Primary Mixed Leukocyte Reaction Research Articles

Functional paralysis of GM-CSF–derived bone marrow cells productively infected with ectromelia virus

Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF–derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

Alloantigen-specific T-cell hyporesponsiveness induced by dnIKK2 gene-transfected recipient immature dendritic cells

Immature dendritic cells (iDCs) have been shown to be able to induce peripheral T-cell tolerance through distinct pathways. Here, we investigated the tolerogenic property of recipient iDCs whose maturation was arrested by a dominant negative mutant of inhibitor of nuclear factor kappa-B kinase 2 (dnIKK2) gene. We found that dnIKK2-iDCs presented a typical semi-mature morphology and expressed lower levels of CD80 and CD86, slightly higher MHC-II than untransfected iDCs. The expression of these molecules had no significant change even dnIKK2-iDCs were pulsed by donor antigen. In primary mixed leukocyte reaction (MLR), dnIKK2-iDCs exhibited impaired ability to stimulate allogeneic T-cells, but induced CD4+CD25− T-cell formation. In co-culture MLR, these CD4+CD25− T-cells suppressed T-cell alloreaction in an antigen-specific manner. Besides, CD4+CD25− T-cells inhibited IL-2 and IFN-γ release, whereas promoted IL-10 and TGF-β secretion. These data suggested recipient dnIKK2-iDCs could maintain peripheral tolerance through down-regulating costimulatory molecule expressions and inducing CD4+CD25− T-cell formation.

Dendritic cells induce antigen-specific regulatory T cells that prevent graft versus host disease and persist in mice

Foxp3(+) regulatory T cells (T reg cells) effectively suppress immunity, but it is not determined if antigen-induced T reg cells (iT reg cells) are able to persist under conditions of inflammation and to stably express the transcription factor Foxp3. We used spleen cells to stimulate the mixed leukocyte reaction (MLR) in the presence of transforming growth factor β (TGF-β) and retinoic acid. We found that the CD11c(high) dendritic cell fraction was the most potent at inducing high numbers of alloreactive Foxp3(+) cells. The induced CD4(+)CD25(+)Foxp3(+) cells appeared after extensive proliferation. When purified from the MLR, iT reg cells suppressed both primary and secondary MLR in vitro in an antigen-specific manner. After transfer into allogeneic mice, iT reg cells persisted for 6 mo and prevented graft versus host disease (GVHD) caused by co-transferred CD45RB(hi) T cells. Similar findings were made when iT reg cells were transferred after onset of GVHD. The CNS2 intronic sequence of the Foxp3 gene in the persisting iT reg cells was as demethylated as the corresponding sequence of naturally occurring T reg cells. These results indicate that induced Foxp3(+) T reg cells, after proliferating and differentiating into antigen-specific suppressive T cells, can persist for long periods while suppressing a powerful inflammatory disease.

ICOS-Ig combined with CsA induces long term survival of cardiac allografts in mouse

