PPD-induced immunoglobulin production in human peripheral blood lymphocytes. II. Separation of PPD-reactive helper T cells from PWM-reactive helper T cells in polyclonal immunoglobulin production of human peripheral blood lymphocytes.

We investigated the relationship bewteen PPD-reactive helper T cells and PWM-reactive helper T cells in polyclonal Ig production of human PBL. Elimination of PPD-reactive T cells by BUdR + light treatment resulted in a loss of helper function in PPD-induced Ig production, but had no effect on helper function in PWM-induced Ig production. On the other hand, elimination of PWM-reactive T cells resulted in a loss of helper function in both PPD-induced Ig production and PWM-induced Ig production. A blast cell-enriched fraction that was generated by PPD and separated by the velocity sedimentation method contained helper function in both responses. On the other hand, blast cell-depleted fractions did not contain PPD-reactive helper function, although the PWM-reactive helper function was evident. These results strongly suggest that PPD-reactive helper T cells are included in PWM-reactive helper T cells.     

Regulatory mechanism of delayed-type hypersensitivity in mice. IV. Effect of suppressor T cells on the development of memory T cells involved in accelerated generation of DTH-effector cells in vitro

The effect of suppressor T cells (Ts) on the induction and the subsequent development of memory T cells for delayed-type hypersensitivity (DTH) was examined. The memory cells were induced in the spleens of mice primed previously with a low dose of reduced and alkylated ovalbumin (Ra-OA), and they generated DTH-effector T cells (DTH-Te) in a significantly accelerated fashion when cultured with OA in vitro. Ts were obtained from the spleens of mice which received OA-coupled spleen cells i.v. 4 days previously, and they inhibited antigen-specifically the induction of DTH responses in the recipient mice sensitized with alum-absorbed OA only when transferred with 5 weeks before sensitization. The spleen cells from mice given Ts together with the priming antigen 7 weeks before culture failed to generate DTH-Te in an accelerated manner on restimulation with OA in vitro. The memory cells from primed mice also did not cause accelerated generation of DTH-Te, when cultured with Ts in the presence of OA in vitro. These results indicate that both the induction of the memory cells by priming with antigen in vivo and the subsequent development of memory cells to DTH-Te by restimulation in vitro are inhibited independently by Ts. This corresponded well with the effect of Ts on the development of DTH-memory in vivo.     

Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. I. Differential helper cell requirement for the generation of cytotoxic effector cells from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Previous studies have shown that both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells capable of developing into alloreactive CTL. In the current study, we have examined the mechanisms involved in the generation of CTL effector cells from these two CD8+ subsets. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic non-T cells, either alone or in the presence of CD4+ Th cells. Although the generation of CTL from CD8+CD45RA- precursor cells consistently required the presence of CD4+ Th cells, cytotoxic effector cells could be generated from CD8+CD45RA+ precursor cells in the absence of CD4+ cells. Several lines of evidence indicated that the helper cell-independent generation of cytotoxic effector cells from CD8+CD45RA+ precursors resulted from the unique ability of this subset to produce and use IL-2 in an autocrine fashion: 1) exogenous IL-2 could replace the effects of CD4+ helper cells for either CD8+ subset; 2) the helper cell-independent functional maturation of CD8+CD45RA+ cells could be blocked by anti-CD25 or anti-IL-2 antibodies; and 3) CD8+CD45RA+ cells produced IL-2 after activation with allogeneic cells. The finding that precursors for helper cell-independent CTL generation are restricted to the CD8+CD45RA+ subset suggests that this capability may vary as a function of the maturation of CD8+ cells.     

Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.     

MHC class II epitope nesting modulates dendritic cell function and improves generation of antigen-specific CD4 helper T cells

CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.     

Antigen-reactive cloned helper T cells. I. Unresponsiveness to antigenic restimulation develops after stimulation of cloned helper T cells

Murine helper T lymphocyte (HTL) clones reactive to ovalbumin (OVA) were maintained in continuous culture in vitro. Clones were propagated by weekly stimulation in the presence of irradiated splenic filler cells, antigen, and supernatant fluid (SF) containing IL 2. By varying the quantity of these reagents in cultures of HTL cells, the reactivity to antigen of the cloned cells was altered markedly. After stimulation by antigen or SF, HTL clones became profoundly unresponsive to antigenic restimulation. Cells remained unresponsive for 2 to 9 days after stimulation, depending on the culture conditions that were chosen for their maintenance. The addition of SF containing a high concentration of IL 2 prolonged the duration of unresponsiveness by 3 days, and the presence of a non-T splenic filler cell increased the period of unresponsiveness by an additional 4 days. The use of a high concentration of OVA in cell cultures also prolonged the time of unresponsiveness. The results described here demonstrate that the response to antigen of HTL cells is down-regulated after stimulation, and appears to be correlated with exposure to SF that contains IL 2.     

