Suppressor T cell circuits in contact sensitivity. III. A monoclonal T cell hybrid-derived suppressor factor specifically suppresses local DTH transfer by a DNP-specific T cell clone

Herein we described the direct suppressive effects of a monoclonal T cell hybridoma-derived, DNP-specific suppressor T cell factor (26.10.2 TsF) on the local transfer of delayed-type hypersensitivity (DTH) by a DNP-specific BALB/c T cell clone (dD1.9). The L3T4+, Lyt-2- dD1.9 T cell clone proliferated in response to DNP-OVA and DNBS, but not TNP-OVA or TNBS, in association with I-Ed determinants present on antigen-presenting cells. Similarly, local injection of histopaque-purified dD1.9 cell blasts resulted in DNP-specific, radioresistant, I-Ed-restricted, mononuclear cell-rich ear swelling responses. Incubation in 26.10.2 TsF specifically suppressed local transfer of DNP-specific DTH by dD1.9, but not local DTH responses transferred by BALB/c T cell clones specific for TNP or GAT. The suppressive effect of 26.10.2 TsF correlated with targeting on DNP-major histocompatibility complex determinants associated with the DTH T cell (TDH) targets. 26.10.2 TsF-mediated suppression was most pronounced after exposure of dD1.9 target cells to antigen (after the stimulation phase of the T cell clone maintenance procedure), and greatly reduced when dD1.9 was cultured for long periods in the absence of DNP (after the rest phase of clone maintenance). In additional support of this hypothesis, GAT-specific TDH, normally resistant to 26.10.2 TsF-mediated suppression, were rendered susceptible to suppression after surface DNPylation. The results demonstrate a direct, antigen-specific, effector phase regulatory effect of a monoclonal TsF on a cloned, antigen-specific T cell target, and strongly suggest that suppression is mediated via targeting on DNP determinants associated with the TDH target. Simplification of complex Ts circuitry operating in suppression of the efferent limb of DTH by the use of monoclonal TsF and cloned T cell targets should provide a basis for the future study of the molecular mechanisms of immune suppression.     

Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H⁺-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.     

miR-125a/b inhibits tumor-associated macrophages mediated in cancer stem cells of hepatocellular carcinoma by targeting CD90

Cancer stem cells promote tumorigenesis and progression of hepatocellular carcinoma (HCC). Recently, emerging evidence indicates tumor-associated macrophages (TAMs) play an important role in tumor progression. However, TAMs often occurs with unknown mechanisms. As an important mediator in intercellular communications, exosomes secreted by host cells mediate the exchange of genetic materials and proteins, which involves tumor aggressiveness. The aim of the study was to investigate whether exosomes derived from TAMs mediate stem cell properties in HCC. TAMs were isolated from the tissues of HCC. microRNA (miRNA) expression profiles of TAMs were analyzed using miRNA microarray. In vitro cell coculture was further conducted to investigate the crosstalk between TAMs and tumor cells mediated by TAMs exosomes. In this study, we showed that TAMs exosomes promote HCC cell proliferation and stem cell properties. Using miRNA profiles assay, we identified significantly lower levels of miR-125a and miR-125b in exosomes and cell lysate isolated from TAMs. Functional studies revealed that the HCC cells were treated with TAM exosomes or transfected with miR-125a/b suppressed cell proliferation and stem cell properties by targeting CD90, a stem cell marker of HCC stem cells. The study indicated that miR-125a/b targeting CD90 played important roles in cancer stem cells of HCC.     

CD40 Activation Rescues Antiviral CD8+ T Cells from PD-1-Mediated Exhaustion

The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naïve HBV-specific CD8⁺ T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naïve CD8⁺ T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (αCD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the αCD40 mediated functional differentiation of HBV-specific CD8⁺ T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8⁺ T cells in αCD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8⁺ T cell exhaustion can be rescued by CD40-mediated mDC-activation.     

Suppression and contrasuppression in athymic nude mice: nude mice produce the antigen-specific component of a T suppressor factor that inhibits the late 24-hr phase of DTH but do not generate suppression nor contrasuppression of the early initiating phase of DTH.

