别为0.5∶1、0.1∶1和0.02∶1时,增殖的CD4+T淋巴细胞比例分别为16.5％、34.1％和62.6％.Ts细胞对CD4+T淋巴细胞的增殖有明显抑制作用,而在I-APC组和Dynabeads组中,此抑制作用不明显.结论 应用含IL-2+ IL-7+ IL-15的培养条件联合异体APC刺激可以在体外大量扩增Ts细胞,扩增所得Ts细胞在体外对供者CD4+T淋巴细胞的增殖有明显抗原特异性抑制作用.     

Engraftment following T cell-depleted bone marrow transplantation. III. Differential effects of increased total-body irradiation on semiallogeneic and allogeneic recipients

Three different doses of total-body irradiation (TBI) (1100, 1300, 1500 cGy) have been analyzed as conditioning regimens for semiallogeneic B6AF1 (H-2b X H-2a) and allogeneic A/J (H-2a) recipients of T cell-depleted C57BL6 (H-2b) bone marrow transplants. Recipient survival and engraftment of both donor erythrocytes and lymphocytes were examined in each group. The large majority of allogeneic mice prepared with 1100 cGy rejected their grafts, which resulted in poor survival (less than 30%); improved survival (up to 80%) and complete donor engraftment were noted as the TBI dose was increased. By contrast, survival in semiallogeneic B6AF1 recipients was independent of TBI dose and was greater than 80% in all groups. Outright failure of marrow grafts (less than 10% donor hematopoiesis) did not occur in these recipients, but mixed chimerism (simultaneous occurrence of both donor and host cells) was frequently observed at lower TBI doses. Complete (greater than 90%) donor engraftment was noted for erythrocytes but not for lymphocytes. Possible mechanisms accounting for these differences between semiallogeneic and allogeneic recipients of marrow transplants are discussed.     

Antithymocyte globulin impairs T-cell/antigen-presenting cell interaction: disruption of immunological synapse and conjugate formation

Antithymocyte globulin (ATG) is employed for the treatment and prevention of acute organ rejection after transplantation. However, the mechanisms underlying its immunomodulatory capacities beyond cellular depletion remains ill defined. A stable interaction between T-cells and professional antigen-presenting cells (APC) and full T-cell stimulation requires a complex molecular rearrangement at the T-cell/APC interface, the so called immunological synapse. Here we investigated, whether ATG affects T-cell/APC interactions. ATG concentration and time-dependently inhibited relocalization of the T-cell receptor/CD3 complex as well as adhesion molecules and cytoskeletal proteins of human peripheral blood T-cells and a human T-cell line towards the APC contact site. Moreover, ATG-treated peripheral blood T-cells were incapable to form conjugates with APCs. In conclusion, ATG impairs T-cell/APC conjugate formation, a mechanism that may help to understand the functional inactivation of peripheral blood T-cells that have escaped cellular depletion after ATG treatment.     

Prolonged allograft survival through conditional and specific ablation of alloreactive T cells expressing a suicide gene

BACKGROUND: Control of antidonor activated T cells involved in allograft rejection while preserving immunocompetence is a challenging goal in transplantation. Engineered T cells expressing a viral thymidine kinase (TK) suicide gene metabolize the nontoxic prodrug ganciclovir (GCV) into a metabolite toxic only to dividing cells. We evaluated this suicide gene strategy for inducing transplantation tolerance in mice. METHODS: Transgenic mice expressing TK in mature T cells were analyzed for (i) specific T-cell depletion under GCV treatment upon various stimulations; (ii) outcome of allogeneic nonvascularized skin or heart allografts under a short 14-day GCV treatment initiated at the time of transplantation; and (iii) the capacities of T cells from such allotransplanted mice to proliferate in mixed lymphocyte reactions and to induce graft-versus-host disease in irradiated recipients with the genetic background of the donor allograft. RESULTS: Upon in vitro or in vivo GCV treatment, only activated dividing TK T cells but not B cells were efficiently depleted. Acute rejection of allogeneic grafts was prevented and a significant prolongation of graft survival was obtained, although associated with signs of chronic rejection. Prolonged skin graft survival correlated with decreased in vitro and in vivo T-cell reactivities against donor alloantigens, whereas overall immunocompetence was preserved. CONCLUSIONS: Efficient and specific depletion of alloreactive TK T cells can be achieved by administrating GCV. These results open new perspectives for the control of allogeneic graft rejection using suicide gene therapy.     

