IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.     

Cell surface comodulation of CD4 and T cell receptor by anti-CD4 monoclonal antibody

In our study we have used anti-CD4 mAb to investigate the cell surface association between CD4 and the Ag-specific TCR complex on mature peripheral T cells. Anti-CD4 mAb was administered in vivo and in vitro and its effects on CD4 and CD3 cell surface expression were determined. In vivo, anti-CD4 mAb reduced cell surface expression of its ligand, CD4, and secondarily also reduced cell surface expression of CD3/TCR on CD4+ splenic T cells. In vitro, multivalent cross-linking of CD4 by anti-CD4 mAb and either FcR+ cells or anti-Ig mAb also resulted in decreased surface expression of CD4 and specific comodulation of CD3/TCR. The secondary reduction in cell surface CD3/TCR expression induced by CD4 cross-linking could be pharmacologically disrupted by high doses of PMA, indicating that the comodulation of CD3 with CD4 was dependent upon intracellular mediators, possibly including protein kinase C. These results demonstrate that, in the presence of anti-CD4 mAb, CD4 is functionally associated with the CD3/TCR complex, and that this association is dependent upon the activity of intracellular mediators. Such intracellular mediators might induce the coordinate down-modulation of physically unassociated CD4 and CD3/TCR molecules, or, alternatively, might promote a physical interaction between CD4 and CD3/TCR molecules.     

Suppression of antibody synthesis by CD4+ T cell clones and normal T cells stimulated with monoclonal anti-CD3 antibody

CD4+ve Th1 clones, as well as normal splenic T cells, were found to suppress LPS-driven antibody secretion in a non-Ag-specific and non-MHC-restricted manner when the T cells were activated with the anti-CD3 mAb, 145-2C11. Suppression was observed with both primed and naive B cells, as well as with purified hapten-specific B cells, a result that suggests a direct effect of anti-CD3-activated T cells on B cell differentiation. Th1 clones activated by cognate Ag also suppressed LPS-driven antibody secretion. Furthermore, suppression of LPS-driven antibody secretion could be achieved across a cell-impermeable porous membrane when T cells were activated with anti-CD3. Suppression by Th1 clones and by normal T cells could not be attributed to a concomitant decrease in B cell proliferation or to a shift in the kinetics or isotype of the antibody response. These data demonstrate that CD4+ve Th1 clones, as well as normal T cells, can effect suppression of polyclonal antibody formation.     

Epigenetic remodeling of the IL-2 and IFN-gamma loci in memory CD8 T cells is influenced by CD4 T cells

Memory T cells (T(M)) are able to rapidly exert effector functions, including immediate effector cytokine production upon re-encounter with Ag, which is critical for protective immunity. Furthermore, this poised state is maintained as T(M) undergo homeostatic proliferation over time. We examined the molecular basis underlying this enhanced functional capacity in CD8 T(M) by comparing them to defective CD8 T(M) generated in the absence of CD4 T cells. Unhelped CD8 T(M) are defective in many functions, including the immediate expression of cytokines, such as IL-2 and IFN-gamma. Our data show that this defect in IL-2 and IFN-gamma production is independent of clonal selection, functional avidity maturation, and the integrity of proximal TCR signaling, but rather involves epigenetic modification of these cytokine genes. Activated Ag-specific CD8 T cells exhibit rapid DNA demethylation at the IL-2 and IFN-gamma loci and substantial histone acetylation at the IFN-gamma promoter and enhancer regions. These epigenetic modifications occur early after infection at the effector stage and are maintained through memory development. However, activated unhelped CD8 T cells, which fail to develop into functional memory and are incapable of rapid cytokine production, exhibit increased DNA methylation at the IL-2 promoter and fail to acetylate histones at the IFN-gamma locus. Thus, CD4 T cell help influences epigenetic modification during CD8 T(M) differentiation and these epigenetic changes provide a molecular basis for the enhanced responsiveness and the maintenance of a "ready-to-respond" state in CD8 T(M).     

