A 16 amino acid synthetic peptide derived from human C3d triggers proliferation and specific tyrosine phosphorylation of transformed CR2-positive human lymphocytes and of normal resting B lymphocytes.

We demonstrate herein that p16, a 16 amino acid synthetic peptide derived from human C3d, which carried LYNVEA sequence of C3d reacting with CR2 and C3d present in trypsin-cleaved C3, triggered "in vitro" and "in vivo" phosphorylations and "in vitro" proliferation of human B lymphocytes, depending on the stage of cell differentiation. Indeed, p16 and C3dT induced "in vivo" tyrosine phosphorylation of pp105 and "in vitro" proliferation only of CR2-positive and not of CR2-negative cell lines. In addition, p16 and C3dT also induced "in vivo" tyrosine phosphorylation of pp100 and "in vitro" proliferation of only small dense resting B lymphocytes and not other B lymphocyte subpopulations nor T lymphocytes. These data suggest that induction of pp100 and pp105 phosphorylation by p16 and C3dT could represent an early event associated with expression of CR2 in the regulation of human B lymphocyte proliferation.     

Human monocyte and macrophage modulation of lymphocyte proliferation.

The effects of human monocytes and mature human macrophages on lymphocyte proliferation in response to PHA and allogeneic lymphocytes were examined. Monocytes enhanced and macrophages markedly suppressed lymphocyte proliferation to both stimuli. Monocyte enhancement of lymphocyte proliferation was, in part, due to a soluble mediator. Macrophage suppression was not due to (a) media depletion, (b) soluble lymphotoxins or inhibitors of proliferation, (c) media depletion, (d) macrophage production of prostaglandins, (e) decreased lymphocyte survival, or (f) induction of suppressor lymphocytes. These data emphasize the dichotomy of human monocyte and macrophage effects on lymphocyte proliferation and suggest, by exclusion, that macrophage suppression may require cell-cell contact.     

Regulation of pp56lck during T-cell activation: functional implications for the src-like protein tyrosine kinases.

The lymphocyte-specific protein tyrosine kinase pp56lck, encoded by a member of the src gene family, is implicated in the control of T-cell growth and differentiation. Purified resting human T lymphocytes contain appreciable levels of lck mRNA and of pp56lck. Upon activation of these T cells, levels of lck mRNA and of pp56lck promptly decline. These reductions in lck mRNA and protein expression are closely correlated with the induction of lymphokine production. Both require identical stimuli and follow a similar time course of response. Down-regulation of lck expression, however, is not correlated with proliferation. Our results provide an example of regulation of a src-like protein tyrosine kinase in a normal fully differentiated cell population and suggest that modulation of lck RNA and protein expression is an important feature of the lymphocyte activation sequence leading to lymphokine production.     

Endotoxin-induced T lymphocyte proliferation

The lymphocyte response to endotoxin (LPS) has been attributed largely to the action of this agent as a polyclonal activator of B lymphocytes. In this study we found that a cloned murine interleukin 2-dependent cytotoxic T cell line, CT 6, proliferates in response to LPS, thus providing the first evidence that T cells can be stimulated directly by LPS. The response was dose and time dependent and was blocked by polymyxin B, an inhibitor of LPS-induced mitogenesis. The fact that this is a cloned T cell line, free of other potentially contaminating lymphoid cell types, precludes the possibility that this proliferation is due to contaminating B lymphocytes or is mediated by macrophage-derived products such as interleukin 1. Moreover, highly purified splenic T lymphocyte populations (purified by negative/positive selection or by a rigorous column purification procedure) contain a small subpopulation (approximately 3%) of T cells that proliferate in response to LPS. This population is missing in the endotoxin-hyporesponsive C3H/HeJ mouse. As was observed in the CT 6 line, proliferation of splenic T cells in response to LPS was inhibited by polymyxin B. Furthermore, treatment of LPS-stimulated T cells with anti-T cell antibodies plus complement blocks the uptake of 3H-thymidine by these cultures. Exogenous interleukin 1 failed to stimulate the T cell cultures comparably to LPS and therefore cannot account for the degree of stimulation observed. These findings support and extend previous findings that suggested a role for an endotoxin-sensitive T cell population in the induction of certain responses, such as LPS-induced adjuvanticity of the lymphocyte-dependent LPS induction of macrophage procoagulant activity.     

Phytohemagglutinin-induced proliferation of guinea pig thymus-derived lymphocytes. I. Accessory cell dependence.