Induction of transplantation tolerance in humans using fetal cell transplants

When engrafted with donor stem cells and lymphoid cells, patients develop transplantation tolerance to donor antigens. We analyzed the mechanism of tolerance induction in immunoincompetent recipients whose immunity has been reconstituted by transplantation of mismatched stem cells. Seven infants or human fetuses received fetal liver transplants as a treatment for severe combined immunodeficiency disease. After reconstitution of immunity by lymphocytes developed from donor stem cells, T-cell clones were produced and analyzed. Because donors and recipients were HLA mismatched, it was easy to demonstrate the donor origin of the T-cell clones. These clones were shown to have developed tolerance to histocompatibility antigens of the stem cell donor via a process of clonal deletion (probably as a result of contact with donor-derived macrophages and dendritic cells). They were also tolerant to histocompatibility antigens of the host but through a different mechanism: many clones recognized these antigens but had no detrimental effect on the target cells exhibiting host antigens, either in vitro or in vivo. Clonal anergy was therefore the cause of this tolerance to host determinants, resulting in a lack of graft-versus-host disease and of autoimmunity. The contact between developing T cells of donor origin and host epithelial cells within the host thymus may explain this colonal anergy. It should be noted that all patients had high serum levels of interleukin-10, which might have contributed to the persistent engraftment and tolerance.     

In vivo T-cell depletion enhances production of anti-GALalpha1,3GAL natural antibodies in alpha1,3-galactosyltransferase-deficient mice

BACKGROUND: It has been reported that T-cell depletion by in vivo treatment with monoclonal antibodies results in polyclonal B-cell activation. However, its effects on B cells responding to Galalpha1,3Gal (Gal) epitopes remain unknown. METHODS: alpha1,3-Galactosyltransferase-deficient (GalT-/-) mice were treated with depleting anti-CD4 and CD8 monoclonal antibodies. The kinetics of anti-Gal natural antibodies (NAb) and total immunoglobulin levels in their sera were evaluated. The frequencies of anti-Gal NAb-producing cells were determined in the various tissues of GalT-/- mice by enzyme-linked immunospot assay. RESULTS: In vivo T-cell depletion led to significant increases in both anti-Gal IgM and total IgM levels in sera of GalT-/- mice, but did not influence either anti-Gal IgG or total IgG levels. An increased frequency of anti-Gal and total IgM-producing cells was observed in the spleens and bone marrow of T-cell-depleted GalT-/-mice but not in peritoneal cavity cells. CONCLUSION: In vivo T-cell depletion facilitates anti-Gal IgM production, suggesting that T cells deliver inhibitory signals to B cells responding to Gal.     

Transient Depletion of Dividing T Lymphocytes in Mice Induces the Emergence of Regulatory T Cells and Dominant Tolerance to Islet Allografts

We previously showed that transient depletion of dividing T cells at the time of an allogeneic transplantation induces long-term tolerance to the allograft. Here we investigated the role of homeostatic perturbation and regulatory T cells (Treg) in such tolerance. Transient depletion of dividing T cells was induced at the time of an allogeneic pancreatic islets graft, by administration of ganciclovir for 14 days, into diabetic transgenic mice expressing a thymidine kinase (TK) conditional suicide gene in T cells. Allograft tolerance was obtained in 63% of treated mice. It was not due to global immunosuppression, permanent deletion or anergy of donor-alloantigens specific T cells but to a dominant tolerance process since lymphocytes from tolerant mice could transfer tolerance to naïve allografted recipients. The transient depletion of dividing T cells induces a 2- to 3-fold increase in the proportion of CD4⁺CD25⁺Foxp3⁺ Treg, within 3 weeks that persisted only in allograft-bearing mice but not in nongrafted mice. Tolerance with similar increased proportion of Treg cells was also obtained after a cytostatic hydroxyurea treatment in normal mice. Thus, the transient depletion of dividing T cells represents a novel means of immuno-intervention based on disturbance of T-cell homeostasis and subsequent increase in Treg proportion.     

