Both the Lyt-2+ and L3T4+ T cell subsets are required for the transfer of diabetes in nonobese diabetic mice

The nonobese diabetic mouse is a model of spontaneous type I diabetes mellitus. It is possible to induce diabetes in young, irradiated nonobese diabetic mice by using adoptive transfer of splenocytes or splenic T cells obtained from diabetic donors. This study demonstrates that the induction of diabetes in the adoptive transfer system is dependent on both the L3T4+ and Lyt-2+ subsets of T cells. Neither of these T cell subsets alone mediates the development of severe insulitis or diabetes when adoptively transferred to young, irradiated recipients. In addition, we show that both the L3T4+ and Lyt-2+ subsets must be obtained from diabetic donors in order to transfer diabetes; neither subset can be replaced with cells obtained from young, nondiabetic donors.     

Cutting edge: differential production of prostaglandin D2 by human helper T cell subsets

Several effector molecules, including cytokines, are differentially produced by Th1 and Th2 cells. We used a gene expression screen method to identify a gene encoding hematopoietic PG D synthase (hPGDS) which was preferentially expressed in human Th2 but not Th1 clones. Studies with anti-hPGDS mAbs confirmed the Th2-dominated expression of hPGDS protein. Upon stimulation with anti-CD3 plus anti-CD28 mAbs, coordinated cyclooxygenase-2 expression and PGD2 production were induced in Th2 lines. hPGDS expression was also observed in a small population (<1.0%) of peripheral blood CD4+ lymphocytes from healthy adults. Most hPGDS-expressing CD4+ lymphocytes showed a typical Th2-type cytokine pattern. Our results suggest that, at the sites of Ag presentation, at least part of the Th2 cell population produces PGD2, which may be involved in various aspects of Th2-related immune responses similar to mast cells.     

Role of interleukin 1 in early T cell development: Lyt-2-L3T4- thymocytes bind and respond in vitro to recombinant IL 1

Thymocytes that bear neither Lyt-2 nor L3T4 differentiation antigens (2-4- thymocytes) contain the precursors for mature L3T4+Lyt-2- and Lyt-2-L3T4+ T cells. In the present study we determined the capacity of 2-4- cells to respond to recombinant interleukin 1 (rIL 1) in vitro. The presence of rIL 1 enhanced IL 2-dependent proliferation to the lectins Con A and PHA by threefold to eightfold. In a second assay, rIL 1 enhanced proliferation and IL 2 production by 2-4- cells in response to phorbol myristate acetate (PMA) and the calcium ionophore, ionomycin. Using a direct IL 1 binding assay, we were able to detect both high-affinity (Kd approximately 5 pM) and low-affinity (Kd approximately 200 pM) classes of IL 1 receptors on freshly isolated 2-4- cells. Bound IL 1 was rapidly internalized, suggesting that such receptors were functional. These results are compatible with a role for interleukin 1 during thymocyte maturation.     

Activation of Lyt-1+ and Lyt-2+ T cell cloned lines: stimulation of proliferation, lymphokine production, and self-destruction

Antigen-induced activation of a chicken gamma-globulin (CGG)-specific Lyt-1+ T cell clone measured both as a function of proliferation and immune interferon (IFN-gamma) production is restricted by a class II determinant of the major histocompatibility complex (MHC) mapped to the I-A subregion, as determined by studies with both recombinant inbred lines and monoclonal antibodies. Activation of Lyt-2+ picryl chloride (PC1)-specific cloned T cell lines by trinitrophenyl (TNP)-coupled spleen cells results in proliferation and the production of at least two lymphokines: lymphotoxin (LT) and IFN-gamma. This antigen-specific activation is restricted to a class I determinant of the MHC complex encoded in the K region. Thus, the common intracellular pathway leading to production of IFN-gamma by Lyt-1+ and Lyt-2+ T cells is mediated and restricted through different surface recognition units. The LT that is produced by antigen-specific activation of T cells not only kills fibroblasts, but it inhibits interleukin 2 (IL 2)-maintained T cells as well. Activation of T cells by concanavalin A (Con A) results in suicidal inhibition of proliferation and cell death by those clones that make LT, but not by those that produce only IFN-gamma under such induction conditions. These results indicate that it is neither Con A nor IFN-gamma that kills T cells, but LT. These results strongly suggest a self-regulatory role of LT in limiting continuing unrestricted T cell response to antigen activation.     