Accessory cells in the in vitro generation of type C virus-specific T killer lymphocytes. II. Demonstration of a T helper cell in the spleen of in vivo primed mice

The existence of a helper T cell cooperating with cytolytic T lymphocytes (CTL) in cell-mediated anti-tumor responses specific for the virus-induced FMR antigens can be demonstrated by using unprimed thymocytes as CTL precursors and in vivo primed irradiated spleen cells as helper. The helper T cells express Thy-1.2 and Lyt-1.2 antigens at their surface, but not Lyt-2.2. The helper function required the presence of macrophages to be detected, is antigen specific, and appears unusually radiosensitive compared with previously described helper T cell function.     

Mitogen-induced IL-2 production and proliferation at defined stages of T helper cell development.

Th cell development inside the thymus can be defined on the basis of qualitative and quantitative CD4 and CD8 marker expression and follows the pathway of CD4-8- cells----CD4+8+ cells----CD4+8low cells----CD4+8- cells, which presumably emigrate to seed the periphery and serve as functionally mature Th cells. The various cell subpopulations at defined developmental stages were isolated by electronic cell sorting and examined for mitogen induced IL-2 production and cell proliferation responses. For TCR-alpha beta-bearing CD4+8+ and CD4+8low thymocytes that are actively engaged in positive and negative selection processes, negligible to low levels of IL-2 production and cell proliferation were observed in response to TCR:CD3 triggering or to the combined activation of protein kinase C and calcium mobilization mediated by PMA and ionomycin, respectively. For CD4-8- TCR-alpha beta early thymocytes that have not yet entered the selection process, PMA + ionomycin induced significant cell proliferation but little IL-2 production, in the absence of added IL-1. However, addition of IL-1 caused a powerful induction of IL-2 production that was accompanied by increased cell proliferation. Triggering of the TCR:CD3 complex had no effect on CD4-8-TCR(-)-alpha beta thymocytes as they do not express detectable levels of TCR-alpha beta. For thymus CD4+8- Th cells, the first cells that have completed TCR repertoire selection, vigorous proliferation was observed in response to TCR:CD3 triggering in the presence of added IL-2. However, the development of IL-2 responsiveness was not accompanied by high level IL-2 inducibility as TCR:CD3 triggering caused only marginal IL-2 production. In contrast, spleen CD4+8- T cells, the most "mature" representatives of Th cells, expressed high levels of IL-2 production as well as IL-2 responsiveness in response to TCR:CD3-mediated stimulation. The lack of anti-TCR-induced IL-2 production by thymus CD4+8- T cells was not due to an intrinsic defect as high levels of IL-2 production was induced by PMA + ionomycin. Possible reasons for the temporal acquisition and differential control of IL-2 inducibility and IL-2 responsiveness are discussed in the context of established Th cell development pathway.     

低聚果糖对AOM/DSS诱导的结肠炎相关结直肠癌小鼠辅助性T细胞的调控作用

探讨益生元低聚果糖（FOS）对结肠炎相关结直肠癌（CAC）小鼠肠道辅助性T细胞的调节作用。

将6周龄SPF级雄性C57BL6小鼠（体重18～20 g）随机分为对照组、模型组和干预组,每组10只。通过偶氮甲烷、葡聚糖硫酸钠构建CAC小鼠模型,干预组每日使用2 mg/kg b.w. FOS灌胃,持续10周。造模期间监测各组小鼠体重变化,造模结束后测量结肠长度和肿瘤数目。采用微球免疫分析法检测血清Th1/Th2/Th17相关细胞因子水平,流式细胞术分析肠系膜淋巴结内Th1、Th2、Th17细胞亚群比例。

与模型组相比,干预组小鼠结肠长度显著增加（t=3.106,P=0.006 4）,肿瘤数目显著减少（U=15.000,P=0.011 1）,血清中IL-17A（t=3.504,P=0.008 8）、TNF-α（t=2.381,P=0.030 0）等促炎细胞因子水平降低,肠系膜淋巴结Th17细胞比例显著降低（t=6.031,P=0.002 7）,Th1/Th2显著升高（t=2.419,P=0.038 7）。

FOS可调控CAC模型小鼠结肠内辅助性T细胞的分化,抑制肿瘤的发生。

     

