Superantigen presentation by airway smooth muscle to CD4+ T lymphocytes elicits reciprocal proasthmatic changes in airway function

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders, including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules, we investigated whether ASM can present SAg to resting CD4(+) T cells, and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg, staphylococcal enterotoxin A (SEA), elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface, indicative of immunological synapse formation, in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine, IL-13, into the coculture medium, which was MHC class II dependent. Moreover, when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues, the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively, these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells, and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that, in turn, evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus, a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and, accordingly, may underlie the reported association between microbial SAg exposure, T cell activation, and severe asthma.     

CD4+ T cells mediate murine syngeneic graft-versus-host disease-associated colitis

Syngeneic graft-vs-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a 21-day course of the immunosuppressive agent cyclosporin A (CsA). Following cessation of CsA, this inducible disease is characterized by weight loss, diarrhea, and development of inflammation in the colon and liver. Although nonspecific effector cells and Th1 cytokines have been shown to participate in disease induction, the role of T cells has not been fully elucidated. Initial studies demonstrated significant increases in CD4+ T cells, but not other T cell populations in the colons of diseased animals relative to transplant control animals. To demonstrate a functional linkage between increases in colonic CD4+ T cells and disease induction, in vivo T cell depletion studies were performed. Beginning on the day of bone marrow transplantation, groups of control and CsA-treated animals were treated with mAb against either CD4 or CD8 for 21 days. Treatment with anti-CD4, but not anti-CD8, eliminated clinical symptoms and colon pathology. Interestingly, neither anti-CD4 nor anti-CD8 therapy affected the development of liver pathology associated with SGVHD. These findings demonstrated that CD4+ T cells initiate development of the intestinal inflammation associated with murine SGVHD.     

Activation of K+ channels induces apoptosis in vascular smooth muscle cells

Intracellular K⁺ playsan important role in controlling the cytoplasmic ion homeostasis formaintaining cell volume and inhibiting apoptotic enzymes in thecytosol and nucleus. Cytoplasmic K⁺ concentration is mainlyregulated by K⁺ uptake viaNa⁺-K⁺-ATPase and K⁺ efflux throughK⁺ channels in the plasma membrane. Carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), a protonophorethat dissipates the H⁺ gradient across the inner membraneof mitochondria, induces apoptosis in many cell types. In ratand human pulmonary artery smooth muscle cells (PASMC), FCCP opened thelarge-conductance, voltage- and Ca²⁺-sensitiveK⁺ (maxi-K) channels, increased K⁺ currentsthrough maxi-K channels [I_K(Ca)], and inducedapoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM)decreased I_K(Ca) by blocking the sarcolemmalmaxi-K channels and inhibited the FCCP-induced apoptosis inPASMC cultured in media containing serum and growth factors.Furthermore, inhibition of K⁺ efflux by raisingextracellular K⁺ concentration from 5 to 40 mM alsoattenuated PASMC apoptosis induced by FCCP and theK⁺ ionophore valinomycin. These results suggest thatFCCP-mediated apoptosis in PASMC is partially due to anincrease of maxi-K channel activity. The resultant K⁺ lossthrough opened maxi-K channels may serve as a trigger for cellshrinkage and caspase activation, which are major characteristics ofapoptosis in pulmonary vascular smooth muscle cells.

     

Rat cerebral microvascular smooth muscle cells in culture

This report describes the development and establishment of long-term serial cultures of adult rat vascular smooth muscle cells (SMC) derived from cerebrocortical resistance vessels (small arteries and arterioles). Electron microscopic examination of microvessels isolated off a 150 microns nylon mesh sieve clearly demonstrated the predominance of these vessel types. Initial outgrowth from collagenase-elastase-treated microvessel fragments yielded both endothelium and smooth muscle cells. However, at confluency (2-3 weeks) these cultures consisted of a homogeneous population of broad, polygonal cells that grew in a multilayered "hill and valley" pattern typical of SMC in vitro. For comparative morphological and functional studies, SMC cultures were also initiated from rat thoracic aortas utilizing ring segments as explants. The smooth muscle origin of cultures derived from both resistance vessel (RV) and aorta (RA) was further demonstrated by positive immunofluorescent staining by the specific smooth muscle alpha-actin and myosin antibodies. Ultrastructural examination of these SMC cultures revealed similar morphologic features consisting of typical cytoplasmic myofilament bundles with associated dense bodies and numerous pinocytotic vesicles. Cell growth studies on early (less than P 15)- and late (greater than P 15)-passage RV- and RA-SMC populations revealed markedly different cell growth responses. Representative growth curves of early- and late-passage RA-SMC showed a significantly higher growth rate (two- to fourfold) than RV-SMC cultures. Both cultures, however, exhibited a marked increase in growth potential at higher passage levels. Heparin, at a concentration of 100 micrograms/ml inhibited the growth of RV-SMC during the first 3 days after addition in both exponential and growth-arrested culture states, whereas RA-SMC cultures showed no inhibitory response. These studies indicate that long-term RV-SMC cultures can serve as a useful model system to study functional and metabolic properties of this cell type and provide the means to explore further the heterogeneity of SMC derived from different vasculatures in normal as well as various disease states.     