Objective To study the synergistic effect of ICOS-Ig combined with cyclosporine (CsA) on mouse heart transplantation and explore its therapeutic potential. Methods ICOS-Ig fusion protein was generated by fusing the extracellular portion of human ICOS and Fc portion of human IgG. To investigate the effect of ICOS-Ig on T-cell proliferation in vitro, ICOS-Ig or IgG was added to the primary MLR cultures (BALB/c spleen T cells as responder cells and irradiated C57BL/6 spleen cells as stimulator cells). The cells responsiveness rates were detected by 3H-TdR methods. Then the T cells of each group in primary MLR were cultured as responder cells for secondary MLR, and irradiated C57BL/6 (donor) or C3H (third party) spleen cells as stimulator cells. To study the effect of ICOS-Ig on T-cell proliferation in vivo, CFSE-labeled C57BL/6 spleen cells were transferred to irradiated BALB/c mice. Mice were then treated with IgG, ICOS-Ig or CsA. Seventy two hours after transfer, the spleen cells of the mice were harvested for the detection of CD4 +CFSE + and CD8 +CFSE + by FACS. C57BL/6 mouse underwent transplantation of the hearts of BALB/c mouse and were then randomly divided into five equal groups: no treatment group, control IgG treated group (250 μg i.p. d2, 4, 6), ICOS-Ig treated group (250 μg i.p. d2, 4, 6), CsA treated group (10 mg/kg i.p. d0-6), ICOS-Ig combined with CsA group. The cardiac allograft survival was monitored by daily palpation. Results In primary MLR, ICOS-Ig inhibited T-cell proliferation, (inhibition ratio 58±8.2% in 50 μg/ml). In secondary MLR, ICOS-Ig specifically inhibited donor spleen cells, which suggested ICOS-Ig could induce donor-specific hyporesponsiveness. In the CFSE dye assay, CD4 +CFSE + and CD8 +CFSE + in ICOS-Ig and CsA group was stronger than those in control group, which showed ICOS-Ig and CsA could inhibit the proliferation of allo-reactive T cells in vivo. In mouse heart transplantation model, survival was significantly prolonged in animals treated with ICOS-Ig or CsA as compared with controls. Moreover, ICOS-Ig combined with CsA group had even longer engraftment (>100 d) than ICOS-Ig or CsA used alone. In histological examination, it was found that there were congestions and edemas in no treatment and IgG treated recipients, together with a lot of inflammatory cells infiltrated. Allogeneic hearts from ICOS-Ig and/or CsA immunized recipients revealed milder histological changes. It was revealed in mechanical analysis that splenic T cells from recipients also exhibited depressed mixed leukocyte reactions (MLR) and cytotoxic lymphocyte reactions (CTL). Conclusion These data suggest that ICOS-Ig combined with CsA induces a long-term survival of mouse cardiac allografts, whereas monotherapy is less effective in this regard. Thus, ICOS-Ig combined with CsA treatment may be a novel regimen to combat allograft rejection.

Molecular Identification of Individual T Cell Clones Specific for Graft- Versus-Leukemia Reaction

Graft-verus-leukemia (GVL) effect in hematopoietic stem cell transplantation (HSCT) is usually complicated by the alloreactivity of donor T cells which leads to acute graft-versus-host (GVH) disease. GVL and GVH reactions are proved to be mediated by different T cell clones. The objective of this study was to identify and characterize T cells clones with specific antileukemia activity without mediating GVHD. We have performed primary mixed leukocyte reaction (MLR) using patient non-leukemic irradiated peripherial blood mononuclear cells (PBMC) as stimulators and donor PBMC as responders. To prepare GVL specific T cells, activated alloreactive T cells were first selectively depleted with an anti-CD25 immunotoxin (Michalek, et al. PNAS 2003, 100: 1180–4). Allodepleted T cells were then stimulated in secondary MLR using irradiated leukemia cells from the same patient. Activated leukemia-reactive cells were purified by immunomagnetic selection or by FACS based on INF-γ or CD25 expression, respectively. Clonotypic assay was used for identification of individual leukemia-specific T cell clones (Michalek, et al. Lancet 2003, 361: 1183–5; Michalek, et al. J Immunol 2007, 178: 6789– 5). This highly sensitive assay is based on detailed analysis of T cell receptor β VDJ unique sequence (TCRB-VDJ). mRNA was extracted from sortred activated cells and cDNA synthetized by anchored reverse transcription. Target TCRB-VDJ gene sequence was amplified by anchor PCR and used to transform bacteria. Bacterial colonies were picked for plasmid isolation and subsequent direct automated sequencing of the TCRBVDJ sequences. We assume that the frequency of particular TCRB-VDJ sequences among bacterial clones after transformation are proportional to the frequency of those sequences in the original population of T cells activated by GVH or GVL reaction. We investigated the presence of individual antileukemic T cell clones in patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), and defined them by the TCRB-VDJ unique sequence. The sequences that occured in more than 10% bacterial colonies are likely to represent the most immunodominant clones. Populations of antileukemic T cell clones were oligoclonal, i.e. we observed limited number of individual immunodominat clones which plays important role in GVL reaction. In first CLL patient who had undergone HSCT, six antileukemic T cell clones were identified, four of them are considered to be immunodominant. In second CLL patient after HSCT, only one highly immunodominat autileukemic T cell clone was observed. This specific clone was further monitored by quantitative real-time PCR in patients peripherial blood.

