Manipulating dendritic cells to induce regulatory T cells

Dendritic cells (DCs) induce and regulate T-cell responses, and tolerogenic DCs can promote the development of regulatory T cells with suppressive activity. The possibility of manipulating DCs using different pharmacological or biological agents, enabling them to exert tolerogenic activities, could be exploited to better control a variety of chronic inflammatory conditions, from autoimmune diseases to allograft rejection.     

Thymus exosomes-like particles induce regulatory T cells

Exosomes released from different types of cells have been proposed to contribute to intercellular communication. We report that thymic exosome-like particles (ELPs) released from cells of the thymus can induce the development of Foxp3(+) regulatory T (Treg) cells in the lung and liver. Thymic ELPs also induce the conversion of thymic CD4(+)CD25(-) T cells into Tregs. Tregs induced by thymic ELPs suppress the proliferation of CD4(+)CD25(-) T cells in vitro and in vivo. We further show that neutralization of TGF-beta in ELPs partially reverses thymic ELP-mediated induction of CD4(+)Foxp3(+) T cells in the lung and liver. This study demonstrates that thymic ELPs participate in the induction of Foxp3(+) Tregs. Also, TGF-beta of thymic ELPs might be required for the generation of Tregs in the peripheral tissues.     

Effects of concanavalin A-induced cells on the proliferative response of T cells. Concanavalin A-induced suppressor and amplifier cells to the proliferative response of human T cells to trinitrophenyl-modified autologous lymphocytes.

Effects of Con A-induced human mononuclear cells on the proliferative response of peripheral T cells were examined by using TNP-modified autologous lymphocytes as stimulator cells. Cells induced by incubation with Con A contained both suppressor cells and amplifier cells. The former were induced from nylon wool-nonadherent T cells and these precursor cells were sensitive to mitomycin treatment. On the other hand, amplifier precursor cells were nylon wool-nonadherent T cells and were resistant to mitomycin treatment. Cell proliferation was required for the induction of suppressor cells but not for the induction of amplifier cells. Con A-induced suppressor effector cells were both nylon wool-adherent and nonadherent cells, on the contrary, Con A-induced amplifier effector cells were nonadherent cells. A small number of macrophages enhanced the suppressive activity of nonadherent T cells when added at the induction phase of suppressor T cells.     

Enrichment and cryopreservation of bone marrow progenitor cells for autologous reinfusion

From 20 patients with solid tumors or acute nonlymphocytic leukemia in remission, hemopoietic progenitor cells were taken and stored in liquid nitrogen, for use in autologous bone marrow transplantation. Bone marrow aspiration resulted in a volume of 920(+/- 170) ml containing 16.8(+/-6.0) x 10(9) nucleated bone marrow cells and 7.2(+/-4.4) x 10(6) myeloid progenitor cells (CFUc). With use of the Haemonetics blood cell separator a progenitor cell-enriched fraction is obtained. This fraction is depleted of 90(+/-6)% of the erythrocytes and 59(+/-15)% of the neutrophils contained in the original. The original aspirate volume is reduced to one-fifth (21 +/- 3%) while containing 88(+/-38)% of the original CFUc's and 52(+/-11)% of the nucleated bone marrow cells. This technique of bone marrow enrichment has the advantage of a minimum of open-air contact, being independent of extensive laboratory facilities and manpower. The enriched fraction is frozen in autologous plasma and a final concentration of 10% (v/v) DMSO, using a program-controlled freezer (L'Air Liquide). Materials are stored at liquid nitrogen temperature in bags (Gambro) and test vials. Total CFUc recovery in test vials after thawing was 81(+/-32)%.     

Feeder cells enhance oncolytic and proliferative activity of long-term human bone marrow interleukin-2 cultures and induce different lymphocyte subsets