CD8+ lymphocytes suppress HIV production by autologous CD4+ cells without eliminating the infected cells from culture

Studies of separated peripheral blood mononuclear cell subsets have indicated that the CD8+ lymphocyte is the primary cell type responsible for suppressing human immunodeficiency virus (HIV) replication by infected CD4+ cells. The effect of this antiviral activity is dose-dependent and does not involve killing of the infected cell. These observations indicate that this response is distinct from the anti-HIV cytotoxic mechanisms also described for human CD8+ cells.     

Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans

Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcalpha2-6Gal sequence, and a corresponding increase in glycans carrying the Galalpha1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, alpha1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.     

Rapid response to Con A by CD4+CD45R- rat memory lymphocytes as compared to CD4+CD45R+ lymphocytes

Functionally distinct subpopulations within the CD4+ subset of T lymphocytes have been described in man, rat, and mouse. In the rat different functions have been assigned to CD45R+ and CD45R- T helper cells. The CD45R+ in contrast to the CD45R- T helper cells have been reported to produce IL-2 and to proliferate well in response both to Con A and in MLR. In the present investigation the kinetics of the response to Con A by the CD45R+ and CD45R- rat T helper subsets have been analyzed. We confirm a strong proliferative response to Con A by CD4+CD45R+ rat T lymphocytes and also that they are the best IL-2 producers. We further demonstrate that CD4+CD45R- cells also produce IL-2, although in order to appreciate this production quantitatively by assays of the culture supernatants it was necessary to block IL-2 absorption by IL-2 receptor (IL-2R) antibodies. This blockage was of importance also in comparisons of the two subsets, since they showed different kinetics of IL-2R appearance. It is demonstrated that the CD4+CD45R- cells respond more rapidly to Con A than the CD4+CD45R+ cells as reflected by phenotypic conversion, IL-2 production, and proliferation. The fast response of the CD4+CD45R- T subset shown in the present study of rat cells and analogous studies of human cells suggests that the memory compartment of T cells besides other characteristics also has the capacity for a more rapid response than naive lymphocytes.     

Activation of inward rectifier K+ channels by hypoxia in rabbit coronary arterial smooth muscle cells

We examined the effects of acute hypoxia on Ba2+-sensitive inward rectifier K+ (K(IR)) current in rabbit coronary arterial smooth muscle cells. The amplitudes of K(IR) current was definitely higher in the cells from small-diameter (<100 microm) coronary arterial smooth muscle cells (SCASMC, -12.8 +/- 1.3 pA/pF at -140 mV) than those in large-diameter coronary arterial smooth muscle cells (>200 microm, LCASMC, -1.5 +/- 0.1 pA pF(-1)). Western blot analysis confirmed that Kir2.1 protein was expressed in SCASMC but not LCASMC. Hypoxia activated much more KIR currents in symmetrical 140 K+. This effect was blocked by the adenylyl cyclase inhibitor SQ-22536 (10 microM) and mimicked by forskolin (10 microM) and dibutyryl-cAMP (500 microM). The production of cAMP in SCASMC increased 5.7-fold after 6 min of hypoxia. Hypoxia-induced increase in KIR currents was abolished by the PKA inhibitors, Rp-8-(4-chlorophenylthio)-cAMPs (10 microM) and KT-5720 (1 microM). The inhibition of G protein with GDPbetaS (1 mM) partially reduced (approximately 50%) the hypoxia-induced increase in KIR currents. In Langendorff-perfused rabbit hearts, hypoxia increased coronary blood flow, an effect that was inhibited by Ba2+. In summary, hypoxia augments the KIR currents in SCASMC via cAMP- and PKA-dependent signaling cascades, which might, at least partly, explain the hypoxia-induced coronary vasodilation.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

Rat peripheral CD4+CD8+ T lymphocytes are partially immunocompetent thymus-derived cells that undergo post-thymic maturation to become functionally mature CD4+ T lymphocytes

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent approximately 40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30-40% of DP T cells expresses CD8beta chain, the remaining cells expressing CD8alphaalpha homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.     