Peripheral blood and synovial fluid T cells differ in their response to alloantigens and recall antigens presented by dendritic cells

Properties of T cells from inflammatory lesions were analysed by comparing the response of peripheral blood (PB) and synovial fluid (SF) T cells from 19 patients with a range of arthropathies to enriched allogeneic dendritic cells (DC) in a primary mixed leucocyte reaction (MLR). In 17 patients the proliferative response of SF T cells was significantly (P less than 0.05) less than that of PB lymphocytes. The reduced response of SF T cells was observed in all disease categories studied and could not be attributed to differences in cell number requirements or response kinetics. Addition of recombinant interleukin-2 enhanced the response of SF T cells in a dose-dependent manner. Cell mixing experiments suggested that active suppression was not the underlying mechanism of the poor MLR response of SF T cells. In contrast to the MLR response. SF T cells were able to mount vigorous proliferative responses to recall antigen presented by autologous antigen-presenting cells. The possibility is discussed that T cells compartmentalized at inflammatory lesions are a unique population with a diminished ability to interact with DC and respond to primary stimuli but an ability to respond to secondary antigenic challenge.

Effect of Seliciclib (CYC202, R-Roscovitine) on Lymphocyte Alloreactivity and Acute Kidney Allograft Rejection in Rat

T cell stimulation by alloantigens is followed by cell cycle progression, an event that is critically dependent on cyclin-dependent kinases. We conducted a study to evaluate whether the cyclin-dependent kinase inhibitor seliciclib affected rat lymph node cells (LNc) activation and proliferation induced by either concanavalin A or allogeneic splenocytes in vitro and studied the mechanisms underlying the suppressive effect. We also investigated the immunosuppressive properties of seliciclib in vivo. Seliciclib completely inhibited in vitro proliferation of LNc and CD8 T cells, in response to either concanavalin A or allogeneic splenocytes. The percentage of activated LNc was lower in mixed leukocyte reactions (MLR) added with seliciclib than in MLR added with vehicle. The percentages of viable and apoptotic cells at the end of MLR with seliciclib were comparable to those of MLR with vehicle. LNc pre-exposed in MLR to seliciclib did not respond to further stimulation with alloantigens, and neither IL-2 nor IL-15 restored proliferation. These data indicate that the inhibitory effect of seliciclib on T cell alloreactivity is not because of cytotoxic effect but is associated with induction of profound T cell anergy. LNc harvested at the end of the primary MLR with seliciclib did not suppress the proliferation of syngeneic LNc cells toward allogeneic splenocytes, thus excluding that seliciclib induced the formation of regulatory cells. Finally, seliciclib partially prolonged grafted animal survival in a rat model of fully major histocompatibility complex-mismatched kidney transplantation. Altogether these results document that seliciclib regulates lymphocyte reactivity and may exert an immunosuppressive effect in vivo in the setting of transplantation.

Safe allogeneic hematopoietic stem cell transplantation for renal cell carcinoma: Novel approach using selective depletion of alloreactive donor T cells

19509 Background: A major challenge in the field of hematopoietic stem cell transplantation (HSCT) is to prevent the alloreactivity of donor T-cells which leads to acute graft-versus-host (GVH) disease while preserving a graft-versus-tumor (GVT) effect. Methods: renal carcinoma cells cells and peripheral blood mononuclear cells (PBMC) of ten patients were used with healthy donor PBMC in a primary mixed leukocyte reaction (MLR) where non-cancer irradiated PBMC from renal cell carcinoma patients served as stimulators and GVH target. GVH-reactive donor T cells were selectively depleted with anti-CD25 IT. Allodepleted donor T cells were then repeatedly stimulated using irradiated shortly cultured renal cell carcinoma tumor cells from the same cancer patient (to simulate GVT effect) or using irradiated PBMC from the same patient (to simulate GVH effect). HLA class I was monitored on the surface of renal cell carcinoma as well. Results: Long-term culture of renal cell carcinoma was succesful in 8 out of ten tumor cell cultures and reached 80–280 million tumor cells in 6–17 weeks. Selective depletion of alloreactive donor T-cells with anti-CD25 IT led to more than 2log depletion (99,2 - 100%, median 99,8%). GVT effect of donor T-cells was preserved (1,8% of donor T cells were GVT-reactive) while GVH reactivation of donor cells was negligible even after 3 rounds of repeated stimulation with patient’s non-tumor PBMC. Immunohistochemical staining for surface HLA class I revealed partial or complete loss of HLA antigens (50 - 100 %) in 4 cultures during 2–6 months of cultivation, 3 cultures remained stable and one culture was negative from the beginning. Conclusions: It is possible to selectively deplete donor alloreactive T-cells with anti-CD25 IT minimizing or eliminating the risk of GVH disease. This strategy eliminates alloreactivity and preserves GVT reactivity in patients with renal cell carcinoma. Thus the allogeneic hematopoietic stem cell transplantation using selectively allodepleted donor T cells seems to be a valuable alternative to recent treatment strategies in patients with renal cell carcinoma.Supported by the Ministry of Education of the Czech Republic, NPVII-2B06058. No significant financial relationships to disclose.