Cytokine secretion by C3H-lpr and -gld T cells. Hypersecretion of IFN-gamma and tumor necrosis factor-alpha by stimulated CD4+ T cells.

Mice homozygous for lpr and gld develop profound lymphadenopathy characterized by the expansion of two unusual T cell subsets, a predominant Ly-5(B220)+ CD4- CD8- double negative (DN) population and a minor CD4 dull+ Ly-5(B220)+ population. The mechanisms promoting lymphoproliferation are unknown, but one possibility is a abnormality in the production of cytokines that regulate T cell growth. In the present report, unfractionated LN cells and sorted T cell subsets from C3H-lpr, -gld, and -+/+ mice were compared for spontaneous and induced secretion of a spectrum of lymphokines. In addition, CD4+, CD4 dull+ Ly-5(B220)+, and DN T cells were examined for expression of CD3 epsilon, TCR-alpha/beta heterodimers, Ly-6C, and CD44 and for proliferative responses to immobilized anti-TCR mAb and cofactors. These studies revealed that sorted DN T cells did not secrete IL-3, IL-4, IL-5, IL-6, GM-CSF, TNF-alpha, or IFN-gamma spontaneously or after TCR-alpha/beta cross-linking. In contrast, stimulated unfractionated lpr and gld LN cells proliferated strongly and secreted high levels of IFN-gamma and TNF-alpha and low levels of IL-3, IL-4, and IL-6. Despite a 5- to 10-fold deficit in the frequency of CD4+ and CD8+ T cells, cytokine secretion by lpr and gld LN generally exceeded that of +/+ LN. Comparisons of cytokine secretion by stimulated CD4+ T cells revealed that +/+, lpr, and gld CD4+ Ly-5(B220)- T cells proliferated strongly, but only lpr and gld cells produced significant levels of IFN-gamma. The lpr and gld CD4+ T cells also produced higher levels of TNF-alpha and IL-2 than +/+ cells. In contrast to normal CD4+ T cells, lpr and gld CD4+ Ly-5(B220)+ T cells proliferated weakly and did not secrete TNF-alpha, IL-2, or, in most experiments, IFN-gamma after stimulation. Phenotypic studies of T cell subsets revealed that unstimulated lpr and gld CD4+ Ly-5(B220)- T cells express significantly higher levels of CD44 than +/+ CD4+ T cells. In addition, CD4 dull+ Ly-5(B220)+ cells closely resembled DN T cells in size and expression of TCR-alpha/beta, CD3epsilon, CD44, and Ly-6C. Since elevated CD44 expression is generally associated with T cell activation and only previously activated normal CD4+ T cells produce high levels of IFN-gamma in vitro, our data suggest that lpr and gld CD4+ Ly-5(B220)- T cells contain a higher than normal proportion of primed or memory T cells and thus may be polyclonally activated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

IL-12 and type-I IFN synergize for IFN-gamma production by CD4 T cells, whereas neither are required for IFN-gamma production by CD8 T cells after Listeria monocytogenes infection

Differentiation of Ag-specific T cells into IFN-gamma producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-gamma production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-gamma in vitro. However, their role in priming Ag-specific T cells for IFN-gamma production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-gamma production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-gamma production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial ( approximately 50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75-95%) reductions in IFN-gamma production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-gamma production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR(+/+) and IFN-IR(-/-) CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-gamma production by CD4 and CD8 T cells in response to bacterial infection.     

Comparison of lymphokine secretion and responsiveness of human T cell clones isolated in IL-4 and in IL-2.