Migration pathways of recirculating murine B cells and CD4+ and CD8+ T lymphocytes

Migration pathways of B cell and CD4+ and CD8+ T cell subsets of murine thoracic duct lymphocytes (TDL) were mapped. Per weight, the spleen accumulated more TDL than any other organ, regardless of lymphocyte subset. Spleen autoradiographs showed early accumulations of TDL in marginal zone and red pulp. Many TDL exited the red pulp within 1 hr via splenic veins. The remaining TDL entered the white pulp, not directly from the adjacent marginal zone but via distal periarterial lymphatic sheaths (dPALS). From dPALS, T cells migrated proximally along the central artery into proximal sheaths (pPALS) and exited the white pulp via deep lymphatic vessels. B cells left dPALS to enter lymphatic nodules (NOD), then also exited via deep lymphatics. T cells homed to lymph nodes more efficiently than B cells. Lymphocytes entered nodes via high-endothelial venules (HEV). CD4+ TDL reached higher absolute concentrations in diffuse cortex than did CD8+ T cells. However, CD8+ TDL moved more quickly through diffuse cortex than did CD4+ TDL. B cells migrated from HEV into NOD. Both T and B TDL exited via cortical and medullary sinuses and efferent lymphatics. A migration pathway across medullary cords is described. All TDL subsets homed equally well to Peyer's patches. T TDL migrated from HEV into paranodular zones while B cells moved from HEV into NOD. All TDL exited via lymphatics. Few TDL entered zones beneath dome epithelium. All subsets were observed within indentations in presumptive M cells of the dome epithelium.     

Induction of CD4+ murine natural killer T-like cells by immunization with syngeneic thymoma expressing embryonic alpha-fetoprotein

In embryo, before the establishment of acquired immunity, a variety of embryonic antigens like alpha-fetoprotein (AFP) are produced and secreted in the sera, which rapidly disappear after the birth. Such embryonic antigens sometimes reappear from various tumor cells and decrease in the case of remission, indicating embryonic antigens may alert immune system to control tumors. In the present study, to examine the evoked immune responses against the tumors expressing embryonic antigen, we administered AFP-gene-transfected EL4 cells into syngeneic C57BL/6 mice and established a killer line against the tumor cells. To our surprise, the killer line was CD4+ NK1.1+, natural killer T (NKT)-like cells and eliminated not only AFP-expressing EL4 but YAC-1 cells. Moreover, the established line uniformly expressed Vbeta11 and secreted IL-4, IL-10, IL-13, and IFN-gamma. In vivo inoculation of the line markedly reduced the tumor growth in SCID mice, suggesting novelty of the NKT-like line for tumor surveillance.     

Activation of intestinal smooth muscle cells by interstitial cells of Cajal in simulation studies

Activation of a two-dimensional sheet network (5 parallel chains of 5 cells each) of simulated intestinal smooth muscle cells (SMCs) by one interstitial cell of Cajal (ICC) was modeled by PSpice simulation. The network of 25 cells was not interconnected by gap-junction channels; instead, excitation was transmitted by the electric field that develops in the junctional clefts (JC) when the prejunctional membrane fires an action potential (AP). Transverse propagation between the parallel chains occurs similarly. The ICC cell was connected to cell E5 of the network [5th cell of the 5th (E) chain] via a high-resistance junction. The stimulating current, applied to the ICC cell interior, was made to resemble the endogenous undershooting slow wave (I(SW)). An I(SW) of 2.4 nA (over a rise time of 4 ms) took the ICC cell from a resting potential (RP) of -80 mV to a membrane potential of -41 mV. The slow wave produced a large negative cleft potential in the JC (V(JC); ICC-E5). The V(jc) brought the postjunctional membrane of E5 to threshold, causing this cell to fire an AP. This, in turn, propagated throughout the SMC network. If the ICC cell was given an RP of -55 mV (like SMC) and a slow wave of 40 mV amplitude (I(SW) of 1.8 nA), it still activated the SMC network. This was also true when the ICC cell was made excitable (developing an overshooting, fast-rising AP). In summary, one ICC cell displaying a slow wave was capable of activating a network of SMC in the absence of gap junctions.     

Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4

The cell types of the gut expressing Toll-like receptor 4, which recognizes specifically bacterial lipopolysaccharides, as well as the functionality of this receptor, have remained controversial. We aimed to clarify these issues. Mouse and human intestinal specimens were stained immunohistochemically to detect Toll-like receptor 4 expression. Smooth muscle and myenteric plexus cells but not enterocytes revealed receptor expression. Murine intestinal smooth muscle and myenteric plexus cells but not enterocytes showed nuclear translocation of nuclear factor-kappaB after in vivo stimulation with lipopolysaccharide. Moreover, lipopolysaccharide added to human jejunum biopsies free of epithelial cells induced release of interleukin-8 (IL-8). We can conclude that Toll-like receptor 4 is not expressed in epithelial layer, but rather on smooth muscle and myenteric plexus cells and that expression is functional. The expression of Toll-like receptor 4 on smooth muscle and myenteric plexus cells is consistent with the possibility that these cells are involved in intestinal immune defense; the low or absent expression of Toll-like receptor 4 on enterocytes might explain the intestinal epithelium hyporesponsiveness to the abundance of LPS in the intestinal lumen.     

Activation of heparin cofactor II by fibroblasts and vascular smooth muscle cells

Inhibition of thrombin by heparin cofactor II (HCII) is accelerated by dermatan sulfate, heparan sulfate, and heparin. Purified HCII or defibrinated plasma was incubated with washed confluent cell monolayers, 125I-thrombin was added, and the rate of formation of covalent 125I-thrombin-inhibitor complexes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fibroblasts and porcine aortic smooth muscle cells accelerated inhibition of thrombin by HCII 2.3-7.5-fold but had no effect on other thrombin inhibitors in plasma. Human umbilical vein endothelial cells and mouse macrophage-derived cells did not accelerate the thrombin-HCII reaction. IMR-90 normal human fetal lung fibroblasts treated with heparinase or heparitinase accelerated the thrombin-HCII reaction to the same degree as untreated cells. In contrast, treatment with chondroitinase ABC almost totally abolished the ability of these cells to activate HCII while chondroitinase AC had little or no effect, suggesting that dermatan sulfate was responsible for the activity observed. [35S]Sulfate-labeled proteoglycans were isolated from IMR-90 fibroblast monolayers and conditioned medium and fractionated into two peaks on Sepharose CL-2B. The lower Mr proteoglycans contained 74-76% dermatan sulfate and were 11-25 times more active with HCII than the higher Mr proteoglycans which contained 68-97% heparan sulfate. The activity of the lower Mr proteoglycans decreased 70-90% by degradation of the dermatan sulfate component with chondroitinase ABC. These results confirm that dermatan sulfate proteoglycans are primarily responsible for activation of HCII by IMR-90 fibroblasts. We suggest that HCII may inhibit thrombin when plasma is exposed to vascular smooth muscle cells or fibroblasts.     

CD40-ligated dendritic cells effectively expand melanoma-specific CD8+ CTLs and CD4+ IFN-gamma-producing T cells from tumor-infiltrating lymphocytes

Professional APC, notably dendritic cells (DC), are necessary for stimulation and expansion of naive T cells. By means of murine models, the interaction between CD40 on DC and its ligand CD154 has been recognized as an important element for conditioning of DC to prime and expand CTL. We translated these findings into the human system, scrutinizing the ability of DC to initiate clonal expansion of single T cells. DC generated under completely autologous conditions from peripheral blood monocytes were cocultured at a rate of 0.3 cell/well with melanoma-infiltrating T cells; this procedure guaranteed that either a CD4+ or a CD8+ cell interacted with the DC, thus avoiding the contact of more than one T cell to the DC. In the absence of further stimulation, this cloning protocol yielded almost exclusively CD4+ T cell clones that predominantly exhibited a Th2 phenotype. However, cross-linking of CD40 on DC resulted in the induction of IFN-gamma-producing Th1 CD4+ T cell clones. In addition, CD40-activated DC were capable of expanding CD8+ CTL clones. The ratio of CD4 to CD8 T cell clones corresponded to the ratio present in the initial tumor-infiltrating lymphocyte preparation. The CTL clones efficiently lysed autologous tumor cells whereas autologous fibroblasts or MHC-mismatched melanoma cells were not killed. Our findings support the critical role of CD40/CD154 interactions for the induction of cellular immune responses.     