Plasmacytoid Dendritic Cell Precursors Induce Allogeneic T-Cell Hyporesponsiveness and Prolong Heart Graft Survival

Dendritic cell (DC) precursors were propagated from C57BL/10 (B10; H2b) mouse bone marrow in fms-like tyrosine kinase 3 ligand. Cosignaling molecule (B7-1/B7-2 and B7-H1) expression and stimulatory capacity of precursor (pre)-plasmacytoid (p)DC (CD11c+B220+CD11b-CD19-) and classic myeloid DC (MDC) for allogeneic (C3H; H2k) T cells were compared. Unstimulated pre-pDC exhibited very low levels of surface MHC class II and classic costimulatory molecules (B7-1/B7-2), whereas a minor population expressed B7-H1 at levels higher than on MDC. The pre-pDC were ineffective T-cell stimulators and induced nonspecific hyporesponsiveness to rechallenge with donor alloantigens in vitro and in vivo. Following stimulation with CpG-oligonucleotide (CpG-ODN), B7 molecule expression was upregulated on pre-pDC, however the ratio between coinhibitory (B7-H1) and costimulatory (B7-1/B7-2) signals was much higher (five- to six-fold) on pre-pDC than MDC. Blockade of B7-H1 expression on pDC increased their T-cell allostimulatory capacity significantly. A single preoperative infusion of C3H hosts with pre-pDC prolonged B10 heart graft survival significantly but nonspecifically compared with untreated mice (median survival times 22 vs. 9 days, respectively). Thus, pre-pDC of donor origin have potential to regulate T-cell responses to alloantigens and can prolong organ graft survival.     

Maturation-Resistant Dendritic Cells Induce Hyporesponsiveness in Alloreactive CD45RA+ and CD45RO+ T-Cell Populations

Dendritic cells (DCs) play a crucial role in the induction of antigen-specific immunity and tolerance. Considering in vivo application of DCs prior to human organ transplantation, a protocol to develop tolerogenic DCs that not only induce unresponsiveness in naive (CD45RA+) T cells, but also in alloreactive memory (CD45RO+) T cells is required. The present study shows that dexamethasone (Dex) alters the differentiation of human monocyte-derived DCs. DexDCs cocultured with allogeneic CD4+ T cells induced low proliferating and low IFNgamma producing T cells. This is caused by lack of both costimulation via CD28 and hampered production of a soluble factor, as well as additional active suppression via B7-H1 and IL-10. T cells primed by DexDCs demonstrated hyporesponsiveness upon restimulation with mature DCs seemingly via the induction of anergy, since these cells showed no enhanced apoptosis and only a limited suppressive capacity. Interestingly, not only cocultures of allogeneic CD45RA+, but also of CD45RO+ T cells with DexDCs rendered T-cell populations hyporesponsive to restimulation with mature DCs. The finding that also alloreactive memory T cells can be regulated supports the rationale of cell-based therapies to obtain allograft-specific tolerance in transplant recipients.     

The Immune Regulatory Effect of Apoptotic Cells and Exosomes on Dendritic Cells: Its Impact on Transplantation

Dendritic cells (DC) are professional antigen (Ag) presenting cells (APC) that trigger the anti-donor T-cell response that causes allograft rejection. During the past decade several laboratories have employed in vitro generated DC with tolerogenic potential for prolongation of allograft survival. This minireview describes the development of a second-generation of DC-based strategies for transplantation tolerance based on the delivery in situ of donor allogeneic (allo)-Ag to quiescent DC of graft recipients by means of donor-derived apoptotic cells or exosomes. Donor leukocytes in early apoptosis are rich in allo-Ag, are internalized efficiently by recipient DC in vivo and deliver immunosuppressive signals to DC. Administration (i.v.) of donor apoptotic leukocytes prolongs bone marrow engraftment and cardiac allografts survival in mice by exerting a profound down-regulatory effect on the anti-donor T-cell response. Exosomes are nanovesicles (<100 nm) produced by different cell types, including APC. DC-derived exosomes are rich in major histocompatibility complex (MHC) molecules that can be employed to target DC in situ. Once i.v. injected, exosomes carrying donor MHC molecules are captured by recipient's DC and prolong allograft survival in rodents. The use of the regulatory functions of apoptotic cells and exosomes may be useful tools to develop new strategies for transplantation tolerance.     