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

Positively selected Lyt-2+ and Lyt-2- mouse T lymphocytes are comparable, after Con A stimulation, in release of IL 2 and of lymphokines acting on B cells, macrophages, and mast cells, but differ in interferon production

Purified mouse T lymphocytes were separated into Lyt-2+ and Lyt-2- populations by the procedure of panning, in which a monoclonal rat anti-Lyt-2 antibody and dishes coated with affinity-purified mouse anti-rat Ig antibodies were used. The populations obtained were 95 to 99% pure as determined by immunofluorescence. Graded doses of these T cells were cultured with optimal mitogenic doses of concanavalin A and the 0 to 24 and 24 to 48-hr culture supernatants were collected. The dose-curve assays of the supernatants of Lyt-2+ and Lyt-2- cells showed comparable activity in interleukin 2 (IL 2) and T cell-replacing factor (TRF), assayed on antigen-stimulated culture of T-depleted spleen cells. Limiting dilution assays of IL 2-secreting precursor cells stimulated by Con A showed a high frequency of precursors in both populations, slightly higher among Lyt-2- cells. The supernatants also contained comparable levels of IPA (inducer of plasminogen activator production by the macrophages), MAF (macrophage-activating factor, assayed by induction of their cytolytic function), and MCGF (mast cells growth factor, assayed on a mast cell line). IPA and MAF were not produced with the same kinetics and in the same T cell concentration conditions as IL 2 and TRF. In contrast, interferon was principally produced by the Lyt-2+ cells.     

Activation of Lyt-2+ (CD8+) and L3T4+ (CD4+) T cell subsets by anti-receptor antibody

The mAb F23.1, specific for V beta 8-related determinants on the TCR, was used to study the requirements for TCR cross-linking and for accessory cells (AC) in the induction of proliferation or IL-2 responsiveness in L3T4+ (CD4+) and Lyt-2+ (CD8+) T cells. T cells were exposed in vitro to soluble native F23.1 antibody, to heteroconjugates composed of the Fab fragments of F23.1 linked to Fab fragments of antibodies specific for Ia determinants on AC, or to F23.1 immobilized on an insoluble matrix. Soluble F23.1 antibody-induced proliferation in naive T cells only in the presence of both AC and exogenous IL-2, and these responses were confined to Lyt-2+ T cells. In contrast, heteroconjugates capable of crosslinking F23.1+ TCR to AC surface Ia determinants were capable of inducing proliferation in both L3T4+ and Lyt-2+ T cells in the absence of added lymphokine. Moreover, binding to and presumably multi-valent crosslinking of the TCR by immobilized F23.1 was sufficient to induce proliferation in both Lyt-2+ and L3T4+ T cells in the absence of AC or exogenous IL-2. Further, it was found that the conditions necessary for T cell growth factor secretion paralleled closely those required for induction of T cell proliferation in the absence of added lymphokine, suggesting that production of endogenous lymphokine might be the limiting process for triggering of T cell proliferation. Taken together, these findings suggest that under optimal conditions of TCR cross-linking, TCR occupancy and cross-linking is sufficient to deliver all of the signals necessary to initiate proliferation in naive populations of both L3T4+ and Lyt-2+ T cells. However, when conditions for TCR signaling are suboptimal, as may be the case for normal Ag-mediated stimulation, a role for second signals delivered by AC or exogenous lymphokines can become critical for T cell activation.     