Phospholipase C activation by prostacyclin receptor agonist in cerebral microvascular smooth muscle cells

The mechanism through which iloprost permits cerebral vasodilation induced by specific stimuli is incompletely understood. Previous study suggests there might be interplay between the adenylyl cyclase and phospholipase C (PLC) systems. Coupling of the prostacyclin receptor with the PLC pathway system was investigated. Iloprost, a stable prostacyclin analog, was used as a prostacyclin receptor agonist. We investigated the effects of iloprost (10-12-10-6 M) on inositol 1,4,5-trisphosphate (IP3) production by piglet cerebrovascular smooth muscle cells in primary culture. Iloprost caused concentration- and time-dependent increases in IP3 production in control cells and in cells pretreated with LiCl (to prevent further IP3 metabolism). Iloprost treatment (10-12 M) of cerebrovascular smooth muscle cells, in the absence and presence of 20 mM LiCl, resulted in 2-fold and 4-fold increases in the formation of IP3, respectively. In contrast, 10-10 M to 10-6 M iloprost, either in the presence or absence of LiCl, induced moderate or no increase in IP3 formation. Iloprost (10-10-10-12 M) strongly stimulated diacylglycerol (DAG) generation, whereas higher concentrations (10-8 M) did not induce an increase. In conclusion, the results suggest that prostacyclin receptors on cerebromicrovascular smooth muscle can couple to PLC, generating the second messengers, IP3 and DAG.     

Human epidermal cells from ultraviolet light-exposed skin preferentially activate autoreactive CD4+2H4+ suppressor-inducer lymphocytes and CD8+ suppressor/cytotoxic lymphocytes

In vivo exposure of human epidermis to UV abrogates the function of T6+DR+ Langerhans cells and induces the appearance of Ag-presenting T6-DR+ OKM5+ cells in the epidermis. Since UV exposure of murine skin results in Ts lymphocyte activation, we investigated the capacity of human epidermal cells (EC) harvested 3 days after in vivo UV exposure to activate regulatory and effector autologous T lymphocyte subsets. T lymphocytes were separated into CD8+ suppressor/cytotoxic lymphocytes and CD4+ helper/inducer lymphocytes by C lysis and panning. The CD4+ subset was further divided by using the 2H4 mAB to obtain CD4+2H4+ lymphocytes (inducers of TS lymphocytes) and CD4+2H4- lymphocytes (inducers of B cell Ig production and inducers of cytotoxic T cells). Unirradiated suction blister-derived EC from control skin (C-EC) and from skin exposed in vivo to UV (UV-EC) were cultured with purified autologous T lymphocyte subsets in the absence of added Ag. The resultant T lymphocyte proliferation was detected by [3H]thymidine uptake. UV-EC were highly effective in the stimulation of CD4+ lymphocytes, whereas C-EC were poor stimulators. The stimulator effect of UV-EC was abrogated after depletion of DR+ UV-EC. When CD4+ lymphocytes were fractionated, UV-EC consistently demonstrated enhanced ability to stimulate suppressor-inducer CD4+2H4+ lymphocytes relative to C-EC. Although less responsive than CD4+2H4+ lymphocytes, CD4+2H4- lymphocytes also demonstrated greater proliferation to UV-EC than to C-EC. Neither UV-EC nor C-EC were able to activate CD8+ lymphocytes devoid of CD4+ lymphocytes. However, after addition of rIL-2 at concentrations that allow binding only to the high affinity IL-2R on T lymphocytes, UV-EC induced vigorous proliferation of CD8+ lymphocytes, whereas C-EC induced only background levels of proliferation. C lysis of leukocytes resident within UV-EC resulted in 66 to 70% reduction of CD8+ lymphocyte proliferation. In conclusion, UV-EC may activate CD8+ lymphocytes by at least two pathways: (1) UV-EC activation of CD4+2H4+ lymphocytes may induce differentiation/proliferation of CD8+ suppressor cells and (2) UV-EC activation of CD4+ cells may induce IL-2 production, that, in combination with UV-induced epidermal leukocytes, stimulates CD8+ cells.