Interleukin (IL)-4 has been shown to be secreted simultaneously with IL-2 and interferon (IFN)-gamma by the majority of CD4+ human T cell clones isolated and cultured using IL-2 as a growth factor. Moreover, IL-4 was found to be as efficient as IL-2 to promote the outgrowth of human T cell clones. In this study we have investigated the pattern of lymphokine production by human T cell clones isolated and cultured in IL-4. Most of the CD4+ T cell clones isolated in IL-4 were found to have the ability to simultaneously secrete IL-2, IL-4, and IFN-gamma upon activation. The T cell clones isolated in IL-4 produced, in general, more IL-4 and less IL-2 than the clones isolated and cultured in IL-2. This tendency did not appear to be a stable feature inasmuch as when representative CD4+ T cell clones were split and cultured in either IL-2 or IL-4, the clones in IL-2 secreted more IL-2 and less IL-4 than the same cells cultured in IL-4. These results indicate that the isolation and culture of human CD4+ T cells in IL-4 did not lead to an "irreversible" development of these cells into Th-1- or Th-2-like cells. Clones isolated in IL-4 responded better to IL-4 than they did to IL-2. On the other hand, T cell clones from the same donor isolated in IL-2 showed the reverse pattern since these latter cells were found to respond better to IL-2 than to IL-4. Furthermore, "nonresponsiveness" of a T cell clones in a [3H]TdR assay to either IL-2 or IL-4 is not a stable feature since clones, unresponsive to a particular lymphokine, could be adapted to become responsive.     

Generation propagation, and targeting of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus recombinant IL-2. An efficient strategy for adoptive tumor immunotherapy.

We developed a culture system for the rapid generation of CD4+ T cells that have both helper and killer functions. CD4+ T cells isolated from human PBL did not proliferate or develop significant cytotoxicity when treated with rIL-2 because of the lack of p75 IL-2R expression. However, culture of isolated CD4+ T cells with immobilized anti-CD3 mAb plus rIL-2 resulted in a marked proliferation (500-fold increase in 14 days) of CD4+ T cells. The proliferating CD4+ T cells produced IL-2 (92 U/ml) and showed strong cytotoxicity against OKT3 hybridoma cells and Daudi, K562, and U937 tumor cells in an anti-CD3 mAb-dependent manner. The CD4+ T cells contained significant amounts of cytolytic granule-related proteins such as serine esterase and perforin. Activated CD4+ helper/killer cells can be generated from both healthy donors and tumor patients and can be propagated in vitro for 14 to 35 days by biweekly restimulation with immobilized anti-CD3 mAb plus rIL-2. This culture yielded about 20,000-fold increase in cell number after a 21-day culture. Bispecific antibody containing anti-CD3 and anti-glioma Fab components enhanced the cytotoxicity of activated CD4+ helper/killer cells against IMR32 glioma cells. Moreover, the activated CD4+ helper/killer cells showed both helper and antitumor activity in vivo and prevented growth of anti-CD3 hybridoma cells in nude mice whether or not IL-2 was administered. These results indicate that anti-CD3 mAb plus IL-2-activated CD4+ helper/killer cells may provide an effective strategy for adoptive tumor immunotherapy of cancer.     

CD30L up-regulates CD30 and IL-4 expression by T cells.

CD30L is frequently expressed on acute myeloid leukemia (AML) blasts. Its presence is associated with the co-expression of interleukin-4 (IL-4) receptor and with the expansion of specific T-helper 2 (Th2) cell subsets producing IL-4 and expressing CD30. Recombinant CD30L-bearing cells up-regulated the expression of surface CD30 and increased the production of IL-4 and soluble (s) CD30 by co-cultured T cells. These findings were confirmed with AML blasts expressing surface CD30L, where blocking anti-CD30 antibodies completely abolished the release of sCD30 and reduced the production of IL-4. Our data indicates a direct role of CD30L(+) neoplastic cells in driving the immune response toward a Th2-polarized non-protective state.     