Summary The effect of feeder cells on oncolytic activity of lymphocyte subsets and their growth was evaluated in long-term human bone marrow interleukin-2 (IL-2) cultures. Two B-lymphoblastoid cell lines (Daudi and Epstein-Barr-virus-transformed BSM) and two human leukemias, AML-M5, were used as feeder cells. The most prominent effects were seen in cultures stimulated with Daudi cells. In these cultures, cytotoxic activity was 100–1000 times increased against a broad range of target cells and the total cellular expansion was more than 40 times higher than in control cultures. This Daudi-related effect appeared to be mediated by natural killer (NK) cells, since cellular expansion occurred mostly in the CD16⁺ and CD56⁺ CD3^– NK cell subset. In cultures stimulated with BSM and acute myelogenous leukemia (AML) feeder cells, the increase in proliferation was similar, but the enhancement of cytotoxicity, even though significant, was less prominent. Although all feeder cells were effective in stimulation of bone marrow reactivity, the highest cytotoxicity was always observed with feeder cells autologous to the targets, indicating some degree of specificity. This was especially evident in cultures stimulated with autologous versus allogeneic AML feeder cells. In contrast to Daudistimulated IL-2 cultures, in which the highest expansion of CD3^– CD56⁺ NK cells was observed, in BSM and AML cultures, the CD3⁺ CD56^+/- T cell subsets were more prolific. This indicates that the response and phenotypic heterogeneity of bone marrow cultures depends on the type of feeder cells used. This observation indicates that the preferential stimulation of a pertinent lymphocyte subset for therapeutic purposes may be possible.Recipient of Florence Maude Thomas Cancer Research Professorship     

Cloned allospecific human helper T cell lines induce an MHC-restricted proliferative response by resting B cells

To analyze helper T (Th) cell-induced B cell proliferation in man, we have cloned allospecific Th cells, grown them as long-term IL 2-dependent T cell lines (TCL), and analyzed their phenotypic and functional properties. The two TCL described in this report, A-7 and A-57, are both composed exclusively of T3+, T4+, T8- T cells blasts. In proliferative assays, with a panel of x-irradiated allogeneic stimulator cells, A-7 was found to proliferate in response to DR3-bearing cells, whereas A-57 responds to DR2-positive stimulators. Both TCL are capable of providing MHC-restricted polyclonal help for allogeneic B cells, as measured in the reverse hemolytic plaque assay. Of greater interest, x-irradiated A-7 and A-57 cells are capable of inducing a proliferative response by allogeneic B cells that is absolutely MHC restricted at the inductive (Th-APC) level. Thus, x-irradiated A-7 cells only trigger proliferation by DR3+ B cells, whereas A-57 cells selectively activate DR2+ B cells. In contrast, after antigen-specific activation, x-irradiated A-7 and A-57 cells can recruit a significant proliferative response by allogeneic B cells bearing "irrelevant" DR antigens. The possibility that Th-induced B cell proliferation may be restricted at the effector (Th-B cell) level was addressed by fractionating B cell populations into "activated" and "resting" subsets by discontinuous Percoll density gradient centrifugation and further purification by employing a monoclonal antibody directed against an antigen expressed on activated B cells (4F2). These studies demonstrate that activated B cells are readily and nonspecifically recruited to proliferate by activated Th cells, whereas optimal proliferative responses by resting B cells require MHC restricted Th-B cell interaction.     

Bone marrow mesenchymal/fibroblastic stromal cells induce a distinctive EMT-like phenotype in AML cells

The development of epithelial-to-mesenchymal transition (EMT) like features is emerging as a critical factor involved in the pathogenesis of acute myeloid leukaemia (AML). However, the extracellular signals and the signalling pathways in AML that may regulate EMT remain largely unstudied. We found that the bone marrow (BM) mesenchymal/fibroblastic cell line HS5 induces an EMT-like migratory phenotype in AML cells. AML cells underwent a strong increase of vimentin (VIM) levels that was not mirrored to the same extent by changes of expression of the other EMT core proteins SNAI1 and SNAI2. We validated these particular pattern of co-expression of core-EMT markers in AML cells by performing an in silico analysis using datasets of human tumours. Our data showed that in AML the expression levels of VIM does not completely correlate with the co-expression of core EMT markers observed in epithelial tumours. We also found that vs epithelial tumours, AML cells display a distinct patterns of co-expression of VIM and the actin binding and adhesion regulatory proteins that regulate F-actin dynamics and integrin-mediated adhesions involved in the invasive migration in cells undergoing EMT. We conclude that the BM stroma induces an EMT related pattern of migration in AML cells in a process involving a distinctive regulation of EMT markers and of regulators of cell adhesion and actin dynamics that should be further investigated. Understanding the tumour specific signalling pathways associated with the EMT process may contribute to the development of new tailored therapies for AML as well as in different types of cancers.     

