Tolerance induction of allo-class I H-2 antigen-reactive Lyt-2+ helper T cells and prolonged survival of the corresponding class I H-2-disparate skin graft

C57BL/6 (B6) mice were i.v. presensitized with class I H-2-disparate B6-C-H-2bm1 (bm1) spleen cells. Such presensitization resulted in almost complete abrogation of bm1-specific Lyt-2+ T cell-mediated proliferative and IL-2-producing capacities as measured by MLC of lymphoid cells from presensitized B6 mice with stimulating bm1 cells. In contrast, comparable magnitude of CTL responses was generated in bulk cultures from presensitized B6 lymphoid cells to that obtained in unpresensitized B6 responding cultures. These differential influences of Lyt-2+ T cell functions were also demonstrated by limiting dilution assays; frequencies of proliferative and IL-2-producing T cell precursors were as low as undetectable in presensitized B6 lymphoid cells, whereas an appreciable frequency of CTL precursors in a portion of the same lymphoid cells was observed. When bm1 skin grafting was performed in B6 mice i.v. presensitized with bm1 cells, the strikingly prolonged survival of bm1 skin grafts was observed. It was also demonstrated that the bm1 skin graft-bearing B6 mice which had been presensitized with bm1 cells not only exhibited a continuing suppressive state of bm1-specific helper (proliferative and IL-2-producing) function but also failed to generate anti-bm1 CTL responses. These results indicate that 1) i.v. presensitization with class I H-2 alloantigens results in selective tolerance of Lyt-2+ Th cells which is adequate for inducing prolonged graft survival, 2) the induction of complete abrogation of CTL potential is not absolute requirement for the prolongation of graft survival, and 3) residual CTL potential is attenuated after grafting so far as Th cells are rendered tolerant.     

Contrasuppressor cells that break oral tolerance are antigen-specific T cells distinct from T helper (L3T4+), T suppressor (Lyt-2+), and B cells

Continuous gastric intubation of mice with the T cell-dependent antigen sheep erythrocytes (SRBC) leads to a state of systemic unresponsiveness to parenteral SRBC challenge, a state termed oral tolerance. The systemic unresponsiveness of mice rendered orally tolerant to SRBC, however, is converted to humoral immune responsiveness by adoptive transfer of effector T contrasuppressor (Tcs) cells. In this study, the authors have isolated and characterized the Tcs cell subset, from the spleens of orally immunized mice, which abrogates oral tolerance. This Tcs cell is a novel cell type, which can be separated from functional T suppressor (Lyt-2+) and T helper (L3T4+) cells, and the effector Tcs cell exhibits a Lyt-1+, 2-, L3T4- phenotype. Furthermore, contrasuppression is not mediated by B cells, including those of the Lyt-1+ phenotype. Adoptive transfer of splenic Lyt-1+, 2-, L3T4- T cells from C3H/HeJ mice given oral SRBC for 21 to 28 days and splenic Lyt-1+, 2-, L3T4- T cells of C3H/HeN mice orally immunized for a shorter interval abrogated oral tolerance. Furthermore, separation of Lyt-1+ T cells into L3T4+ and L3T4- subsets by flow cytometry resulted in Lyt-1+, L3T4+ T cells with helper but not contrasuppressor function, whereas the Lyt-1+, L3T4- T cell fraction abrogated oral tolerance even though it was without helper activity. This Tcs cell subset was also effective when added to cultures of tolerized spleen cells derived from SRBC-fed mice. The effector Tcs cells are antigen-specific, because Tcs cells from SRBC-immunized mice reverse tolerance to SRBC but not to horse erythrocytes (HRBC), and Tcs cells from HRBC-immunized mice reverse tolerance to HRBC but not to SRBC. When splenic T3 (CD3)-positive T cells (Lyt-1+, 2-, and L3T4-) were separated into Vicia villosa-adherent and nonadherent subpopulations, active contrasuppression was associated with the T3-positive and Vicia villosa-adherent T cell fraction. Thus, a distinct Lyt-1+, 2-, L3T4- T cell subset that contains a T3-T cell receptor complex, which can regulate oral tolerance, is present in spleens of orally immunized mice.     

