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.     

Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

CD4+ T cell subsets. Lymphokine secretion of memory cells and of effector cells that develop from precursors in vitro

We have studied the properties of several developmentally defined subpopulations of CD4+ T cells from normal animals which can be stimulated to secrete lymphokines. We find that the Th cells responsible for direct secretion of lymphokines after stimulation are from a resting, very long lived subpopulation of CD4+ T cells which persists for over 25 wk after adult thymectomy. These T cells are depleted by in vivo administration of antithymocyte serum and they are enriched among T cells which express high levels of Pgp-1. This phenotype suggests that the T cells responsible are most likely memory T cells which have resulted from antigen exposure in vivo. T cells in this subset secrete predominantly IL-2 with small quantities of IL-3, granulocyte/macrophage CSF, and IFN-gamma. In contrast, the CD4+ T cells which require in vitro culture and restimulation before they develop into an effector population with the ability to secrete lymphokines after restimulation, differ dramatically by most of these criteria. The precursors we study are resting Th cells which are considerably shorter lived after adult thymectomy (5 to 10 wk) and resistant to the same doses of antithymocyte serum which deplete the putative memory population. We hypothesize that this precursor population represents naive helper cells which have not yet encountered Ag. The effectors derived from such precursors can be stimulated to secrete high levels of both Th cell types 1 and 2 lymphokines (IFN-gamma, IL-4, IL-5, granulocyte/macrophage CSF, and IL-3). Generation of effectors requires proliferation and differentiation events which occur during a mandatory culture with lymphokines and antigen presenting cells for 3 to 4 days. We discuss the striking phenotypic and functional differences among these subpopulations of helper cells--the precursor population and the two types--memory and cultured effector Th which secrete lymphokines. We also discuss the relationship of these populations to CD4+ T cell subsets defined by other studies of patterns of lymphokine secretion and by cell surface phenotype.     

Functional and phenotypic differences between CD4+ and CD4- T cell receptor-gamma delta clones from peripheral blood.

CD4+ TCR-gamma delta+ T cells comprise a very small subset of TCR-gamma delta+ T cells. CD4+ TCR gamma delta+ T cell clones were established to study the phenotypical and functional characteristics of these cells. Thirty-four CD4+ TCR-gamma delta+ T cell clones were established after sorting CD4+ T cells from a pre-expanded TCR-gamma delta+ T cell population. These clones as well as the CD4- TCR-gamma delta+ T cells from the same donor used V gamma 2 and V delta 2. In a second cloning experiment CD4+ TCR-gamma delta+ T cells were cloned directly from freshly isolated TCR-gamma delta+ T cells using a cloning device coupled to a FACS sorter. Forty-three clones were obtained, which all expressed CD4 and TCR-gamma delta. Eleven of these clones used V delta 1 and three of them coexpressed V gamma 2. The other CD4+ TCR-gamma delta+ T cell clones used both V delta 2 and V gamma 2. CD4+ TCR-gamma delta+ T cell clones expressed CD28 irrespective of the V gamma or V delta usage, and were CD11b negative. Three CD4-CD8+ TCR-gamma delta+ clones expressed CD8 alpha but not CD8 beta and were CD11b positive. CD28 expression among CD4-CD8+ and CD4-CD8- was variable but lower than on CD4+ T cell clones. CD4- TCR-gamma delta+ T cell clones using V gamma 2 and V delta 2 specifically lyse the Burkitt lymphoma cell line Daudi and secrete low levels of IFN-gamma and granulocyte-macrophage-CSF upon stimulation with Daudi. In contrast, most CD4+ T cell clones that use V gamma 2 and V delta 2 had a very low lytic activity against Daudi cells and secrete high levels of IFN-gamma and granulocyte-macrophage-CSF after stimulation with Daudi cells. The NK-sensitive cell line K562 was killed efficiently by the CD4- TCR-gamma delta+ T cell clones, but not by CD4+ TCR-gamma delta+ T cell clones, and could not induce cytokine secretion in CD4+ or CD4- T cell clones. CD4+ TCR-gamma delta+ T cell clones, but not the CD4- clones, could provide bystander cognate T cell help for production of IgG, IgM, and IgA in the presence of IL-2 and IgE in the presence of IL-4. Thus, CD4+ TCR-gamma delta+ T cells are similar to CD4+ TCR-alpha beta+ T cells in their abilities to secrete high levels of cytokines and to provide T cell help in antibody production.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of an IL-1-dependent IL-4-producing bovine CD4+ T cell clone.