Bone marrow stromal proteoglycans regulate megakaryocytic differentiation of human progenitor cells

Adherence of hematopoietic progenitor cells (HPCs) to stroma is an important regulatory step in megakaryocytic differentiation. However, the mechanisms through which megakaryocytic progenitors are inhibited by stroma are poorly understood. We examined the role of sulfated glycoconjugates, such as proteoglycans (PGs), on human bone marrow stroma (hBMS). To this end, PG structure was altered by desulfation or enzymatic cleavage. PGs participated in adhesion of human HPC, as desulfation resulted in about 50% decline in adhesion to hBMS. Heparan sulfate proteoglycans (HSPGs) were found to be responsible by showing about 25% decline in adhesion after pre-incubation of HPC with heparin and about 15% decline in adhesion after enzymatic removal of HSPGs from hBMS. Furthermore, PGs were involved in binding cytokines. Both desulfation and enzymatic removal of stromal HSPGs increased release of megakaryocytopoiesis-inhibiting cytokines, that is, interleukin-8 (IL-8, 1.9-fold increase) and macrophage inflammatory protein-1alpha (MIP-1alpha, 1.4-fold increase). The megakaryocytic output of HPC grown in conditioned medium of desulfated stroma was decreased to 50% of the megakaryocytic output in CM of sulfated stroma. From these studies, it can be concluded that PGs in bone marrow, in particular HSPGs, are involved in binding HPC and megakaryocytopoiesis-inhibiting cytokines. Bone marrow stromal PGs thus reduce differentiation of HPC toward megakaryocytes.     

Chemically modified antigen-presenting cells induce T lymphocyte allospecific hyporesponsiveness.

We have investigated the interaction between murine T lymphocytes and allogeneic APC in an in vitro proliferative mixed leukocyte reaction. Our results demonstrate that freshly isolated potentially alloreactive murine splenic T lymphocytes, in primary culture, can be induced to develop a state of allospecific proliferative hyporesponsiveness in vitro by exposure to 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-modified allogeneic APC, a method similar to that previously used to induce nonresponsiveness in murine Ag-specific self-MHC-restricted T lymphocyte clones. This hyporesponsiveness was: specific for the allohaplotype of inducing APC, maintained for 96 h in vitro, not due to cellular inhibitory mechanisms, and associated with reduced ability to secrete IL-2 but not IL-3. Induction of this hyporesponsiveness was not due to altered expression of class II MHC gene products on the APC but was associated with markedly reduced T lymphocyte-APC adhesive interactions despite the lack of a detectable immunophenotypic change in lymphocyte function-associated Ag 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) expression on the modified APC. Therefore, we propose that TCR occupancy in the absence of normal T lymphocyte-APC adhesive clustering may induce T lymphocyte tolerance.     

Tac antigen-positive T cells activated in autologous mixed lymphocyte reaction regulate the generation of killer T cells against hapten-modified autologous cells

T cells that proliferate in the autologous mixed lymphocyte reaction (auto-MLR) have been shown to acquire some suppressor or regulatory activities. In the present study, we examined the suppressive effects of T cells activated in the auto-MLR on the induction of hapten-specific cytotoxic T cells. NRFT (depletion of ARFT from UT) were used as the responder cells of TNP-MLR. After primary and secondary TNP-MLR, the cells were harvested and tested for their cytotoxic activities against TNP-modified autologous cells by 51Cr-release assay. When UT cells cultured for 1 wk in auto-MLR were added to primary TNP-MLR at the beginning of culture, the cytotoxic activity tested at the end of the culture was suppressed from 15.6% +/- 2.7 to 5.8% +/- 1.1 (percent cytotoxicity, mean +/- SE). However, these auto-MLR-activated UT cells had little suppressive activity against cytotoxic T cells when they were added to the final assay of TNP-CTR. Suppressive activities of these cells on the generation of cytotoxic T cells during secondary TNP-MLR were also tested. The addition of auto-MLR-activated UT cells to the secondary TNP-MLR at the beginning of the culture reduced the cytotoxic activities of NRFT from 23.8% +/- 2.3 to 9.7% +/- 1.7 after secondary TNP-MLR. Allo-activated T cells, PHA blasts, and fresh autologous T cells were used as the controls, but none of the cells had suppressive effects on the generation of CTL. Characteristics of these suppressor cells were examined. Auto-MLR-activated cells from ARFT fractions exhibit very powerful suppressor activity. Treatment of the auto-MLR-activated T cells with mitomycin C eliminated their suppressive effects on the generation of CTL; 21.2% +/- 6.3 of UT cells became anti-Tac positive after 1 wk of auto-MLR. Treatment of auto-MLR-activated UT cells with anti-Tac antibody plus complement eliminated their suppressive activities on the induction of CTL. Thus, T cells stimulated in auto-MLR were shown to have suppressive effects on the induction of cytotoxic T cells against TNP-modified autologous cells. These cells were mitomycin C sensitive. Because anti-Tac antibody is reactive to activated T cells, activation of T cells during auto-MLR was thought to be necessary for the acquisition of the suppressive activity.     