Lyt-2- T cell-independent functions of Lyt-2+ cells stimulated with antigen or concanavalin A

Approximately 30% of cytolytic Lyt-2+ clones from primed mice are able to proliferate autonomously, i.e., independent of IL 2 derived from Lyt-2- cells after antigenic stimulation. H-2K- or -D-restricted induction of Lyt-2+ cells to autonomous proliferation requires Ia+ stimulator cells. A strict correlation was observed between the ability of Lyt-2+ clones to proliferative autonomously and to induce DH. Eventually, the growth of all Lyt-2+ cytolytic clones becomes dependent on exogenous IL 2, and their ability to induce DH is lost. Small Lyt-2+ cells can also be induced in primary cultures by antigen or concanavalin A to proliferate in the absence of exogenous IL 2. The frequency of autonomously proliferating small Lyt-2+ cells is the same as that of small Lyt-2+ cells proliferating in the presence of exogenous IL 2. IL 2 derived from Lyt-2- cells can augment proliferation of Lyt-2+ cells, but is not obligatory.     

Lyt-2+ suppressor T cells are resistant to anti-Lyt-2 antibody blocking

Lyt-2 molecules play a role in antigen recognition by cytotoxic T lymphocytes (CTL). In an attempt to determine whether Lyt-2 molecules play a similar role in suppressor T cell (Ts) functions, the effect of anti-Lyt-2 antibodies on Ts generation and effector activity was studied. Allospecific Ts were induced in allogeneic mixed lymphocyte cultures (MLC). Anti-Lyt-2 antibodies added to MLC in the absence of complement abolished CTL generation, but had no effect on concomitant induction of Ts. In a different experimental system, allospecific Ts were induced in cultures treated with pyrilamine, which blocks generation of CTL but allows differentiation of Ts. The addition of anti-Lyt-2 antibodies to pyrilamine-treated MLC resulted in unaffected induction of Ts. It was further demonstrated that the effector activity of Ts was as resistant to anti-Lyt-2 antibodies as their induction, in contrast to the cytolytic activity of CTL, which was inhibited by the same antibodies. Ts in the present experimental system were Lyt-2+ antigen-specific cells. It therefore appears that Lyt-2 molecules, although expressed on both CTL and Ts, are involved in CTL activity, but do not play an essential role in Ts function.     

Dual regulation of resistance against Toxoplasma gondii infection by Lyt-2+ and Lyt-1+, L3T4+ T cells in mice

Studies were performed to attempt to define the T cell subset responsible for resistance to Toxoplasma gondii. A temperature-sensitive mutant (ts-4) strain of T. gondii was used for immunization because it causes infection but does not persist in the host. Immunization with this strain induced marked resistance against lethal challenge infection with virulent strains of T. gondii in mice. The resistance could be transferred to normal recipient mice by i.v. injection of spleen cells from ts-4-immunized mice. Marked inhibition of cyst formation in the recipient mice was also noted. The protective activity of immune spleen cells was removed by pretreatment of the spleen cells with anti-Thy-1.2 and C, indicating that T cells are responsible for the observed protection. Pretreatment of immune spleen cells with anti-Lyt-2.2 and C completely ablated their protective effect; pretreatment with anti-Lyt-1.2 or anti-L3T4 and C had lesser effects on their ability to transfer resistance. The effect of anti-Lyt-1.2 was the same as that obtained with anti-L3T4. This suggested that one T cell subset that is partially responsible for protection has both Lyt-1.2 and L3T4 markers on the cell surface. These results indicate that there are substantial roles for both the Lyt-2+ and Lyt-1+, L3T4 T cell subsets in dual regulation of resistance against toxoplasma infection and that Lyt-2+ T cells are the principal mediator of the resistance.     

Lyt phenotype of cytotoxic T cells: shift from Lyt-1+2+3+ to a mixed population of Lyt-1+2+3+ and Lyt-1-2+3+ during in vitro culture

The Lyt phenotype of cytotoxic T cells generated in the primary H-2 response was investigated kinetically. The cytotoxicity generated in the early stage of culture was abolished by treatment with alpha Lyt-1,2,3, and complement (C), whereas that generated in the late stage was only partially eliminated by alpha Lyt-1, but was abolished by alpha Lyt-2, 3, and C. This suggested late expansion of the Lyt-1-2+3+ population. Lack of Lyt-1 antigen was confirmed with cells that were depleted of Lyt-1+ from primary culture and then stimulated in the secondary response by elimination of cytotoxicity and by direct Lyt typing. Results indicated that the response of proliferative and cytotoxic T cells of the Lyt-1+2+3+ phenotype in the early stage of culture was followed by activation of Lyt-1-2+3+ T cells. Cytotoxic T cells in the late stage were shown to be a mixture of Lyt-1+2+3+ and Lyt-1-2+3+ cells. This was confirmed with cytotoxic T cells from secondary culture and uncloned long-term T cell lines.     