Antigen-specific helper factor production by immortalized clone of OVA-specific T cells. I. In vitro activity

Immortalized clones of virally transformed OVA-specific T cells produce antigen-specific helper factor upon stimulation in vitro. The helper factor activate DNP-primed B cells to multiply and synthesize IgG anti-DNP antibodies. The trigger of the helper clone is antigen specific and the B cell-stimulating hapten must be coupled to the specific T cell carrier in order to transfer the help signal from the activated T clone to the B lymphocytes. Activation of the helper clone is performed by antigen-pulsed macrophages and cannot be achieved by the free soluble antigen. However, cell-free supernatant of the antigen-pulsed macrophages can stimulate the helper cells. Thus the antigenic determinant must be presented to the helper cell in the form of macrophage-processed antigen. These requirements for antigenic stimulation and the activity of the secreted helper factor demonstrate that the immortalized helper clone preserved the cellular components which control the antigen-specific immune function of the normal T lymphocyte.     

Cellular basis of tolerance to serum albumin in adult mice. I. characterization of T suppressor and T helper cells.

The surface markers and size of suppressor cells were determined in adult (BALB/c x C57BL/Ka)F1 mice which were tolerized with a single injection of deaggregated bovine serum albumin (BSA). Suppressor cells from the spleens of tolerazided donors were assayed in a cell transfer system in which graded numbers of cells were injected into irradiated syngeneic mice along with limiting numbers of T cells primed to BSA and an excess of B cells primed to DNP-BSA. Adoptive hosts were challenged with DNP-BSA in saline, and the anti-DNP response was measured. Suppressor cells were antigen specific as shown by the inhibitory activity of BSA-tolerant spleen cells on the response to DNP-BSA, but not to DNP-BGG. Suppressor cells were eliminated by in vitro treatment with anti-Thy 1.2, anti-Ly-2.2, anti-I-J subregion antisera and C, but not with anti-Ly-1 or anti-I-A subregion antisera. Neither unprimed nor primed helper T cells were detected in the spleen of tolerized donors after in vitro treatment with anti-Ly-2.2 antisera. Both helper and suppressor T cells from the spleens of primed or tolerized donors, respectively, showed a rapid sedimentation velocity (S greater than 3.7 mm/hr).     

Tolerance to Mls-disparate cells induces suppressor T cells that act at the helper level to prevent in vivo generation of cytolytic lymphocytes to hapten-altered self

We have been examining the mechanisms that control in vivo development and down regulation of cytolytic T lymphocytes (CTL) to trinitrophenyl (TNP)-altered self antigens. In vivo generation of hapten-specific CTL requires an auxiliary antigenic stimulus, which can be provided by H-2 compatible but Mls-disparate cells. These experiments were designed to study the effect of tolerization with such Mls-disparate cells on CTL development. C3H/HeN (H-2k, Mlsc ) mice sensitized in the footpads with C3H-TNP spleen cells plus CBA/J (H-2k, Mlsd ) spleen cells develop CTL in the draining lymph nodes that will lyse 51Cr-labeled TNP-modified C3H targets. However, we have found that if C3H/HeN mice are given tolerizing doses of CBA/J spleen cells 5 to 7 days before sensitization, a splenic suppressor T cell (Ts) appears. This Ts will suppress CTL development in its tolerant host, and can be transferred adoptively to function in naive mice. Ts and its precursor are cyclophosphamide insensitive and therefore different from the naturally existing suppressor cell present in mice. When triggered by cells with Mlsd , the Ts produces a factor (TsF) that hinders helper factors from functioning in an in vitro CTL assay. Furthermore, TsF acts to prevent utilization of IL 2 by an IL 2-dependent cell line. Thus, evidence has been provided that the in vivo generation of CTL toward hapten-altered self can be down regulated at the level of helper signals by a Ts. The latter is inducible by the Mls-disparate cells that are needed at a different site to trigger the helper factors in this CTL system.     

Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.

To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.     

Regulatory mechanism of cell division : I. Colchicine-induced endoreduplication

Experiments were carried out to study the induction of endoreduplication by colchicine in Chinese hamster cells cultivated in vitro. The cells that endoreduplicate are those that, at the moment of treatment, are in late-S and, in particular, in G2. The endoreduplication cycle consists of two periods of synthesis (S1 and S2), as already noted by Schwarzacher & Schnedl [42], separated by an intervening period that we call G? The S2 synthesis begins in a highly synchronous manner, without the cells having gone into a c-mitosis. The quantity of endoreduplicated cells induced is proportional to the 3.5th root of the colchicine concentration, above a threshold value, and does not depend on the duration of the treatment. When the cultures are treated twice with colchicine, the second treatment is also able to induce endoreduplication and, after it, there appear double endoreduplicated cells (with quadruplochromosomes).     