Selective Depletion of Alloreactive T Cells and Study of Anti-Tumor Activity of Specific T Cell Clones in Patients with Leukemia and Renal Carcinoma.

A major challenge in the field of hematopoietic stem cell transplantation (HSCT) is to prevent the alloreactivity of donor T-cells which leads to acute graft-versus-host disease (GVHD) while preserving a graft-versus-tumor (GVT) effect. Selective depletion using anti-CD25 immunotoxin (IT) can eliminate harmful alloreactive T-cells while preserving other donor T-cells with antileukemic/antitumor reactivity. We have used irradiated peripheral blood mononuclear cells (PBMC) from cancer patients and healthy donor PBMC as responder cells in primary mixed leukocyte reaction (MLR).To prepare GVL/GVT-specific T-cells, alloreactive T-cells in primary MLR were depleted with anti-CD25 IT. The remaining T-cells had insignificant alloreactivity in secondary MLR. Allodepleted donor cells were then repeatedly stimulated using purified leukemia/tumor cells from the same cancer patient. Leukemia/tumor-reactive donor T-cells were purified by immunomagnetic separation on the basis of INF-g production. 23 MLRs (12 with acute myeloid leukemia cells, 3 with acute lymphocytic leukemia cells and 8 with renal carcinoma cells) were performed. Selective depletion of alloreactive donor T-cells with anti-CD25 IT led to more than 2log depletion (99.2–100%, median 99.7%). Graft-versus-leukemia (GVL) effect of donor T-cells was well preserved (4.7% of donor T cells were GVL-reactive) while the graft-versus-host (GVH) reactivation of donor cells was negligible even after repeated stimulation with patient's non-leukemic PBMC. In the case of renal cell carcinoma, GVT effect was less dominant (1.8% of donor T cells were GVT-reactive clones) and GVH reactivation appeared in 2.4% of donor T cells. In conclusion, it is possible to selectively deplete donor alloreactive T-cells with anti-CD25 IT. In case of patients with leukemia, a strong GVL reactivity was noticed in allodepleted donor T cells while in case of renal cell carcinoma the GVT effect was less dominant and associated with mild GVH-reactivation.

Vaccination with Host Dendritic Cells Induces Graft-Versus-Leukemia Responses without Severe Graft-Versus-Host Disease in a Preclinical Mouse Model for Allogeneic Stem Cell Transplantation.