IL-1 as a co-factor for lymphokine-secreting CD8+ murine T cells

Immunologically important among the known biologic activities of IL-1 is its ability to function as a co-factor for responses mediated by lymphokine secreting CD4+ Th cells. In contrast to its known effects in CD4+ T cell responses, IL-1 is not known to play a role in CD8+ T cell responses. In the present study, we have assessed the ability of murine recombinant IL-1 to function as a co-factor for stimulating CD8+ T cells to secrete lymphokines such as IL-2. We found that, in conjunction with either Ag or mitogen, IL-1 is able to stimulate lymphokine-secreting CD8+ T cells. Furthermore, we found that, as a consequence of its stimulation of lymphokine-secreting CD8+ T cells, IL-1 is able to reconstitute MHC class I allospecific cytolytic T lymphocyte responses by cell populations depleted of both accessory cells and CD4+ T cells. These results demonstrate that the biologic activity of IL-1 is not restricted to CD4+ cell responses, and suggests that IL-1 can function as a co-factor for the stimulation of lymphokine-secreting Th cells regardless of their CD4/CD8 phenotype. If IL1 acts directly on lymphokine-secreting T cells or on the APC with which they interact is not yet certain.     

Role of lymphokine-secreting CD8+ T cells in cytotoxic T lymphocyte responses against vaccinia virus

The present study has examined the relative role of CD4+ and CD8+ Th cells in the generation and reactivation of antivaccinia virus memory CTL responses. We show that mice primed in vivo to vaccinia virus generate in vitro antivaccinia virus memory CTL responses through both CD4+ and CD8+ Th cell pathways, with the CD4+ Th pathway being the more prominent of the two. In addition, we show that vaccinia virus-specific CD8+ Th cell function is mediated through production of lymphokines, including IL-2, and that the CD8+ Th cell component in the CTL response is labile, decreasing progressively with increasing time after in vivo priming. Thus, this study demonstrates the existence of two phenotypically distinct Th cell pathways in the generation of antivirus CTL responses.     

Cloned Listeria monocytogenes specific non-MHC-restricted Lyt-2+ T cells with cytolytic and protective activity

Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given together with rIL-2. These data suggest that non-MHC-restricted Lyt-2+ killer cells generated during listeriosis are cytolytic T lymphocytes that interact with their target Ag via the T cell receptor/T3 complex and the Lyt-2 molecule and, furthermore, that these cells play a role in anti-listerial resistance. The possible relevance of IFN-gamma secretion and target cell lysis for antibacterial protection is discussed.     

Phenotypic identification of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells

Murine peripheral Lyt-2+ T cells could be subdivided according to surface expression of the Pgp-1 glycoprotein into major (71%) Pgp-1- and minor (29%) Pgp-1+ subsets. A striking correlation was observed between Pgp-1 expression and enrichment for antigen-specific memory cytolytic T lymphocyte precursors (CTLp). After immunization with the male minor transplantation antigen H-Y, virtually all the H-Y-specific CTLp were found in the minor Pgp-1+ subset of Lyt-2+ cells. In addition, after alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-Lyt-2 antibody was markedly enriched within the Pgp-1+ cells, suggesting an enrichment for CTLp bearing high avidity antigen receptors. Taken together, these data suggest that surface Pgp-1 expression is stably acquired at the time of primary antigenic stimulation by virgin T cells. As such, Pgp-1 represents an important marker for identifying a subset of Lyt-2+ T cells with the quantitative and qualitative properties of memory CTLp.     