An Ag-specific interleukin 1 (IL-1)-dependent bovine CD4+ Th cell clone, termed 300B1, was isolated and found to resemble the previously described IL-1-dependent murine CD4+ Th2 cell clone, D10.G4.1. Both the 300B1 and the D10.G4.1 T cell clones proliferated to bovine (Bo) IL-1 beta, human (Hu) IL-1 alpha and IL-1 beta, and murine IL-1 alpha when cells were costimulated with concanavalin A (Con A). Proliferation of the 300B1 clone, when costimulated with Con A, appeared to be IL-1-specific in that proliferation could not be promoted by BoIL-2, HuIL-3, HuIL-4, HuIL-5, or HuIL-6. The 300B1 clone produced interferon-gamma (IFN-gamma), but not IL-2 following stimulation with either Con A, Con A plus phorbol 12-myristate 13-acetate or Ag plus antigen-presenting cells. Upon stimulation with Con A, the 300B1 clone expressed IL-4 mRNA and produced an autocrine growth factor (AGF) that could be inhibited by anti-HuIL-4 but not by anti-HuIL-2 Ab. The clonal derivation of the 300B1 clone was confirmed by isolating five 300B1 subclones, all of which produced IFN-gamma and an AGF but not IL-2. Collectively, these results suggest the IL-1-dependent bovine 300B1 Th cell clone produces IL-4, but not IL-2, as an AGF. Furthermore, the bovine Th cell clone appeared to share many characteristics of previously described murine Th2 cell clones except that the bovine clone produced IFN-gamma.     

Prostaglandin E2 selectively inhibits human CD4+ T cells secreting low amounts of both IL-2 and IL-4

PGE2 is a potent inflammatory mediator with profound immune regulatory actions. The present study examined the effects of PGE2 on the activation/proliferation of CD4+ T cells using 37 cloned CD4+ T cell lines. Ten T cell clones sensitive to PGE2 and 10 T cell clones resistant to PGE2, as measured by proliferation in response to anti-CD3 Ab, were selected for comparison. It was found that the PGE2-sensitive T cells were characterized by low production (<200 pg/ml) of both IL-2 and IL-4, while PGE2-resistant T cells secreted high levels (>1000 pg/ml) of IL-2, IL-4, or both. The roles of IL-2 and IL-4 were confirmed by the finding that addition of exogenous lymphokines could restore PGE2-inhibited proliferation, and PGE2-resistant Th1-, Th2-, and Th0-like clones became PGE2 sensitive when IL-2, IL-4, or both were removed using Abs specific for the respective lymphokines. In addition, we showed that the CD45RA expression in PGE2-sensitive T cells was significantly lower than that in PGE2-resistant cells (mean intensity, 1.2 +/- 0.6 vs 7.8 +/- 5.7; p = 0.001). In contrast, CD45RO expression in PGE2-sensitive T cells was significantly higher that that in PGE2-resistant cells (mean intensity, 55.7 +/- 15.1 vs 33.4 +/- 12.9; p = 0.02). In summary, PGE2 predominantly suppressed CD45RA-RO+ CD4+ T cells with low secretion of both IL-2 and IL-4.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

A common precursor for CD4+ T cells producing IL-2 or IL-4.

To investigate whether CD4+ T cells are predetermined to produce a given pattern of lymphokines, we have used a culture system that allows the controlled induction of either IL-2- or IL-4-producing CD4+ T cells. Single, freshly isolated murine CD4+ T cells were activated with Con A, rIL-2, and APC; the developing clones were split and then cultured for an additional 14 days with either rIL-2 alone or with rIL-2 and anti-CD3 stimulation. Subclones expanded in the presence of rIL-2 alone produced predominantly IL-2, although subclones derived from the same precursor and expanded in the presence of rIL-2 and a mitogenic antibody to CD3 released predominantly IL-4. Subclones expanded for 2 wk in the presence of rIL-2 plus a mitogenic mAb to CD3 released up to 60 times more IL-4 but only 1/90 the amount of IL-2 released by subclones derived from the same precursor cell and expanded with rIL-2. Both phenotypes can be derived from IL-2-producing precursor cells. These results demonstrate that IL-2-producing clones can be derived from the same cells as IL-4-producing clones and are most consistent with the view that the IL-2-producing Th1 or the IL-4-producing Th2 phenotype of a T cell clone is acquired during T cell differentiation and is not secondary to the expansion of distinct subpopulations that are predetermined to produce a specific cytokine pattern.     