Monocyte-derived human macrophages mediate anergy in allogeneic T cells and induce regulatory T cells

Activation of alloreactive T cells by APCs such as dendritic cells (DC) has been implicated as crucial step in transplant rejection. In contrast, it has been proposed that macrophages (Mphi) maintain tolerance toward alloantigens. It was therefore the aim of this study to further analyze the T cell-stimulatory capacity of mature DC and Mphi in vitro using the model of allogeneic MLR. There was a strong proliferative response in T cells cocultured with DC, which was further increased upon restimulation in a secondary MLR. In contrast, T cells did not proliferate in cocultures with Mphi despite costimulation with anti-CD28 and IL-2. Cytokine analysis revealed considerable levels of IL-10 in cocultures of T cells with Mphi, whereas high amounts of IL-2 and IFN-gamma were present in cocultures with DC. There was only minimal T cell proliferation in a secondary MLR when T cells were rescued from primary MLR with Mphi and restimulated with DC of the same donor, or DC of an unrelated donor (third party), whereas a strong primary proliferative response was observed in resting T cells, demonstrating induction of T cell anergy by Mphi. Functional analysis of T cells rescued from cocultures with Mphi demonstrated that anergy was at least partly mediated by IL-10-producing regulatory T cells induced by Mphi. These results demonstrate that Mphi drive the differentiation of regulatory T cells and mediate anergy in allogeneic T cells, supporting the concept that Mphi maintain peripheral tolerance in vivo.     

Cytotoxic T cells generated in the autologous mixed lymphocyte reaction. I. Primary autologous mixed lymphocyte reaction

In the course of the culture of an autologous mixed lymphocyte reaction (AMLR), T cells proliferated in response to autologous non-T cells, and differentiated to cytotoxic T cells (AMLR killers). DNA synthesis was necessary to generate AMLR killers, as the elimination of autoreactive proliferating cells with BUdR and UV light completely abrogated AMLR killer cytolysis. Amlr killers lysed various lymphoid cell lines, including autologous B cell lines, autologous or allogeneic mitogen blasts stimulated by Con A, PHA, or pokeweed mitogen, variious nonlymphoid cell lines derived from human, mouse, or rat, and weakly normal autologous or allogeneic non-T cells. KMT-17, methylcholanthrene-induced rat fibrosarcoma, was the only resistant cell line to have been tested. AMLR killers had characteristics similar to NK cells, Major histocompatibility antigens were not the target antigens for AMLR killers. AMLR killers distinguished the blasts stimulated by alloantigens as self from the blasts stimulated by mitogens as non-self.     

Rapid changes in the regulatory potential of autologous anti-idiotopic T cells during an antigen-driven primary response

The antibody response of C57BL/6 strain mice to Streptococcus pneumoniae R36a (Pn) is dominated by the T15 idiotype, but the responding cells appear to be idiotypically heterogeneous, in that individual antibody plaque-forming cells (PFC) may express some but not all idiotopes (Id) of the T15 complex. The presence of these distinct Id on the PFC was detected by a plaque-inhibition assay with three different monoclonal anti-Id antibodies, designated AB1-2, MaId5-4, and B36-82. A periodic change in the expression of AB1-2 and MaId5-4 Id was observed during primary (IgM) antibody response to Pn in the spleen. Those two Id were poorly expressed in the log phase of the response between day 2 and day 4 after immunization (few PFC in the spleen bore the Id), but they became detectable on the majority of PFC at the peak of the response, day 5 to day 7. The proportion of the Id-(AB1-2 or MaId5-4) positive PFC declined, again at day 10 after immunization. In contrast, the B36-82 Id was expressed on greater than or equal to 80% PFC throughout the entire primary response. The possibility that the apparent changes in the Pn-reactive cell populations are regulated by autologous anti-Id T cells was tested in vitro. Normal, unimmunized B cells were cultured with Pn, either alone or in the presence of syngeneic T cells isolated from the spleen of mice at the appropriate intervals after immunization: day 2 (T2), day 5 (T5), and days 10 to 14 (T10 to T14); T cells from unimmunized donors (T0) served as a control. The specific response after 4 days in culture was determined in regard to the total PFC as well as the proportion of PFC expressing the Id. Pn-stimulated B cells, alone or with the control T0 cells, produced moderate, variable levels of AB1-2+ and MaId5-4+ PFC. The expression of these two Id in the assay cultures was suppressed by addition of either T2 cells or T10-14 cells, but it was enhanced if T5 cells were added. However, these various T cell populations did not differ in their effect on the total PFC response. Also, the proportion of PFC bearing the third Id, B36-82 was high, and it was not consistently influenced by the added T cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Myelopeptides: Bone marrow regulatory mediators