Allogeneic stem cell transplantation is a potent treatment for patients with leukemia. This therapeutic effectiveness is largely attributed to the graft-versus-leukemia (GVL) response during which alloreactive donor CD8+ T cells eradicate minor histocompatibility antigens (MiHA)-expressing leukemic cells. Unfortunately, recognition of MiHA expressed in healthy tissues, including the liver, skin and gut, often results in morbidity and even mortality due to the development of graft-versus-host disease (GVHD). Antigen presenting cells (APC) play an essential role in the activation of alloreactive T cells and thus in the induction of GVL responses and GVHD. Using several in vivo models it has been suggested that host APC, which present endogenous MiHA to donor CD8+ T cells, are more potent inducers of alloreactive responses than donor APC. Here, we compared the ability of host APC versus donor APC to activate alloreactive T cells in vitro and to induce alloreactions in vivo. For this purpose, we isolated T cells and cultured bone marrow-derived dendritic cells (DC), the most potent APC, from B6.SJL ‘host' mice and MHC (H-2b)-matched but MiHA-mismatched C3.SW ‘donor' mice. Following their activation by the TLR4 ligand lipopolysaccharide for no longer than 5 hours, both host and donor DC showed a fully mature phenotype (MHCIhigh MHCIIhigh CD40+ CD80high CD86high), and comparable migratory behavior towards lymph node-specific chemokines. In primary mixed leukocyte reactions host DC were 2–3 times more potent than donor DC in inducing donor CD8+ T cell proliferation. To determine the role of host and donor DC in the development of GVL and GVHD, we established a preclinical mouse model (C3>B6) for allogeneic stem cell transplantation. Repopulation analyses demonstrated that T cells, B cells and myeloid cells from blood and spleen of transplanted mice were for more than 95% of donor origin. Transplanted mice were subsequently vaccinated intravenously with 3 weekly doses of 3 × 105 host or donor DC. DC vaccination, which did not lead to significant GVHD, was followed by an intravenous challenge with 106 GFP-expressing mouse AML (C1498) cells. While sham-vaccinated mice died quickly due to leukemia progression, vaccination with both host and donor DC resulted in diminished leukemia growth. However, only vaccination with host DC, and not donor DC, resulted in a (partial) rescue from challenge. These results suggest that host DC are the most potent inducers of GVL reactions without causing significant GVHD. We believe that these data will be instrumental in designing appropriate DC-based immunotherapies in transplanted leukemia patients.

The Direct Pathway of Human T‐Cell Allorecognition is not Tolerized by Stimulation with Allogeneic Peripheral Blood Mononuclear Cells Irradiated with High‐Dose Ultraviolet Ba

Transfusions of high-dose (> or =10,000 Joule/m(2)) ultraviolet-B (UVB)-irradiated allogeneic leukocytes in rodent models have been shown to induce immunologic tolerance that is mediated by allospecific regulatory CD4(+) T cells. Whether these regulatory T cells recognize alloantigens through the direct or indirect pathway of allorecognition is controversial. Here, we demonstrate that the proliferative response obtained in standard primary mixed leukocyte reactions (MLRs) with human peripheral blood mononuclear cells (PBMCs) reflected a CD4(+) T-cell-dependent direct pathway of allorecognition and that high-dose UVB irradiation of PBMCs totally inhibited their capacity to induce a proliferative alloresponse. Re-stimulation with gamma-irradiated PBMCs from the same allogeneic donor (secondary MLR) elicited a proliferative and Th1-deviated response that was similar to the response induced in unprimed PBMCs. Finally, high-dose UVB was found to induce a rapid and massive apoptosis of irradiated PBMCs. Collectively, these data indicate that leukocytes irradiated with high-dose UVB are unable to prime for unresponsiveness or immune deviation in T cells directly recognizing allogeneic major histocompatibility complex molecules. Because it is well-established that antigens within transfused apoptotic cells are captured by resident tolerogenic spleen dendritic cells, we propose that tolerance induced by transfusions of high-dose UVB-irradiated leukocytes primarily involve T cells indirectly recognizing alloantigens.

Activation of purified allogeneic CD4 + T cells by rat bone marrow-derived dendritic cells induces concurrent secretion of IFN-γ, IL-4, and IL-10

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play a key role in the initiation and regulation of immune responses. The ability of DCs to process antigens and the outcome of their interaction with T cells are largely dependent on phenotype as well as maturation state of DCs. In this study, we generated DCs from rat bone marrow precursors. Bone marrow cells cultured in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and Flt-3 ligand (FL) produced immature DCs that expressed intermediate levels of major histocompatibility complex (MHC) class II, low levels of CD80 and CD86 molecules and displayed a high capacity of endocytosis. Bone marrow-derived DCs (BMDCs) matured in the presence of lipopolysaccharide (LPS) upregulated expression of MHC class II, CD80 and CD86, while their phagocytic capacity was dramatically reduced. Mature BMDCs stimulated vigorous proliferation of purified allogeneic CD4 + T cells in a primary mixed leukocyte reaction (MLR) and elicited a mixed cytokine profile in allogeneic CD4 + T cells: DCs activated CD4 + T cells to produce interferon (IFN)-γ, IL-4, and IL-10. Thus, rat BMDCs effectively internalize antigens and stimulate T cell proliferation but fail to induce an unidirectional polarization of T helper (T H) cells and in this respect differ from both human and mouse DCs.