TNP-specific Lyt-2+ cytolytic T cell clones preferentially respond to TNP-conjugated epidermal cells

A most effective method for the induction of hapten-specific allergic contact sensitivity (CS) is via epicutaneous application of the hapten. Another effective method is by the administration of haptenated epidermal cells (EC) subcutaneously. The latter method induces more intense and longer lasting CS than does the subcutaneous administration of haptenated spleen cells (SC). Thus, there may be something unique about EC which, when haptenated, allows them to generate effector cells more effectively than do SC. We therefore attempted to generate T cell clones that were both hapten- and epidermal-specific. Four days after painting mice with 7% trinitrochlorobenzene, draining lymph node cells were obtained and T cells were purified. These cells were co-cultured with trinitrophenylated (TNP) Langerhans cell-enriched EC. After 4 days, cells were harvested and rested on non-TNP-conjugated EC. The cells were restimulated and rested three times, and were then cloned by limiting dilution with added interleukin 2, which was then continually added. Proliferation of T cells was assessed by [3H]-thymidine incorporation. Cytotoxicity assays utilized TNP-conjugated concanavalin A SC blasts or EC as targets. Clones A-2 and E-4 are Thy-1+, Lyt-2+, and L3T4-, and TNP-specific. In contrast to noncloned TNP-specific T cells, the clones proliferate preferentially in response to TNP-EC rather than TNP-SC. Also in contrast to noncloned T cells, the clones were preferentially cytotoxic for TNP-EC; compared to TNP-SC, there was an eight- to 32-fold increase in killing when TNP-EC were used as targets. Clones A-2 and E-4 therefore exhibit hapten and epidermal specificity. The epidermal-specific epitope that is recognized is unknown, but genetic restriction and antibody inhibition studies indicate that it is co-recognized with H-2K.     

NK cells suppress the generation of Lyt-2+ cytolytic T cells by suppressing or eliminating dendritic cells

Cells highly enriched for natural killer activity suppress the generation of Lyt-2+ cytolytic T cells in one-way mixed lymphocyte cultures. Suppression occurs because natural killer cells suppress or eliminate dendritic cells, which are required for proliferation of both Ly-1+ and Lyt-2+ lymphocytes.     

In vitro induction and expression of interleukin 2 receptor in a clonal T helper cell differentiation model

Induction and expression of interleukin 2 (IL 2) receptor have been studied using a poly( Glu60 Ala30 Tyr10 ) (GAT)-specific T cell clone of mouse origin. This clone (52-3) has been characterized and it exhibits functional properties of T helper (TH) cells: it leads to a specific anti-DNP response in the presence of DNP-GAT and DNP-primed B cells and it secretes biological activities which can induce polyclonal B cell proliferation and IgM secretion. In vitro this clone mimics the activation stages of normal T lymphocytes and can be obtained under two states of differentiation. depending on the antigen-specific signal provided by antigen-presenting cells (APC). The expression of IL 2 receptor by this clone has been studied by (i) its response to IL 2, (ii) its capacity to absorb IL 2 bioactivity, and (iii) its reactivity with monoclonal antibody 7D4 specific for mouse IL 2 receptor. All the results indicate that the unstimulated state does not express the IL 2 receptor while the activated state does. Clone 52-3 has been compared with clone 14-1.6 that derives from a TH cell line and expresses the IL 2 receptor constitutively. 52-3 offers a good experimental model for studying in vitro, in a clonal TH cell population, the detailed mechanism of IL 2 receptor induction.     

The role of L3T4+ and Lyt-2+ T cells in the IgE response and immunity to Nippostrongylus brasiliensis

The role of L3T4+ and Lyt-2+ T cells in protective immunity to Nippostrongylus brasiliensis (Nb) was studied in BALB/c mice that were depleted of either the L3T4+ or Lyt-2+ T cell population by injection with rat mAb specific for the appropriate determinant. Host responses to Nb infection including spontaneous elimination of adult worms, development of intestinal mucosal mast cell hyperplasia and the generation of a polyclonal IgE response were all completely blocked by 0.5 mg anti-L3T4 antibody administered simultaneously with Nb inoculation. However, administration of 0.5 mg of anti-Lyt-2 antibody at the same time and 7 days after inoculation with Nb had no effect on any of these responses. Injection of anti-L3T4 antibody as late as 9 days after Nb inoculation interfered with spontaneous cure of Nb infection and anti-L3T4 antibody injection 11 days after Nb inoculation inhibited serum IgE levels measured on day 13 by 50%. In addition, administration of anti-L3T4 antibody at the time of the peak serum IgE response, 13 days after Nb inoculation, accelerated the decline in serum IgE levels. Injection of previously Nb-infected mice with anti-L3T4 antibody at the time of a second Nb inoculation prevented the development of a secondary IgE response but did not affect immunity to Nb infection based on finding no adult worms in the intestines of these mice. These data indicate that 1) L3T4+ T cells are required for spontaneous cure of Nb infection, development of intestinal mucosal mast cell hyperplasia, and the generation and persistence of an IgE response during primary infection with Nb and 2) L3T4+ T cells are required for a considerable time after inoculation for optimal development of these responses. However, L3T4+ T cells are not required for all protective responses in immune mice. In contrast, our data indicate that considerable depletion of the Lyt-2+ T cell population has no significant effect on either worm expulsion or the generation of serum IgE responses.     