Functional differentiation of T cell precursors. I. Parameters of carrier-specific tolerance in murine helper T cell precursors.

Irradiated mice reconstituted with bone marrow from sheep gamma-globulin- (SGG) tolerant syngeneic donors display reduced IgG responsiveness to challenge with trinitrophenylated (TNP)-SGG compared with recipients of normal marrow. This effect is SGG-specific and is due neither to suppressor T cells nor to antigen carryover. "Helper T cell precursor tolerance" can be induced with as little as 40 micrograms tolerogen (SGG). Unlike mature helper T cells, these precursors show both a rapid induction and rapid waning patterns, suggesting a high rate of turnover. Our results imply that marrow helper T cell precursors bear antigen-specific receptors and that the T cell repertoire must be at least partially generated before residence in the thymus.     

The role of macrophages in the generation of T-helper cells. I. The requirement for macrophages in helper cell induction and characteristics of the macrophage-T cell interaction.

The role of macrophages in the induction of helper cells in vitro was investigated. Using either soluble or particulate antigens, macrophages were found to be essential. This was true regardless of the anatomical source of the T cells (spleen, lymph node or the cortisone resistant pool of the thymus), or of the method of macrophage depletion, (adherence to polystyrene or nylon wool, or by the use of carbonyl iron). There were some differences, however, depending on the physical nature of the antigen used. With soluble antigen, 2-mercaptoethanol or allogeneic macrophages would not overcome the macrophage deficit, whereas they did with particulate antigen. The nature of the interaction between macrophages and T cells was investigated using flasks with double chambers, separated by a nucleopore membrane with 0.2 μm pores. Since there was effective interaction, contact between T cells and macrophages is not essential for helper cell induction.     

Regulatory mechanism of delayed-type hypersensitivity in mice. V. Augmentor cells for DTH responses

Mice primed with 1 microgram of reduced and alkylated ovalbumin (RA-OA) developed not only long-lived memory cells for delayed-type hypersensitivity (DTH), capable of differentiating into DTH-effector T cells (DTH-Te) against ovalbumin (OA) when restimulated in vitro with OA, but also spleen cells capable of augmenting recipients' DTH responses to OA when transferred into cyclophosphamide (CY)-pretreated mice. The augmenting activity in spleen cells, upon transfer, was found 7 days, but not 21 or 91 days, after priming with RA-OA, although memory DTH-Te were present throughout the period of observation. The loss of augmenting activity after day 7 of priming was not due to the presence of suppressor cells; spleen cells taken 21 days after priming failed to suppress, upon transfer, the augmenting activity in 7-day-primed spleen cells as well as induction and expression of DTH responses to OA. When 7-day-primed spleen cells were fractionated on a discontinuous bovine serum albumin density gradient, the augmenting activity was found only in the medium-density-cell layer, although memory DTH-Te were separated in the high-density layer. Augmentation of DTH-Te generation could also be demonstrated in vitro when 7-day-primed spleen cells, but not 21-day-primed spleen cells, were added to cultures of spleen cells from CY-pretreated mice. These results indicate that, in the 7-day-primed spleen, there is an augmentor cell population which is different from memory DTH-Te and interacts with CY-resistant unprimed cells to facilitate DTH-Te generation.     

Regulatory T cells suppress T cell activation at the pathologic site of human visceral leishmaniasis

Suppression of T cell response is thought to be involved in the pathogenesis of visceral leishmaniasis (VL). Regulatory T cell (Treg) mediated immune-suppression is reported in animal models of Leishmania infection. However, their precise role among human patients still requires pathologic validation. The present study is aimed at understanding the frequency dynamics and function of Treg cells in the blood and bone marrow (BM) of VL patients. The study included 42 parasitologically confirmed patients, 17 healthy contact and 9 normal bone marrow specimens (NBM). We show i) the selective accumulation of Treg cells at one of the disease inflicted site(s), the BM, ii) their in vitro expansion in response to LD antigen and iii) persistence after successful chemotherapy. Results indicate that the Treg cells isolated from BM produces IL-10 and may inhibit T cell activation in IL-10 dependent manner. Moreover, we observed significantly higher levels of IL-10 among drug unresponsive patients, suggesting their critical role in suppression of immunity among VL patients. Our results suggest that IL-10 plays an important role in suppression of host immunity in human VL and possibly determines the efficacy of chemotherapy.