Dendritic Cells Genetically Engineered with Adenoviral Vector Encoding dnIKK2 Induce the Formation of Potent CD4+ T-Regulatory Cells

Immature dendritic cells (DC), characterized by low expression of both major histocompatibility complex class II antigens and co-stimulatory molecules, can be instrumental in the induction of peripheral tolerance. Because nuclear factor (NF)-kappa B is central to DC maturation, the authors engineered DC with an adenoviral vector (Adv) encoding for a kinase-defective dominant negative form of IKK2 (dnIKK2) to block NF-kappa B activation and inhibit DC maturation. DC were obtained by culturing bone marrow from Brown Norway (BN) rats with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 11 days. To block NF-kappa B activation, at day 9, cells were transfected with AdV-dnIKK2. At day 11, cells were used as stimulators in primary mixed leukocyte reaction (MLR) with naive Lewis rat lymphocytes as responders. CD4+ T cells were purified from primary MLR and tested in secondary MLR with allogeneic mature DC and in co-culture MLR with naive lymphocytes. The tolerogenic potential of dnIKK2-DC was evaluated in vivo in a model of rat kidney allotransplantation. DnIKK2-DC were immature and lacked any allostimulatory activity. T cells preexposed to allogeneic dnIKK2-DC were hyporesponsive to a secondary stimulation with mature DC and acquired potent regulatory properties, inhibiting naive T-cell proliferation toward allogeneic stimuli. Pretransplant infusion of allogeneic donor dnIKK2-DC prolonged the survival of a kidney allograft from the same allogeneic donor, without the need for immunosuppressive therapy. Allogeneic DC, rendered immature by dnIKK2 transfection, induce in vitro differentiation of naive T cells into CD4+ T-regulatory cells, effective at low ratios with target cells, rendering them applicable for cellular therapy of immune-mediated abnormalities and for preventing transplant rejection.

Allogeneic T-cell apoptosis induced by interleukin-10–modified dendritic cells: A mechanism of prolongation of intestine allograft survival?

Background Genetic modification of donor dendritic cells (DC) is a potential therapy for allograft rejection. We hypothesized that in vitro interleukin-10 (IL)-10–transfected DC (DC-IL-10) may induce allogeneic T-cell apoptosis, resulting in prolonged allograft survival rat small intestine. Methods Myeloid DC from Wistar–Furth rats ( RT-1 u ) were propagated with rrGM-CSFand rrIL-4,then genetically modified to express the hIL-10 gene. Secretion of IL-10 was quantitated by enzyme-linked immunosorbent assay (ELISA). Allogeneic T cells from Lewis (LEW; RT-1 l ) at proliferative responses were determined by MTT assay in primary mixed leukocyte reactions. We then used a combination of DNA agarose gel electrophoresis, acridine orange staining, and Annexin V/propridium iodide assays to examine apoptosis of allogeneic T cells exposed to DC-IL-10. Then 5 × 10 6 donor-derived DC-IL-10 or untransduced DC were injected intravenously 7 days before small intestine transplantation (WF→LEW). Results DC-IL-10 showed pronounced impairment of T-cell allostimulatory activity. Apoptotic T cells were detected in the DC-IL-10 group. Flow cytometry counting at 72 hours showed 45.1% apoptotic T cells in response to DC-IL-10, whereas the untransduced group did not undergo significant apoptosis ( P < .01). DC-IL-10 pretreated recipients showed moderate prolongation of allograft survival compared with controls (20.7 ± 6.0 days vs 7.5 ± 2.2 days, P < .01). Conclusions DC-IL-10 induced allogeneic T-cell hyporesponsiveness in vitro, possibly due to apoptosis. DC-IL-10 pretreated recipients displayed prolonged intestinal allograft survival rates.

T-cell receptor beta-chain usage in directly activated alloreactive CD4+ T cells: unrestricted Vbeta gene usage with CDR3 size limitations.