Superantigen-induced CD4 T cell tolerance mediated by myeloid cells and IFN-gamma

We have previously shown that systemic staphylococcal enterotoxin A (SEA) injections cause CD4 T cells in TCR-transgenic mice to become tolerant to subsequent ex vivo restimulation. An active IFN-gamma-dependent mechanism of suppression was responsible for the apparent unresponsiveness of the CD4 T cells. In this study, we analyze the response of CD4 T cells isolated throughout the first 10 days of the in vivo response to injected SEA. We show that CD4 T cells isolated at the peak of the in vivo response undergo very little activation-induced cell death after sterile FACS sorting or restimulation in the presence of neutralizing Abs to IFN-gamma. We also show that the IFN-gamma-dependent tolerance develops soon after SEA injection in the spleens of both normal and TCR-transgenic mice. This suppression is dependent upon myeloid cells from the SEA-treated mice and is optimal when inducible NO synthase activity and reactive oxygen intermediates are both present. The data indicate that IFN-gamma, myeloid cells, and a combination of NO and reactive oxygen intermediates all contribute to a common pathway of T cell death that targets activated or responding CD4 T cells. Sorted Gr-1(+) cells from SEA-treated mice also directly suppress the response of naive CD4 T cells in mixed cultures, indicating that this tolerance mechanism may play a role in down-regulating other vigorous immune responses.     

Characterization of tumor-infiltrating CD4+ T cells as Th1 cells based on lymphokine secretion and functional properties

Recent studies have subdivided the Th cells into mutually exclusive Th1 subset producing IL-2 and IFN-gamma and Th2 cells producing IL-4 and IL-5. The relative role played by these two subsets in the antitumor immunity is not clear. We earlier demonstrated that treatment of C57BL/6 mice bearing a syngeneic Ia- T cell lymphoma, LSA, with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) resulted in 90 to 100% survival of the mice. Furthermore, host's T cell responses were critical for successful BCNU-mediated cures. In our study we observed that immediately after BCNU treatment, there was a dramatic increase in the percentage of CD4+ T cells at the site of tumor growth in the peritoneal cavity. The percentage of CD4+ T cells increased from approximately 3 to 4% found in normal or tumor-bearing mice to approximately 41% in BCNU-treated tumor-bearing mice. The percentage of CD8+ T cells also increased although to a lesser degree. Also, these alterations were primarily restricted to the site of tumor-growth inasmuch as they were not seen in the thymus and were less pronounced in the spleen. The tumor-infiltrating CD4+ T cells obtained after BCNU-treatment, when further characterized, were found to secrete only IL-2 and IFN-gamma but not IL-4, after tumor-specific stimulation. Furthermore, the supernatants from LSA-activated CD4+ T cell cultures failed to provide help to the B cells but were able to activate the macrophages to inhibit the tumor cell proliferation. The CD4+ T cells when adoptively transferred could also protect the nude mice from LSA tumor challenge and induced tumor-specific delayed-type hypersensitivity reaction. Together our data suggest that in the LSA tumor model, the tumor-infiltrating CD4+ T cells have the properties of Th1 cells and these cells can mediate tumor-rejection independent of the CD8+ T cells by activating the macrophages.     

Effects of in vivo administration of anti-CD3 monoclonal antibody on T cell function in mice. II. In vivo activation of T cells

Anti-CD3 mAb are known to be both immunosuppressive and mitogenic to T cells in vitro. However, only immunosuppression has been observed after in vivo administration of these mAb. The present study demonstrates that T cell activation does occur after in vivo administration of anti-CD3 mAb to mice, evidenced by increased IL-2R expression on T cells, CSF secretion, and extra-medullary hematopoiesis in the spleen. These effects required multivalent cross-linking of the mAb, since F(ab')2 fragments failed to induce them. However, the F(ab')2 fragments did induce modulation of CD3/TCR from the surface of T cells, demonstrating that TCR modulation is not sufficient to induce activation. In addition, interaction of the TCR with either intact or F(ab')2 fragments of the mAb led to increased expression of CD8 in vivo, suggesting that the F(ab')2 fragments of anti-CD3 mAb might be capable of inducing a T cell to undergo some, but not all, of the changes involved in reaching a fully activated state. Further study of the activating effects of anti-CD3 mAb might increase the understanding of the mechanisms of in vivo T cell activation and might also be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myelodysplastic disorders.     