Previous studies demonstrated that the initiation of murine delayed-type hypersensitivity (DTH), as exemplified by contact sensitivity induced by picryl chloride (PCI) or oxazolone (OX), is due to antigen-specific, T cell-derived, DTH-initiating factors called, respectively, PCl-F and OX-F. These factors participate in the extravascular recruitment of CD4+, Th-1, DTH effector T cells in the elicitation of DTH. Related factors also participate, together with nonantigen binding factors derived from CD8+ T cells, to constitute an antigen-specific T cell-derived suppressor factor (TsF) that can down regulate the ability of Th-1 effector T cells to mediate DTH. Since it was shown recently that athymic nude mice can make antigen-specific, DTH-initiating T cell factors, the current study tested whether nude mice also could produce the antigen-specific component of the TsF that suppresses DTH effector T cells. We found that antigen-specific factors from nu/nu mice could complement the nonantigen-binding subfactor produced in normal mice to constitute the whole antigen-specific TsF. Additional studies showed that the successful adoptive cell transfer of DTH-initiating T cell activity from nude mice into normal mice required cyclophosphamide treatment of the recipient. In contrast, transfer of DTH-initiating cell activity from nu/+ mice did not require cyclophosphamide treatment of the recipients. We hypothesized that nude mice lacked contrasuppressor cells. Although nude mice were able to manifest the early, initiating phase of DTH, we found that there was no suppression of early DTH-initiating T cells in nude mice, compared to nu/+. Therefore the production of DTH-initiating T cell factor could be boosted in nude mice. The ability to boost DTH-initiating cells in nude mice should facilitate the development of cell lines and clones with the ability to initiate DTH.     

Reconstitution of an inactive antigen-specific T cell suppressor factor by incubation of the factor with prostaglandins

Experiments were performed to test the hypothesis that prostaglandins are crucial to the ability of an antigen-specific T cell suppressor factor to deliver a suppressive signal. In the system employed, T suppressor cells release an antigen-specific factor (TsF) that suppresses the ability of effector cells to transfer contact sensitivity (CS) skin swelling responsiveness to adoptive recipients. Culture of TsF-producing cells in the presence of indomethacin caused production of an inactive TsF that could be reconstituted by incubation of this inactive factor with low concentrations of certain prostaglandins such as PGE2 or PGE1. Subsequently, nearly all the prostaglandins were removed by dialysis, and the reconstituted TsF then acted as an antigen-specific suppressor of CS effector cells. Neither the inactive factor nor prostaglandins were suppressive alone. Furthermore, the prostaglandins are crucial to the constitution of TNBSA-F, the non-antigen-binding subunit of the TsF that probably delivers the ultimate suppressive signal. These results provide a new type of antigen-specific role for prostaglandins in immunoregulation and indicate that simple, local, hormonal molecules in physiologic concentrations can have a crucial and long-lasting role in constituting the suppressive activity of antigen-specific regulatory macromolecules released by suppressor T cells.     

Selected MicroRNAs Define Cell Fate Determination of Murine Central Memory CD8 T Cells

During an immune response T cells enter memory fate determination, a program that divides them into two main populations: effector memory and central memory T cells. Since in many systems protection appears to be preferentially mediated by T cells of the central memory it is important to understand when and how fate determination takes place. To date, cell intrinsic molecular events that determine their differentiation remains unclear. MicroRNAs are a class of small, evolutionarily conserved RNA molecules that negatively regulate gene expression, causing translational repression and/or messenger RNA degradation. Here, using an in vitro system where activated CD8 T cells driven by IL-2 or IL-15 become either effector memory or central memory cells, we assessed the role of microRNAs in memory T cell fate determination. We found that fate determination to central memory T cells is under the balancing effects of a discrete number of microRNAs including miR-150, miR-155 and the let-7 family. Based on miR-150 a new target, KChIP.1 (K ⁺ channel interacting protein 1), was uncovered, which is specifically upregulated in developing central memory CD8 T cells. Our studies indicate that cell fate determination such as surface phenotype and self-renewal may be decided at the pre-effector stage on the basis of the balancing effects of a discrete number of microRNAs. These results may have implications for the development of T cell vaccines and T cell-based adoptive therapies.     

miRNA profiling of naïve, effector and memory CD8 T cells

microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific na?ve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to na?ve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to na?ve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.     

Spontaneous membrane transfer through homotypic synapses between lymphoma cells

Formation of an immunological synapse by T, B, or NK cells is associated with an intercellular transfer of some membrane fragments from their respective target cells. This capture is thought to require effector cell activation by surface recognition of stimulatory ligand(s). However, spontaneous synaptic transfers between homotypic lymphoid cells has never been described. In this study, we show that without adding Ag, resting healthy lymphoid cells and several tumor cell lines are inactive. Conversely, however, some leukemia cell lines including the Burkitt's lymphoma Daudi continuously uptake patches of autologous cell membranes. This intercellular transfer does not involve cytosol molecules or exosomes, but requires cell contact. In homotypic Daudi cell conjugates, this occurs through immunological synapses, involves constitutive protein kinase C and mitogen-activated protein/extracellular signal-regulated kinase kinase activity and strongly increases upon B cell receptor activation. Thus, spontaneous homosynaptic transfer may reflect the hitherto unsuspected autoreactivity of some leukemia cell lines.     