Functional analysis of human T cell subsets defined by monoclonal antibodies. III. Regulation of helper factor production by T cell subsets

In the present report we extended our previous studies demonstrating that obligatory T-T interactions are important in regulating human immune responses in vitro. Functionally distinct human T cell subsets were isolated by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Evidence was obtained that during allogeneic interactions, OKT4+, but not OKT8+, responder T cells are required to generate helper factor(s) capable of polyclonally activating human B cells independent of additional T cell help. Importantly, the alloantigen-induced helper factor(s) production and/or release was found to be suppressed by addition of graded numbers of radiosensitive OKT8+ cells. On the other hand, no evidence was obtained that supernatant derived from alloactivated OKT8+ cells could counterbalance the helper activity generated in the presence of supernatant from alloactivated OKT4+ cells. Furthermore, OKT8+ cells, known to suppress PWM-driven B cell differentiation in the presence of OKT4+ cells, do not suppress B cell differentiation induced by preformed helper factor even in the presence of OKT4+ cells. These data further underscore the importance of functional T-T interactions in immunoregulation in vitro and support the idea that the target of suppression of B cell differentiation, induced either by alloantigen-triggered helper factor or PWM, are OKT4+ cells and not B cells themselves.     

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.     

Enhancing effect of IFN-gamma on helper T cell activity and IL 2 production

A single injection of young murine immune interferon (IFN-gamma) in young (3 mo) or old (14 to 24 mo) mice 3 days before carrier-priming significantly enhances helper T cell activity of their spleen cells. Maximal enhancement is attained when IFN-gamma is injected once immediately before priming or for 4 consecutive days from the time of priming. Helper activity for anti-TNP antibody response was titrated in vitro by adding graded numbers of spleen cells from HRBC-primed mice of a given age to cultures containing a constant number of spleen cells from 3-mo-old normal mice and TNP-HRBC. When T cell-enriched spleen cells from HRBC-primed young or old mice, uninjected or injected with IFN-gamma, were separated by nylon wool filtration into passed (Thi) and adherent (Th2) cells, the helper activity of both T cell subpopulations was found to be enhanced by IFN-gamma injection. Helper activity of purified Th1 and Th2 cells was also increased by their in vitro preincubation with IFN-gamma. Furthermore, interleukin 2 (IL 2) production by mitogen-activated spleen cells from young and old mice is enhanced by addition of IFN-gamma to cultures. These data altogether indicate that IFN-gamma plays an important role in immunoregulation of helper T cell activity.     

A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones

Hybridoma H129 .19 was derived by fusion between spleen cells of a Lou / Ws1 rat immunized with an Lyt-1+,2- anti-I-Ak cytolytic T lymphocyte (CTL) clone and the nonsecreting myeloma X63-Ag8.653. The monoclonal antibody (mAb) H129 .19 (IgG2a, kappa) was selected for its capacity to inhibit the lytic potential of the immunizing clone. H129 .19 identified a monomorphic determinant on a 55 m.w. murine T cell differentiation antigen, which appeared to be homologous to the human T4 molecule in that: 1) H129 .19 reacted with 80% adult thymocytes, with a subset of splenic T cells, and with the interleukin 2 (IL 2)-producing EL4 thymoma; 2) The mAb bound to and inhibited the IL 2 production and the proliferation of various allo- or soluble antigen-reactive T cell clones that recognized restriction or activating determinants on the I-A or I-E molecules, respectively; 3) H129 .19 did not inhibit the proliferation and/or cytolysis of Lyt-2,3+ T cells specific for class I MHC antigen; and 4) Among six anti-Iak CTL clones examined in this study, the mAb H129 .19 reacted with two I-Ak-specific, Lyt-2,3- clones on which it exerted strong cytolysis inhibiting effect at the effector cell level. By contrast, two other anti-I-Ak and two anti-I-Ek CTL clones were found to express the Lyt-2,3+,T4- cell surface phenotype. The cytolytic potential of the latter clones was not inhibited by anti-Lyt-2,3 mAb. These studies strongly suggest that the mouse T4 molecule facilitates the recognition of class II MHC antigen by most but not all T cells.     