IFN-gamma production is specifically regulated by IL-10 in mice made tolerant with anti-CD40 ligand antibody and intact active bone

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.     

Simultaneous production of IL-2, IL-4, and IFN-gamma by activated human CD4+ and CD8+ T cell clones

In the present study, we have investigated the ability of human T cells to secrete IL-2, IL-4, and IFN-gamma. IL-4 and IFN-gamma were quantified with enzymatic immunoassays and IL-2 with a biologic assay by using the murine IL-2-dependent cell line CTLL-2. PBL, stimulated with Con A or with a combination of the phorbol ester 13-O-tetradecanoylphorbol-12-acetate and the Ca2+ ionophore A23187 secreted IL-2, IL-4, and IFN-gamma. The kinetics of the secretion of the three lymphokines was investigated with two CD4+ clones; one (GEO-2) that produced IL-2, IL-4, and IFN-gamma and another (HY640), that produced only IL-2 and IFN-gamma. Significant IL-2, IL-4, and IFN-gamma production was observed after only 8 h of activation. Maximal levels of IL-2 and IL-4 were found 20 h after the onset of the stimulation which subsequently decreased. In contrast, IFN-gamma levels continued to increase in a period up to 40 h and then leveled off. In spite of these differences in secretion, the kinetics of accumulation of mRNA did not differ. The IL-2, IL-4, and IFN-gamma mRNA were detectable 2 h after stimulation and continued to accumulate for a period up to 20 h. In a series of 22 CD4+ clones, 21 were able to secrete all three lymphokines upon stimulation. Almost all CD8+ clones were able to produce IL-2 and IFN-gamma, but only six of the 23 CD8+ T cell clones secreted IL-4. In addition, five CD4+ (allo)antigen-specific T cell clones were tested for IL-2, IL-4, and IFN-gamma secretion upon specific stimulation. Two alloantigen-specific and two tetanus toxoid-specific T cell clones secreted IL-2, IL-4, and IFN-gamma simultaneously, whereas one alloantigen-specific T cell clone secreted IL-2 and IFN-gamma, but not IL-4. A supernatant of the CD4+ T cell clone GEO-2, that contained high levels of IFN-gamma and IL-4, was unable to induce the low affinity receptor for IgE, CD23, on a Burkitt lymphoma cell line. However, after separation of IL-4 from IFN-gamma by using HPLC, the IL-4-containing fraction-induced CD23, which could be blocked by the fraction that contained IFN-gamma and by a polyclonal rabbit anti-IL-4 antiserum. Finally, the partly purified IL-4, that was devoid of IL-2, promoted the growth of the clone GEO-2.     

Distinct IL-2 receptor signaling pattern in CD4+CD25+ regulatory T cells

Despite expression of the high-affinity IL-2R, CD4(+)CD25(+) regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4(+)CD25(+) T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4(+)CD25(+) Tregs to IL-2. IL-2R stimulation results in a G(1) cell cycle arrest, cellular enlargement and increased cellular survival of CD4(+)CD25(+) T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4(+)CD25(+) T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.     

Generation of mature dendritic cells from a CD14+ cell line (XS52) by IL-4, TNF-alpha, IL-1 beta, and agonistic anti-CD40 monoclonal antibody.

We established a model system to generate mature dendritic cells (DC) from a GM-CSF-dependent cell line, XS52, which had been isolated from the epidermis of newborn BALB/c mice. Screening of various soluble factors revealed that IL-4 induces phenotypic maturation of XS52 (as evaluated by enhanced expression of class II, CD40, CD80, CD86, CD11c, and loss of expression of CD14) in a time-dependent manner. The addition of TNF-alpha, IL-1 beta, and agonistic anti-CD40 mAb further enhanced expression of these maturation markers. Consistent with their phenotypic maturation, these cells (termed XS-DC) exhibited potent Ag-presenting capacity to both naive and primed T cells. In addition, injection of hapten-conjugated XS-DC induced contact hypersensitivity in vivo, suggesting their potential as tools for vaccination. Expression of CD14 by the starting cell population, the requirement for GM-CSF and IL-4, and the relatively long culture period are the common characteristics shared between our cells and human monocyte-derived DC, whose analogues in mice have not been identified. Because large numbers of skin-associated mature DC devoid of other cell lineages are easily obtained, this model system may facilitate the study of molecular events associated with maturation of DC and the use of DC for immunization.     