Concurrent Generation of Effector and Central Memory CD8 T Cells during Vaccinia Virus Infection

It is generally thought that during the contraction phase of an acute anti-viral T cell reponse, the effector T cells that escape activation-induced cell death eventually differentiate into central memory T cells over the next several weeks. Here we report that antigen-specific CD8T cells with the phenotype and function of central memory cells develop concomitantly with effector T cells during vaccinia virus (vv) infection. As soon as 5 days after an intraperitoneal infection with vv, we could identify a subset of CD44^hi and CD62L⁺ vv-specific CD8 T cells in the peritoneal exudate lymphocytes. This population constituted approximately 10% of all antigen-specific T cells and like central memory T cells, they also expressed high levels of CCR7 and IL-7R but expressed little granzyme B. Importantly, upon adoptive transfer into naïve congenic hosts, CD62L⁺, but not CD62L⁻ CD8 T cells were able to expand and mediate a rapid recall response to a new vv challenge initiated 6 weeks after transfer, confirming that the CD62L⁺ vv-specific CD8 T cells are bonafide memory cells. Our results are thus consistent with the branched differentiation model, where effector and memory cells develop simultaneously. These results are likely to have implications in the context of vaccine design, particularly those based on vaccinia virus recombinants.     

Loss of exosomal miR-148b from cancer-associated fibroblasts promotes endometrial cancer cell invasion and cancer metastasis

Cancer-associated fibroblasts (CAFs) play crucial roles in tumor progression, given the dependence of cancer cells on stromal support. Therefore, understanding how CAFs communicate with endometrial cancer cell in tumor environment is important for endometrial cancer therapy. Exosomes, which contain proteins and noncoding RNA, are identified as an important mediator of cell–cell communication. However, the function of exosomes in endometrial cancer metastasis remains poorly understood. In the current study we found that CAF-derived exosomes significantly promoted endometrial cancer cell invasion comparing to those from normal fibroblasts (NFs). We identified a significant decrease of miR-148b in CAFs and CAFs-derived exosomes. By exogenously transfect microRNAs, we demonstrated that miR-148b could be transferred from CAFs to endometrial cancer cell through exosomes. In vitro and in vivo studies further revealed that miR-148b functioned as a tumor suppressor by directly binding to its downstream target gene, DNMT1 to suppress endometrial cancer metastasis. In endometrial cancer DNMT1 presented a potential role in enhancing cancer cell metastasis by inducing epithelial–mesenchymal transition (EMT). Therefore, downregulated miR-148b induced EMT of endometrial cancer cell as a result of relieving the suppression of DNMT1. Taken together, these results suggest that CAFs-mediated endometrial cancer progression is partially related to the loss of miR-148b in the exosomes of CAFs and promoting the transfer of stromal cell-derived miR-148b might be a potential treatment to prevent endometrial cancer progression.     

Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation of this is that antigen-specific host suppressor T cells (Ts) are activated. Alternatively, donor Lyt-2+ T cells, introduced in the priming inoculum, may inactivate host CTL precursors (CTLp) that recognize the priming (donor) alloantigens. Donor cells that act in this way are termed veto T cells. The experiments described here exclude veto T cell participation in transferable alloantigen-specific suppression, and demonstrate the operation of an alloantigen-specific host-derived T suppressor (Ts) cell. The origin of the Ts has been studied directly by using Thy-1-disparate BALB/c mice. The cell responsible for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally non-cross-reactive-third party antigen (H-Y), provided the two antigens were expressed on the same cell membrane. Such third-party suppression is incompatible with the operation of veto T cells. Depletion of Thy-1.2+ or Lyt-2+ cells from the suppression-inducing donor SC inoculum did not abrogate suppression induction in BALB/c mice; instead, suppression was enhanced. The demonstration of veto cell activity in similarly primed mice by other groups of investigators indicates that both types of suppression may operate. However, our results show that only antigen-specific Ts can mediate the transferable suppression of CTL responses to alloantigens.     

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).     

Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells

Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1 week. After the treatment with PC12-derived exosomes, MSCs developed neuron-like morphology, and gene and protein expressions of neuronal markers were upregulated. Microarray analysis showed that the expression of miR-125b, which is known to play a role in neuronal differentiation of stem cells, was much higher in PC12-derived exosomes than in exosomes from B16-F10 melanoma cells. These results suggest that the delivery of miRNAs contained in PC12-derived exosomes is a possible mechanism explaining the neuronal differentiation of MSC.     

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8⁺ and CD4⁺ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression.     

MHC II in Dendritic Cells is Targeted to Lysosomes or T Cell-Induced Exosomes Via Distinct Multivesicular Body Pathways

Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide-loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen-loaded DCs, which is triggered by cognately interacting antigen-specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent-resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion.     