Bone marrow cells of various animal species and men produce a group of bioregulatory peptides called myelopeptides (MPs). A highly purified MP fraction and some individual molecules have been isolated from the supernatant of porcine bone marrow cell cultures by reverse phase chromatography.MPs have a wide spectrum of functional activities: immunoregulatory, differentiating and opiate-like. They evoke 2–5-fold stimulation of antibody production to various antigens. They correct some immune defects in MRL/lpr mice with spontaneous autoimmune disorders that results in 2-fold prolongation of the life span of these mice. MPs influence the differentiation of bone marrow and peripheral blood cells derived from healthy and leukemic donors. They induce terminal differentiation in the leukemic human HL-60 cell line. MPs also show an effect on pain sensitivity.A new immunocorrective drug Myelopid has been developed on the basis of MP mixtures. This drug is effectively used in Russia both in medicine and veterinary practice for prophylaxis and treatment of diseases accompanied by immunodeficiency.Two individual MPs were isolated and identified: Phe-Leu-Gly-Phe-Pro-Thr (MP-1) and Leu-Val-Val-Tyr-Pro-Trp (MP-2). MP-1 displays immunoregulatory activity; MP-2 abolishes the inhibitory effect of leukemic cells on T-lymphocyte functional activity.MPs seem to provide not only immunoregulation but also to participate in complex interactions between different systems in the organism.     

Bone marrow T cells. II. Thymic dependency

Mouse bone marrow contains cells capable of responding in vitro to the T cell mitogens PHA and Con A. These cellular responses are not demonstrable in the marrow of athymic nude mice (when compared with heterozygous littermates) and are depressed 47% in the marrow of neonatally thymectomized LAF₁ mice (when compared with sham-operated littermates). Therefore, the population of “bone marrow T cells” is thymus dependent.     

Human lymphocyte proliferative response to a sporozoite T cell epitope correlates with resistance to falciparum malaria

To identify vaccine relevant T cell epitopes on the circumsporozoite (CS) protein of Plasmodium falciparum, the lymphocyte proliferative responses to 10 CS protein derived peptides were studied in 28 adult Kenyans, and correlated with resistance to malaria. Eight peptides, six of which were not overlapping, induced proliferation of lymphocytes from one to five volunteers, suggesting either genetic restriction of response to each of the T epitopes, or dominance of some T sites on the immunizing sporozoites. The 28 volunteers were radically cured of malaria and during the next 126 days 25 of the 28 were reinfected. Resistance to malaria was not correlated with antibodies to malaria Ag, but was significantly correlated with lymphocyte responses to CS protein residues 361-380 and 371-390. Among the 25 volunteers who became re-infected with malaria, lymphocytes from only two responded to a peptide including residues 361-380 of the P. falciparum CS protein, and only one to peptide 371-390. In contrast, lymphocytes from all three volunteers who did not become infected responded to peptide 361-380 (p = 0.003), and lymphocytes from two of the three responded to peptide 371-390 (p = 0.023). The significant correlation between proliferation to peptides 361-380 and 371-390 and resistance to malaria suggests that at least one epitope within these overlapping peptides is involved in a protective cellular immune response. The data support inclusion of these residues in future CS protein vaccines.     

Vitamin A metabolites induce gut-homing FoxP3+ regulatory T cells

In this study, we report a novel biological function of vitamin A metabolites in conversion of naive FoxP3- CD4+ T cells into a unique FoxP3+ regulatory T cell subset (termed "retinoid-induced FoxP3+ T cells") in both human and mouse T cells. We found that the major vitamin A metabolite all-trans-retinoic acid induces histone acetylation at the FoxP3 gene promoter and expression of the FoxP3 protein in CD4+ T cells. The induction of retinoid-induced FoxP3+ T cells is mediated by the nuclear retinoic acid receptor alpha and involves T cell activation driven by mucosal dendritic cells and costimulation through CD28. Retinoic acid can promote TGF-beta1-dependent generation of FoxP3+ regulatory T cells but decrease the TGF-beta1- and IL-6-dependent generation of inflammatory Th17 cells in mouse T cells. Retinoid-induced FoxP3+ T cells can efficiently suppress target cells and, thus, have a regulatory function typical for FoxP3+ T cells. A unique cellular feature of these regulatory T cells is their high expression of gut-homing receptors that are important for migration to the mucosal tissues particularly the small intestine. Taken together, these results identify retinoids as positive regulatory factors for generation of gut-homing FoxP3+ T cells.