Loss of IL-4 secretion from human type 1a diabetic pancreatic draining lymph node NKT cells

Altered frequency and function of peripheral invariant NKT (iNKT) cells have been implicated in the regulation of murine and human type 1a diabetes. To examine regulatory cells from the site of drainage of autoinflammatory tissue and autoantigenic T cell priming in diabetes, we directly cloned iNKT cells from human pancreatic draining lymph nodes (PLN). From 451 T cell clones from control and diabetic PLN, we derived 55 iNKT cells by two methods and analyzed function by cytokine secretion. iNKT cell clones isolated from control PLN secreted IL-4 and IFN-gamma upon TCR stimulation. For type 1a diabetic subjects, PLN iNKT cell clones from three samples secreted IFN-gamma and no IL-4. In a rare recent onset diabetic sample with islet-infiltrating CD4+ T cells, the phenotype of PLN iNKT cell clones was mixed. From normal and diabetic PLN, one-third of CD1d tetramer+-sorted T cell clones were reactive with CD1d transfectants or proliferated/secreted cytokine in response to alpha-galactosylceramide-pulsed PBMCs; tetramer-staining T cell clones from diabetic PLN did not secrete IL-4. This is the first report directly examining iNKT cells from lymph nodes draining the site of autoimmunological attack in humans; iNKT cells were altered in cytokine secretion as previously reported for circulating iNKT cells in human type 1a diabetes.     

Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation

IL-4 is known to promote the differentiation of CD4+ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor alpha-chain (IL-4Ralpha-/-) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a requirement for prior IL-4-mediated stimulation. To address whether naive, conventional CD4+ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4Ralpha-/-mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4Ralpha-/- DO11.10/scid mice secreted approximately 50 pg of IL-4 per10(6) cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4+ T cells into Th2 cells, particularly if IFN-gamma and IL-12 were neutralized during the priming cultures. CD4+ cells from the IL-4Ralpha-/- mice gave rise to a minor proportion (approximately 2%) of IL-4-producing cells upon stimulation in the presence of anti-IFN-gamma and anti-IL-12. These data show that conventional, naive CD4+ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-gamma and IL-12, this IL-4 can induce Th2 polarization.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

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)     

Changes in the precursor frequencies of IL-4 and IFN-gamma secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis.

Limiting dilution analysis was used to estimate the frequency of clonogenic Ag-specific CD4+ T lymphocytes in draining lymph nodes of mice over the course of infection with Leishmania major, and to measure the production of IL-2, IL-3, IL-4, IFN-gamma, and TNF by the resultant clones. Infection of both genetically susceptible BALB/c ("non-healer") and resistant C57BL/6 ("healer") mice resulted in at least a fourfold increase in the frequency (to about 0.3%) and at least a 10-fold increase in the total number of lymph node CD4+ cells that formed clones when cultured with L. major Ag in vitro. At 1 wk after infection, the majority of clones from BALB/c mice secreted IL-4 (precursor frequency 0.15%) and fewer secreted IFN-gamma (0.05%); this pattern remained constant for at least 8 wk after infection. In C57BL/6 mice, however, a high precursor frequency of IL-4-secreting clones was measured in the first 1 to 2 wk when the mice had lesions, but resolution of infection was associated with a decrease in the frequency of IL-4-secreting clones (from 0.13% at 2 wk to 0.03% at 4 wk) and an increase in the frequency of IFN-gamma-secreting clones (from 0.08% to 0.22%). At all stages of infection, most clones from either mouse strain secreted IL-3 and very few secreted TNF. Analysis of PCR-amplified cDNA from draining lymph nodes of infected mice also revealed that IL-4 and IFN-gamma mRNA were expressed in both mouse strains early in infection. IL-4 mRNA was the major species at 2 and 6 wk after infection in BALB/c mice, but declined relative to IFN-gamma mRNA over this time in C57BL/6 lymph nodes. Precursor frequency estimates of lymphokine-secreting CD4+ cells in draining lymph nodes therefore correlated with lymphokine expression patterns in vivo. Analysis of a panel of individual short term clones derived from mice 1 wk after infection revealed marked heterogeneity in lymphokine production patterns. In BALB/c mice, 49% secreted IL-4 without IFN-gamma, 18% secreted IFN-gamma without IL-4, and 14% secreted both IL-4 and IFN-gamma. Similarly in C57BL/6 mice, 39% secreted IL-4, 20% secreted IFN-gamma, and 17% secreted both lymphokines. Many of the clones also produced IL-3 and/or IL-2. Together the data suggest that both IL-4 and IFN-gamma are synthesized early in infection of susceptible and resistant mice as assessed by mRNA and precursor frequency analyses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential interleukin secretion by in vitro activated human CD45RA and CD45RO CD4+ T cell subsets.