Lymphotoxin production by subsets of T cells

Lymphotoxin is produced by T cells sensitized to antigen upon re-exposure in vitro. It is also elicited by mitogen treatment. Its production has been correlated with delayed-type hypersensitivity and it may be a mediator of that phenomenon. We have examined the Ly phenotype of the subset(s) of T cells that produce lymphotoxin in order to investigate the relationship of lymphotokin killing to allo-killing mediated by Ly2+ T cells. We have found that Ly1 T cells sensitized to ovalbumin secrete more lymphotoxin than Ly2 cells. The ovalbumin-sensitized T cells do not lyse their target in a short term 51Cr-release assay even when "glued" to the target with Con A. Thus, lymphotoxin-producing cells differ phenotypically from the previously defined cytotoxic T cells that bear the Ly2 differentiation marker.     

Heterologous antisera to Lyt-1+, 2- -derived antigen-binding factor detect a subfactor of an antigen-specific suppressor factor and cell surface proteins on Lyt-1+, 2- and Lyt-1-, 2+ T cells

Rabbits were immunized with TNP-specific Lyt-1+, 2- T cell-derived, antigen-binding proteins (PCI-F) released by T cells sensitized by skin painting with picrylchloride. The resulting antiserum (anti-PCI-F) bound to PCI-F and TNP-specific factors that suppressed delayed hypersensitivity (TSF) known to be comprised of PCI-F and Lyt-2+ -derived polypeptides released by cells sensitized by injection of trinitrobenzenesulfonic acid (TNBSF). Anti-PCI-F bound to T lymphocytes and 68,000 to 72,000 m.w. T cell surface proteins but not B cells on their surface proteins. Anti-PCI-F bound to both Lyt-1+ and Lyt-2+ T cells and surface proteins. A comparison of anti-PCI-F with anti-TSF indicates that anti-TSF contains specificity for Ly-2+ T cell-derived components of TSF and T cells not present in anti-PCI-F. The possibility of multiple isotypes of T cell receptors and antigen-binding molecules is discussed.     

Human CD4+ T cells are predominantly distributed among six phenotypically and functionally distinct subsets

Human T cells are heterogeneous, varying in terms of their phenotype, functional capabilities, and history of Ag encounter. The derivation of a functionally relevant model for classifying CD4+ T cells has been hampered by limitations on the numbers of parameters that may be measured using classical four-color flow cytometry. In this study we have taken advantage of the introduction of reagents for five-color flow cytometry to develop a detailed, functionally meaningful scheme for classifying human CD4+ T cells. We show that CD4+ T cells are predominantly distributed among six of eight possible compartments, identified by the expression of CCR7, CD45RA, and CD28. We demonstrate novel phenotypic and functional correlates that justify the choice of these three molecules to define CD4+ T cell compartments. We note that CD4+ T cells with different Ag specificities are distributed differently among the six described subsets. On the basis of these results, we propose a cross-sectional model for classification of peripheral CD4+ T cells. Knowledge of where T cells lie on this model informs about their functional capacity and can reflect their history of Ag exposure.     

Proliferation in vitro and interleukin production by 14 day fetal and adult Lyt-2-/L3T4- mouse thymocytes

When 14 day fetal mouse thymocytes, which are phenotypically Lyt-2-/L3T4-(2-4-), were stimulated in vitro with a combination of phorbol myristate acetate (PMA) and the calcium ionophore ionomycin, they proliferated without addition of exogenous interleukins and/or growth factors. Addition of exogenous IL 2 resulted in a slight enhancement of fetal thymocyte proliferation. By using factor-dependent indicator cell lines, this proliferation was shown to be accompanied by the production of IL 2 and IL 3. However, phenotypic analysis by using flow microfluorometry and monoclonal antibodies to Lyt-2 and L3T4 showed little differentiation among proliferating 2-4-fetal thymocytes. Interestingly, the in vitro growth of PMA + ionomycin-stimulated fetal thymocytes appeared to be IL 2 dependent in that it was inhibited by a monoclonal antibody to the IL 2 receptor. The results obtained with fetal thymocytes were compared with those obtained when using 2-4- thymocytes from adult mice.     