Signal transduction and co-stimulatory pathways

Using specific cell surface receptors lymphocytes continuously sample their environment. Maturation of the immune system and initiation of a specific immune response rely on an array of extracellular cues that elicit complex intracellular biochemical signals. Essential molecules involved in signal transduction from immunoreceptors have emerged. After immunoreceptor engagement a core signaling complex is assembled comprising cytoplasmic immunoreceptor chains, kinases of the Src and ZAP70 families and various cytoplasmic and transmembrane adaptor molecules. Further effectors nucleate onto this complex evoking the characteristic responses of lymphocyte activation. Successful maturation of T cells into effector cells relies on the presence of a persistent stimulus presented in an appropriate extracellular environment. Encounter of MHC presented antigenic peptides and their cognate T cell receptors (TCRs) results in the formation of a nanometer intercellular gap between T cells and antigen presenting cells, which is now commonly referred to as the immunological synapse. The synapse is believed to sustain persistent TCR engagement. Its formation requires massive changes in T cell cytoskeletal architecture which essentially relies on signals provided by costimulatory molecules. The well orchestrated interplay between TCR and costimulatory signals decides about successful immune response and tolerance induction or immune failure and autoimmunity.     

Photodynamic therapy and anti-tumor immunity

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) efficacy appears to be enhanced in the presence of an intact immune system and PDT has been shown to augment anti-tumor immunity. The mechanisms leading to the enhancement of the host immune response to tumor are unclear. Anti-tumor immunity depends upon the presence of activated antigen presenting cells (APCs). These cells are activated by their recognition of components released by pathogens, viruses, dead cells, and the presence of pro-inflammatory mediators. Activated APCs stimulate the generation of cytokine secreting effector cells. Therefore, we have hypothesized that PDT generated inflammatory mediators and components released from tumor cells killed by PDT results in the activation of APCs capable of stimulating effector T-cell proliferation and cytokine secretion. STUDY DESIGN/MATERIALS AND METHODS: To determine the effect of PDT on APCs, tumor draining lymph nodes (TDLNs) of EMT6 or Colo 26 tumor bearing mice were isolated 24 hours after Photofrin-PDT and flow cytometry was used to detect the presence of APCs secreting the T cells stimulatory cytokine, IL-12. APCs were also isolated from TDLNs and used to stimulate T-cell proliferation and secretion of interferon-gamma (IFN-gamma). RESULTS: PDT results in an increase in IL-12 expressing APCs in the TDLN. This increase was accompanied by an increase in the ability of APCs isolated from TDLNs of PDT-treated mice to stimulate T-cell proliferation and T-cell secretion of IFN-gamma. CONCLUSIONS: Our results indicate that APCs isolated from PDT-treated mice exhibit an enhanced ability to stimulate T-cell proliferation and IFN-gamma secretion, suggesting that PDT results in increased APC activity.     

The novel immunosuppressant FK778 inhibits formation of the immunologic Synapse

The malononitrilamide FK778 is a derivative of A77 1726, the active metabolite of the antirheumatic drug leflunomide. A77 1726 inhibits de novo pyrimidine synthesis and activity of Src-family kinases; thus, it may interfere with T-cell proliferation as well as with early T-cell signaling. Formation of a stable interaction between T cells and antigen-presenting cells (APC)--the immunologic synapse--has emerged to be of crucial importance for T-cell activation. Here in we show that FK778 inhibits formation of the immunologic synapse by blocking superantigen-stimulated relocalization of adhesion (LFA-1), and signaling molecules (CD3) to the T-cell/APC contact site. These data show that FK778 affects T-cell/APC interactions, particularly events crucial for T-cell adhesion and formation of stable conjugates underlying sustained and effective T-cell activation. Thus, in this model system close to physiologic T-cell stimulation, FK778 affects critical events in the course of T-cell-mediated immune responses earlier than T-cell proliferation, which may contribute to its immunosuppressive potential.     