Alloreactive immune responses may engage both direct and indirect antigen allorecognition. This study focuses on T-cell receptor (TCR) beta-chain usage by in vitro generated alloreactive CD4+ T cells involved in direct allorecognition pathway. We have established Lewis anti-Brown Norway rat CD4+ T-cell lines and confirmed their reactivities against cell-surface, but not soluble, alloantigens. TCR Vbeta-specific reverse-transcriptase polymerase chain reaction detected all 22 Vbeta genes in these cell lines at all stages, regardless of the lengths of in vitro stimulation. By using spectrotyping, we found that Vbeta complementarity determining region (CDR)3 length distribution pattern altered dramatically after repeated allostimulation. Such a skewed CDR3 distribution occurred in most Vbeta genes without any obvious preference, indicating that expansion occurred in all Vbeta expressing cells by allostimulation. Fluorescence-activated cell sorter analysis of Vbeta expression in alloreactive CD4+ T-cell lines with anti-Vbeta-specific monoclonal antibodies showed, quantitatively, similar percentages of individual Vbeta-expressing cells in the alloreactive pool after repeated allostimulation. To test whether preferential TCR Vbeta gene usage occurred in "high responder" cells, we sorted CD4+ T cells that underwent three or more divisions from primary mixed leukocyte reactions. Unlimited Vbeta usage with CDR3 alterations was observed, as in unsorted alloreactive T cells. TCR Vbeta gene usage in directly alloreactive CD4+ T-cell population is unrestricted. Clonal expansion occurs in all Vbeta expressing T cells by allostimulation.

Definitive separation of graft-versus-leukemia- and graft-versus-host-specific CD4+ T cells by virtue of their receptor beta loci sequences.

Although graft-versus-host (GVH) disease (GVHD) is usually associated with graft versus leukemia (GVL), GVL can occur in the absence of clinical GVHD. There is evidence to suggest that GVL and GVH are mediated by different clones of T cells. The objective of this study was to identify the two types of T cells based on their receptor sequences. To this end we used irradiated nonleukemic cells from recipients as stimulator cells in a primary mixed leukocyte reaction (MLR). The activated CD4(+) donor T cells that expressed CD25 were purified by cell sorting. To prepare GVL-specific T cells, alloreactive T cells in the primary MLR were first depleted with an anti-CD25 immunotoxin. The remaining T cells had negligible alloreactivity in a secondary MLR. The allodepleted cells were then stimulated by using purified leukemia cells from the same individual as stimulator cells, and the CD25(+)-activated cells were purified by cell sorting. The GVL- and GVH-specific T cells were analyzed for their T cell receptor (TCR) clonality by using anchored RT-PCR of all the TCRbeta locus complementarity-determining region 3 (CDR3) sequences. By comparing TCRbeta CDR3 sequences from transformed bacterial colonies, we were able to demonstrate that T cells mediating GVH were different from those mediating GVL in each of the eight HLA-mismatched and one HLA-matched donor/recipient pairs. By using the appropriate TCRbeta CDR3-specific primers and probes, the GVH- and GVL-specific clones were monitored in a recipient undergoing an allogeneic stem cell transplant from her HLA-matched related donor.

Mechanisms of dendritic cell-induced T cell proliferation in the primary MLR assay

DC are unique antigen presenting cells, and their ability to induce proliferation of T cells in a mixed leukocyte reaction (MLR) assay is commonly used for the evaluation of their function. To determine the mechanisms involved in DC-induced T cell activation in a primary MLR assay, a variety of different agents were examined in this study that interfere with DNA synthesis, membrane organization, protein synthesis, and maturation induced by bacterial products. While only live DC were able to induce T cell proliferation in the MLR assay, irradiation of DC did not influence their stimulatory capacity. Fixation of DC membrane with paraformaldehyde resulted in a loss of DC capacity to induce T cell proliferation demonstrating that physical organization of the plasma membranes plays an important role in the induction of T cell activation. In addition, the pretreatment of DC with cycloheximide revealed that protein synthesis was not critical for the ability of DC to activate T cells. Finally, Staphylococcus aureus-mediated activation of DC significantly increased T cell proliferation and this effect was not dependent on IL-12 production of DC since DC generated from IL-12 knockout mice were not different from wild type DC. In summary, these data suggest that DC membrane structures are responsible for the antigen presentation and co-stimulation and play a key role in T cell recognition and activation by DC.

Characterization of murine dendritic cells derived from adherent blood mononuclear cells in vitro.