Thymic stroma-derived T cell growth factor (TSTGF). IV. Capacity of TSTGF to promote the growth of L3T4- Lyt-2- thymocytes by synergy with phorbol myristate acetate or various IL

The present study investigates the role of thymic stroma-derived T cell growth factor (TSTGF) in promoting the growth of L3T4- Lyt2- (double-negative) thymocytes. Partially purified TSTGF samples were prepared from the culture supernatant of a newly established thymic stromal cell line, MRL104.8a. The TSTGF alone induced only marginal proliferation of double-negative thymocytes, whereas this factor exerted a potent growth-promoting effect on these cells in combination with PMA. Because such an enhanced proliferation was not inhibited by anti-IL-4 or anti-IL-2R antibody, this was not due to the stimulation of an autocrine mechanism involving the production and utilization of IL-4 or IL-2. In scrutinizing PMA-equivalent physiologic substance(s), IL-1 was revealed to be capable of replacing the role of PMA in the above co-stimulation cultures and including enhanced proliferation of double-negative thymocytes in combination with TSTGF. Although TSTGF plus IL-2 or IL-4 also exhibited an appreciable or moderate synergistic effect on the growth of double-negative thymocytes, its magnitude was weaker compared with that obtained by TSTGF plus IL-1. More important, the strikingly enhanced proliferation was induced in the combinations of TSTGF, IL-1, and IL-2 or IL-4 under conditions in which the proliferation induced by IL-1 plus IL-4 or IL-1 plus IL-2 was marginal or slight. Furthermore, such strongly enhanced proliferation was also observed in the double-negative thymocyte population which was additionally depleted of T3+ cells (namely, the L3T4- Lyt-2- T3- or dull population). These results indicate the crucial role of TSTGF in the proliferation of immature thymocytes by synergy with various cytokines.     

IL 2 restores memory B cell activation by antigen-specific T cell clone variants

It has recently been demonstrated that there are at least two separate pathways by which a single keyhole limpet hemocyanin (KLH) reactive T cell clone can induce B cell differentiation. With the use of the high-dose antigen-driven system (10 micrograms/ml trinitrophenyl (TNP)-KLH), a KLH-specific T cell clone was able to induce a primary anti-TNP response in unprimed B cells. In the presence of aliquots of the same T cell clone, a low-dose of antigen (5 X 10(-2) micrograms/ml TNP-KLH) induced an immunoglobulin (Ig)G response in primed B cells. It has also been demonstrated that there are variant subclones of such KLH-specific helper T cell clones that are unable to provide antigen-specific help in the presence of low-dose antigen but maintain the high-dose antigen-driven helper response. This study was undertaken to investigate whether interleukin 2 (IL 2) had some activity in the low-dose, antigen-driven response induced by the T cell clone. With the use of a variant T cell clone (which lost low-dose, antigen-driven helper activity), it was demonstrated that IL 2 was capable of reconstituting the low-dose, antigen-driven helper activity. To investigate whether accessory cells were required in this system, we removed the adherent cell population from the primed spleen cells added to culture. Interestingly, removal of the G10-adherent cells eliminated the low-dose, antigen-driven response induced by IL 2. Additionally by add-back experiments, we were able to demonstrate that the necessary adherent cell population did not require major histocompatibility complex (MHC) restriction for reconstitution of the IL 2-dependent, low-dose, antigen-driven response. Furthermore, 1% concanavalin A (Con A) supernatant (Sn), but not interleukin 1 (IL 1), could replace this adherent cell function. These data suggest that in this system, IL 2 bypasses the MHC-restricted interaction between T cells and antigen-charged adherent cells; B cells can present antigen to cloned helper T cells efficiently for primary responses but need an added factor(s) to induce IgG production; and adherent cells are essential for IgG production in primed B cells, possibly through the release of soluble factor(s) included in Con A Sn.