The CD45RA and CD45RO isoforms have been reported to define complementary subsets among CD4+ T cells: CD45RA CD4+ T cells are considered "virgin T cells" and CD45RO "primed T cells." We investigated the secretion of lymphokines by human CD4+ CD45RO and CD4+ CD45RA T helper cells after mitogen stimulation. CD45RA and CD45RO CD4+ T cells were isolated by negative immunoselection using magnetic beads. CD45RO cells, but not CD45RA cells, proliferate well in response to pokeweed mitogen (PWM) or insoluble anti-CD3. Both subpopulations produced interleukin (IL)-2, IL-6, and interferon (IFN)-gamma when stimulated with PWM for 1-4 days. Only Day 1 supernatants from CD45RO cells contained moderate amounts of IL-4. After 14 days of continuous culture and stimulation with PWM, the CD45RA subset had lost the expression of CD45RA and gained that of CD45RO. When long-term cultured CD45RA or CD45RO cells were treated with insoluble anti-CD3, they incorporated [3H]thymidine at similar levels, but only CD45RO cells secreted IL-4 and significantly increased their secretion of IFN-gamma. These data indicate that despite phenotype conversion, the two subpopulations maintain functional differences in the secretion of lymphokines, thus suggesting that circulating CD45RA and CD45RO cells may represent different lines of differentiation.     

Protein vaccines induce uncommitted IL-2-secreting human and mouse CD4 T cells, whereas infections induce more IFN-gamma-secreting cells

Mouse and human CD4 T cells primed during an immune response may differentiate into effector phenotypes such as Th1 (secreting IFN-gamma) or Th2 (secreting IL-4) that mediate effective immunity against different classes of pathogen. However, primed CD4 T cells can also remain uncommitted, secreting IL-2 and chemokines, but not IFN-gamma or IL-4. We now show that human CD4 T cells primed by protein vaccines mostly secreted IL-2, but not IFN-gamma, whereas in the same individuals most CD4 T cells initially primed by infection with live pathogens secreted IFN-gamma. We further demonstrate that many tetanus-specific IL-2+IFN-gamma- cells are uncommitted and that a single IL-2+IFN-gamma- cell can differentiate into Th1 or Th2 phenotypes following in vitro stimulation under appropriate polarizing conditions. In contrast, influenza-specific IL-2+IFN-gamma- CD4 cells maintained a Th1-like phenotype even under Th2-polarizing conditions. Similarly, adoptively transferred OTII transgenic mouse T cells secreted mainly IL-2 after priming with OVA in alum, but were biased toward IFN-gamma secretion when primed with the same OVA peptide presented as a pathogen Ag during live infection. Thus, protein subunit vaccines may prime a unique subset of differentiated, but uncommitted CD4 T cells that lack some of the functional properties of committed effectors induced by infection. This has implications for the design of more effective vaccines against pathogens requiring strong CD4 effector T cell responses.     

Expression of CD161 (NKR-P1A) defines subsets of human CD4 and CD8 T cells with different functional activities

A subset of T cells in human peripheral blood expresses CD161 (NKR-P1A) receptors that are primarily associated with NK cells. In the current study we isolated blood T cell subsets according to the expression of CD161 and examined their contents of naive, central memory, and effector memory cells and their capacities for proliferation, cytokine secretion, and natural cytolysis. We found that CD4+CD161- and CD8+CD161- subsets contained predominantly naive T cells that secreted high levels of IL-2 after in vitro stimulation, and CD4+CD161int and CD8+CD161int subsets contained predominantly effector and central memory T cells that secreted high levels of IFN-gamma and TNF-alpha. All of these subsets showed vigorous proliferation after stimulation in vitro, but none had NK lytic activity. Unexpectedly, the CD8+CD161+ cells contained an anergic CD8alpha+CD8betalow/-CD161high T cell subset that failed to proliferate, secrete cytokines, or mediate NK lytic activity.     

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.     

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.