Mechanism of action of a T suppressor factor (TsF) in contact sensitivity: the T cell target for TsF activity in adoptive transfer of immunity is not effector cell

The passive transfer of contact sensitivity (CS) by immune cells into normal animals requires the interaction of two distinct Ly-1+ T cells, one which is Vicia villosa lectin (VV)-nonadherent, the other which adheres to VV. Functional deletion of either cell type abrogates the adoptive transfer of CS into normal animals, whereas VV-nonadherent cells alone can transfer CS into animals pretreated with cyclophosphamide (Cy). An antigen-specific T suppressor factor, designated TNP-TsF, inhibits the transfer of CS into normal adoptive recipients. TNP-TsF mediates its suppressive activity by inducing an I-J+ subfactor (designated I-J2) from the assay population by the interaction of PC1-F (a TNP-binding subfactor of TNP-TsF) with antigen-primed Ly-2+ T cells. This I-J+ subfactor then complements TNBS-F (an antigen-nonbinding subfactor of TNP-TsF) to form an antigen-nonspecific effector molecule which suppresses DTH responses in an antigen-nonspecific fashion. We report here that TNP-TsF suppresses the adoptive transfer of CS into normal animals but not into animals pretreated with Cy. TNBS-F + I-J2, the effector complex of TNP-TsF, also suppresses the transfer of CS into normal but not Cy-treated animals. When the Ly-1 immune cells were separated into VV-adherent and -nonadherent populations, the TNBS-F + I-J2 suppressor complex suppressed the functional activity of the VV-adherent cell population, but not the VV-nonadherent cells. This suppressive activity correlates with the need for VV-adherent cells in the transfer of CS into normal but not Cy-treated recipients. When an I-J+ molecule (I-J1) from an SRBC-specific TsF was used in place of I-J2 to form a suppressor complex with TNBS-F, this TNBS-F + I-J1 TsF suppressed the transfer of CS into both normal and Cy-treated recipients. This difference in functional suppressive activity correlated with a difference in target cell specificity: TNBS-F + I-J1 suppressed the VV-nonadherent TDTH cell, whereas TNBS-F + I-J2 suppressed the VV-adherent T cell of CS. Immune cells which are transferred under conditions which do not require the VV-adherent cell for transfer are not suppressed by TNBS-F + I-J2 or TNP-TsF, but are suppressed by the TNBS-F + I-J1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressor T cell circuits in contact sensitivity. II. Induction and characterization of an efferent-acting, antigen-specific, H-2-restricted, monoclonal T cell hybrid-derived suppressor factor specific for DNFB contact hypersensitivity

This report defines a methodology for the production and characterization of an antigen-specific, monoclonal T cell hybrid-derived suppressor T cell factor (TsF) that suppresses the passive transfer of 2,4-dinitrofluorobenzene (DNFB) contact hypersensitivity. Fusion of T cells from BALB/c (H-2d) mice tolerized with syngeneic DNP-spleen cells to BW 5147 thymoma cells resulted in several hybrids that constitutively produce a soluble regulatory molecule. One of these hybrids, 26.10.2, was subsequently cloned, and its soluble factor was characterized with respect to its antigen specificity, biochemical nature, MHC restriction pattern, and identity of its target cell. 26.10.2 TsF suppresses the passive transfer of delayed-type hypersensitivity (DTH) mediated by DNP- but not trinitrochlorobenzene- or oxazalone-primed DTH T cells (TDH) after a 1 hr incubation at 37 degrees C. In contrast, 26.10.2 TsF had no suppressive effect on secondary in vitro DNP-specific T cell proliferative responses. 26.10.2 TsF therefore represents an antigen-specific factor with effector (efferent-acting) function. The monoclonal TsF was shown to consist of a two-chain, disulfide-bonded molecule, and to bear a receptor(s) specific for DNP and determinants encoded by the I region of the H-2 complex. Effector suppressive activity of 26.10.2 TsF was restricted by Class I H-2Dd determinants. One cellular target of this monoclonal factor was shown to be the DNP-specific TDH cell, because DNFB-primed lymph node cells from cyclophosphamide-pretreated donors (lacking Ts-auxiliary (Ts-aux) cells) were efficiently suppressed. The TsF appears to focus on passively bound, TDH receptor-associated, DNP-Class I determinants, as suggested by the observation that freshly prepared, but not overnight cultured, DNP-specific TDH cells were susceptible to suppression.     

In Vivo Induction of Functionally Suppressive Induced Regulatory T Cells from CD4+CD25- T Cells Using an Hsp70 Peptide

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4⁺CD25^- naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25⁺ cells prior to immunization, allowing the in vivo formation of Tregs from CD4⁺CD25^- precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4⁺CD25⁺ T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4⁺CD25^- T cells.