The therapeutic potential of dendritic cells loaded with tumour antigens for the induction of effective immune responses against cancer is currently being tested in numerous clinical trials. In most cases, the dendritic cells are generated in vitro from peripheral blood monocytes. Many aspects of dendritic cell-based vaccination have not yet been examined in detail, and homologous mouse model systems may prove very valuable for optimizing clinical procedures. In the murine system, however, dendritic cells are usually isolated from either lymphoid tissues or bone marrow cultures. To date, murine monocyte-derived dendritic cells have been described only sporadically. Here, we describe a culture system for the generation of murine dendritic cells from adherent peripheral blood mononuclear cells by culturing in the presence of granulocyte-macrophage colony stimulating factor and interleukin-4. After 7 days of culture the nonadherent cells were harvested from the cultures. Most of these cells exhibited well-accepted characteristics of mature dendritic cells (e.g. veiled appearance, high expression of major histocompatibility complex class II and CD86) and stimulated vigorous proliferation of allogeneic T cells in a primary mixed leucocyte reaction following stimulation with bacterial lipopolysaccharide. Interestingly, staining the cells for expression of the putative antigen-uptake receptor DEC-205 revealed a distinct bimodal distribution.

MHC class II molecules transferred between allogeneic dendritic cells stimulate primary mixed leukocyte reactions.

Presentation of antigen to T cells is generally restricted by MHC type but the mixed leukocyte reaction (MLR) was thought to involve direct stimulation by dendritic cells (DC) of allogeneic T cells. However, here we showed that DC bearing allogeneic MHC class II acted synergistically with responder-type DC. Removal of residual DC from 'purified' responder T cell populations was achieved using treatment with DC-specific antibody and complement. These DC-depleted cells showed a significantly reduced response to allogeneic DC which was restored by addition of DC syngeneic with responder T cells. The studies support the concept that a major component of the MLR is the secondary presentation of alloantigens acquired from stimulator DC by DC of responder type. To investigate the reasons why DC and not other cells stimulate an MLR, synergy between DC and other cell types was investigated. Synergy was found exclusively between DC; macrophages, B cells or L cells transfected with MHC class II molecules did not contribute. When allogeneic DC were mixed in culture, transfer of MHC molecules between DC was observed as assessed by flow cytometry. Freshly obtained cell-free supernatants from cultured DC contained MHC class II and stimulated primary allogeneic MLR. DC of responder type acquired allogeneic MHC molecules from the supernatants and stimulated proliferation in syngeneic T cells. The capacity of DC both to shed and to acquire MHC molecules may contribute to their potency in stimulating primary responses, and could explain why passenger DC within allografts provide a potent stimulus for graft rejection.

CD40‐deficient dendritic cells producing interleukin‐10, but not interleukin‐12, induce T‐cell hyporesponsiveness in vitro and prevent acute allograft rejection

The induction of an immune response or tolerance is mediated by corresponding subsets of dendritic cells (DC). However, the property of tolerogenic DC is not clear. Recently, we have characterized a population of CD11c+ splenic DC derived from long-term mixed leucocyte culture (LT-MLC), which are able to proliferate upon stimulation and have a strong primary mixed leucocyte reaction (MLR)-stimulating activity in conventional MLR. In this study, we show that, in contrast to the irradiated ones, non-irradiated LT-MLC-derived DC induce polyclonal antigen-specific T-cell hyporesponsiveness when cocultured with allogeneic splenocytes for 3-11 days. The degree of the hyporesponsiveness increased with the length of coculture. Although these DC expressed major histocompatibility complex class II and B7 costimulatory molecules, which are down-regulated during coculture, they expressed very low or undetectable CD40 before and after coculture, respectively. The CD40-deficient DC spontaneously produce interleukin-10 (IL-10), but not IL-12. The skewed balance between IL-10 and IL-12 is associated with their capability to induce T-cell hyporesponsiveness, because a neutralizing antibody to IL-10, exogenous recombinant IL-12 or lipopolysaccharide (LPS) significantly blocked the hyporesponsiveness. Accordingly, infusion of a small number of non-irradiated LT-MLC-derived DC (5x105) significantly prolonged the survival of a vascularized heterotopic murine heart transplant, whereas irradiated DC accelerated graft rejection. These data suggest that CD40-deficient DC producing IL-10, but not IL-12 can induce T-cell hyporesponsiveness in vitro and in vivo.