Frequency analysis of lymphokine-secreting CD4+ and CD8+ T cells activated in a graft-versus-host reaction

Lymphokine secretion by in vivo-activated T cells was analyzed at the population and single-cell levels in lymphocytes from mice undergoing an acute allogeneic graft-vs-host reaction (GVHR). Three observations were made. First, constitutive lymphokine production by these cells was very low but could be dramatically up-regulated by TCR ligation. Thus, even when harvested at the peak of the GVHR, fewer than 0.1% of lymphocytes secreted detectable granulocyte-macrophage (GM)-CSF, IFN-gamma, or IL-3 in the first 24 h in vitro, and average production of these lymphokines in bulk cultures was less than 10(-5) U/cell. However, when cultured for 24 h with anti-CD3 antibody under conditions which activated less than 0.1% of normal cells, about 30% of GVHR T cells secreted GM-CSF, IFN-gamma, and/or IL-3, and average production levels were increased by 10(3)- to 10(4)-fold. Together with evidence that host alloantigen-induced lymphokine secretion was 10 to 100 times lower than the anti-CD3 response, these data suggest that physiologic lymphokine synthesis by most T cells is low (less than 10(-18) mol of IL-3 per cell) but can be raised above the threshold of detection by TCR cross-linking. Second, individual GVHR lymphocytes varied markedly in their total and relative production of different lymphokines in response to anti-CD3 stimulation, with some cells secreting IL-3 alone, some secreting IL-3 accompanied by other lymphokines (GM-CSF and/or IFN-gamma), and some secreting other lymphokines without detectable IL-3. Finally, both CD4+ and CD8+ T cells from GVHR mice responded to anti-CD3 antibody by secreting IL-3 and other lymphokines: purified CD4+ cells contained an average of 16% and CD8+ cells an average of 10% anti-CD3-inducible lymphokine-secreting cells. By contrast, only 2 to 3% of cells of either subset formed clones in cultures with host allogeneic cells and IL-2, suggesting that clonogenic alloreactive cells were a minority of the T cells activated in the GVHR.     

滤泡辅助性T细胞分化和功能的研究进展

滤泡辅助性T细胞(Tfollicular helper cells,TFH细胞)是新近发现的一种CD4+辅助性T细胞亚群,具有不同于其他亚群的独特功能:迁移到B淋巴滤泡并辅助B淋巴细胞产生抗体。在短短的几年里面,已有大量针对滤泡辅助性T细胞分化与功能的研究。该文将对滤泡辅助性T细胞的最新研究进展作一综述。     

T helper 2 and T follicular helper cells: Regulation and function of interleukin-4

Type 2 immunity is characterized by expression of the cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13, which can function in mediating protective immunity in the host or possess a pathogenic role. T helper (Th) 2 cells have emerged to play a beneficial role in mediating anti-parasitic immunity and are also known to be key players in mediating allergic diseases. In addition to the Th2 cells, recent studies have identified T follicular helper (Tfh) cells as an alternative source of IL-4 to regulate type 2 humoral immune responses, indicating that Th2 and Tfh cells exhibit overlapping phenotypical and functional characteristics. Th2 and Tfh cells appear to utilize distinct mechanisms for regulation of IL-4 expression; however unlike Th2 cells, the regulation and function of Tfh-derived IL-4 is not yet fully understood. Understanding of the molecular mechanisms for IL-4 expression and function in both cell subsets will be beneficial for the development of future therapeutic interventions.