Lectin-mediated induction of IL-4-producing CD4+ T cells.

Cultured murine CD4+ T cells have been shown to differentiate into IL-2 or IL-4-producing subsets. The factors responsible for the development of CD4+ T cells which produce IL-2 but not IL-4 and cells capable of producing IL-4 but not IL-2 are unknown. Here we describe a system that allows the controlled induction of IL-2- or IL-4-producing T cells after one single round of activation. Freshly isolated CD8-depleted T cells were activated with various polyclonal T cell activators for 48 h, washed, and then expanded under different conditions. IL-2 and IL-4 production were induced by restimulation of T cells and were measured with CTLL cells that respond to both cytokines and mAb to IL-2 and IL-4. T cells produced mainly IL-2 and small amounts of IL-4 when restimulated after expansion culture for 12 days with rIL-2 alone. However, after expansion for 12 days in the presence of rIL-2 plus Con A, we observed a 30- to 100-fold up-regulation of IL-4 activity and a 100-fold down-regulation of IL-2 when assessed by responses of CTLL cells incubated with the supernatant of restimulated T cells and by responses of CTLL cells cocultured with restimulated cells. An increase of IL-4 and decrease of IL-2 was also observed when the results were based on the cell numbers at the beginning of the expansion culture. The induction of IL-4 and the down-regulation of IL-2 1) were not reproduced with alpha-methyl-mannoside-treated supernatant of Con A-stimulated spleen cells, 2) were not dependent on the presence of large numbers of APC, 3) did not result from differential consumption of lymphokines after restimulation, 4) were not due to a difference in the time course of IL-2 or IL-4 release in either T cell population, and 5) were obtained regardless of the agents used to activate or to restimulate the T cells. Because Con A remained detectable on the T cell surface and because expansion of activated T cells with IL-2 plus Con A for several days was necessary, our results indicate that mainly IL-4-producing CD4+ T cells can be induced by prolonged engagement of T cell surface molecules.     

Roles of CD4+ and CD8+ T cells in murine contact sensitivity revealed by in vivo monoclonal antibody depletion

mAb specific for murine CD4+ and CD8+ T cell subsets were utilized to determine the populations participating in delayed-in-time, cutaneous hypersensitivity responses in BALB/c mice. In vivo depletions of these T cell phenotypes revealed that delayed-type hypersensitivity to cellular and protein Ag were mediated by CD4+ effector cells, whereas CD8+ cells down-regulated such responses. Similar depletions in mice prior to sensitization with the hapten 1-fluoro-2,4-dinitrobenzene demonstrated a more complex pattern of cell participation in contact sensitivity (CS) responses. Depletion of CD4+ cells resulted in strikingly enhanced ear swelling, indicating not only an important effector role for CD8+ cells but also a down-regulatory role for some CD4+ cells; depletion of CD8+ cells revealed that some CD4+ cells also act as CS effectors. In vitro depletion of immune lymph node cells with the same mAb before adoptive transfer confirmed CS effector roles for both subsets, and also suggested that at least some CD4+ suppressors act on the efferent limb of the CS response, perhaps by down-regulating the activity of CD8+ effector cells. Partial in vivo depletion with small amounts anti-CD4 mAb and subsequent flow cytometric analysis of residual CD4+ cells was consistent with the hypothesis that CD4+ CS effector cells express a higher density of the CD4 antigen than do CD4+ suppressor cells, raising the possibility that these two functionally distinct CD4+ populations might be separable on the basis of their surface expression of CD4.