Immunosuppressive Activity of Adipose Tissue-Derived Mesenchymal Stem Cells in a Rat Model of Hind Limb Allotransplantation

Many reports have shown that bone marrow–derived mesenchymal stem cells exhibit immunosuppressive effects in allogeneic transplantation. However, few reports have evaluated the immunosuppressive properties of adipose tissue–derived mesenchymal stem cells (ASCs) in vitro and in vivo. In this study, we investigated the immunosuppressive characteristics of ASCs, and investigated whether ASCs originating from donor rats prolong allotransplant survival in a rat hind limb allotransplantation model. T-cell proliferation stimulated by allogeneic stimuli or mitogen with or without ASCs originating from the donor was assessed in vitro. The effects of cellular contact or soluble factors on the inhibition of T-cell proliferation were also evaluated. In the in vivo study, cultured ASCs (1 × 10⁵) that originated from the donor were injected into recipient animals intravenously immediately after operation, followed by 1 dose per day for 3 consecutive days post-transplantation. When immune rejection occurred, the survival time of allotransplants was determined and rejected tissue was histologically and immunochemically assessed for determining regulatory T-cell infiltration. ASCs inhibited the T-cell proliferation stimulated by alloantigen or mitogen in a dose-dependent manner, and recipient T cells proliferated less in animals treated with ASCs than in controls. Although ASCs were separated from T cells, ASCs persisted to elicit a suppressive effect. ASC culture supernatants did not inhibit T-cell proliferation; however, supernatants obtained from the mixed lymphocyte reaction in the presence of ASCs suppressed T-cell proliferation. ASCs prolonged allotransplant survival time, reduced inflammatory cell infiltration, and induced regulatory T cells. In conclusion, ASCs can exhibit in vitro immunosuppressive properties and prolong allotransplant survival time in a rat hind limb composite tissue allotransplantation model, possibly through the induction of regulatory T cells.     

Allogeneic stimulation of naturally occurring CD4+CD25+ T cells induces strong regulatory capacity with increased donor-reactivity

Current therapies in transplantation require continuous immunosuppression and do not result in transplantation tolerance. It is increasingly appreciated that CD4(+)CD25(+) regulatory T-cell (T(REG)) activation is pivotal for the induction and maintenance of peripheral tolerance. To optimally exploit T(REG) in allograft tolerance, we investigated how to further harness their function. In vitro, CD4(+)CD25(+)T cells were expanded by allogeneic bone-marrow derived DC or polyclonal stimulation and were compared in suppressive capacity and phenotype. In vivo, naive allogeneic CD4(+)CD25(+)T cells were analyzed in wild type hosts for proliferative capacity and suppressive capacity upon priming by alloantigen. DC of donor origin were found to potently stimulate alloreactive T(REG)in vitro. This was accompanied by a substantial enhancement of the suppressive capacity of the T(REG) population as a whole, likely due to a proportional rise of alloreactive T(REG) as indicated by CFSE analysis. In vivo analysis of infused naturally occurring allogeneic T(REG) revealed a robust proliferative capacity for T(REG) upon stimulation. Moreover, allogeneic skin transplantation resulted in enhanced capacity of the T(REG) population to suppress the response towards donor antigens. Combining, activation of alloreactive T(REG) is an intrinsic part of the regular alloimmune response and this feature can be exploited for therapeutic purposes. We propose that selectively favoring the effects of alloreactive T(REG) is a pivotal element in inducing graft acceptance.     

Generation of NO by Bystander Human CD8 T Cells Augments Allogeneic Responses by Inhibiting Cytokine Deprivation-Induced Cell Death

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in bystander human CD8 T cells, augments the accumulation of allogeneically activated human CD8 T cells in vitro and in vivo . Here, we report that iNOS-derived NO does not affect T-cell proliferation but rather inhibits cell death of activated human CD8 T cells after activation by allogeneic endothelial cells in culture. Exogenous NO did not affect activation-induced cell death of human CD8 T cells but specifically reduced death of activated T cells due to cytokine deprivation. NO-mediated inhibition of T-cell death did not involve cGMP signaling, and NO did not affect the expression of Bcl-2-related proteins known to regulate cytokine deprivation-induced cell death. However, NO inhibited the activity of caspases activated as a consequence of cytokine deprivation in activated T cells. This protective effect correlated with S-nitrosylation of caspases and was phenocopied by z-VAD.fmk and z-LEHD.fmk, pharmacological inhibitors of caspases. In summary, our findings indicate that NO augments the accumulation of activated human T cells principally by inhibiting cytokine deprivation-induced cell death through S-nitrosylation of caspases.