The accessory cell requirement for mitogen-induced T lymphocyte proliferation has been investigated by using a population of guinea pig lymph node lymphocytes enriched in T cells and markedly depleted of macrophages and B lymphocytes. We have found that effective phytohemagglutinin-induced proliferation of T cells is dependent on the participation of accessory cells. Augmentation of PHA responsiveness was noted when cultural conditions were manipulated to increase cell density, suggesting that physical proximity between T cell and accessory cell is required for efficient triggering. Both syngeneic and allogeneic macrophages, as well as syngeneic fibroblasts, serve as accessory cells in this response whereas polymorphonuclear leukocytes or thymocytes do not. Thus, although PHA-induced T lymphocyte proliferation requires accessory cells, the specificity of these cells is strikingly less stringent than for antigen-mediated triggering of immune guinea pig T cells, a response which is dependent upon participation of syngeneic macrophages.     

Development and regulation of chlamydia-responsive murine B lymphocytes

We have examined characteristics of chlamydia-stimulated mouse B cells as well as cells that regulate polyclonal responses in vitro. B lymphocyte proliferation stimulated by chlamydia arises at a similar time as Escherichia coli lipopolysaccharide (LPS)-induced proliferative responses during ontogeny. In contrast, development of immunoglobulin (Ig)-secreting cells after chlamydia stimulation is delayed by several weeks relative to ontogeny of LPS-inducible plaque-forming cells (PFC). The lack of Ig secretion by immature B cells is not due to a deficiency of Lyb5+ B lymphocytes, since X-linked immunodeficient (xid) NBF1 mice that lack this B lymphocyte population respond well to chlamydia stimulation. Adherent cells are important for chlamydia-stimulated B lymphocyte differentiation, but are not as necessary for their proliferation. Neither adult adherent cells nor T cells can correct the inability of immature spleen cells to develop into Ig-secreting cells; spleen cells from 2-wk-old mice (i.e., immature B cells) will not suppress adult B lymphocyte responses to chlamydia. When B lymphocytes are separated according to their buoyant densities, chlamydia stimulates low density (activated) B cells to proliferate and differentiate better than high density (resting) cells. Proliferative responses to chlamydia arise earlier during ontogeny, do not require adherent cells, and can proceed to a relatively greater extent in resting B cell population (compared with activated B cells) than induction of Ig-secreting cells.     

Lymphocyte transformation induced by autologous cells.

Human peripheral blood T lymphocytes are stimulated to proliferate when cultured with autologous B-lymphoblastoid cell lines, autologous mitogen-induced lymphoblasts, or autologous non-T blood lymphocytes. This reaction, the autologous mixed lymphocyte reaction, has attributes of an immune response possessing both memory and specificity. The capacity to stimulate autologous T lymphocyte proliferation depends on the lineage of the lymphoid cell and not on its establishment in continuous culture or carriage of the EB viral genome. The determinant on non-T lymphocytes which stimulates the autologous mixed lymphocyte reaction appears to be an Ia determinant. Thus, allogeneic graft rejection and the allogenic mixed lymphocyte reaction are very likely extensions of an immune response expressed within the host.     

Potentiation of lymphocyte activation by colchicine.

Proliferation of human lymphocytes induced by IO4- is potentiated by 30 min exposure to colchicine (10(-6)M), whereas the response to Con A is inhibited. Treatment with colchicine before or after IO4- modification has similar enhancing effects. Lumicolchicine does not alter proliferative responses. In addition to the proliferation of IO4--oxidized cells, irradiated IO4- modified lymphocytes induce proliferation when mixed with untreated lymphocytes. Enhancement occurs in both these conditions only when IO4--modified cells are treated with colchicine. Preliminary data indicate that proliferation in mixed lymphocyte cultures is also potentiated when either stimulating or responding cells are pretreated with colchicine. These findings suggest a selective stimulatory effect of colchicine on lymphocyte responses induced by cell-cell contact. Agents that modify microtubular assemblies might regulate the induction of immune responses that involve cellular interactions.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Effect of cyclosporin A on human lymphocyte responses in vitro. III. CsA inhibits the production of T lymphocyte growth factors in secondary mixed lymphocyte responses but does not inhibit the response of primed lymphocytes to TCGF

The mechanism whereby Cyclosporin A (CsA) inhibits secondary mixed lymphocyte responses was assessed. CsA added to secondary MLR cultures inhibited proliferation and induction of cytolytic lymphocyte activity. This inhibition was found to be associated with the inhibition of T lymphocyte stimulating growth factor(s) (TCGF) production in the supernatants of secondary MLR cultures. As little as 1.0 micrograms/ml of CsA added to secondary MLR cultures resulted in no measurable TCGF activity. In contrast, moderate doses of CsA (1.0, 2.5 micrograms/ml), which completely inhibited the secondary MLR response to alloantigen, did not inhibit the proliferative and CML response of alloantigen-primed lymphocytes to these stimulating growth factors. Even at high doses of CsA (20 micrograms/ml), substantial levels of proliferation (50% of control response) and CML induction (60% of control response) were observed when the primed cells were exposed to secondary MLR supernatants containing TCGF activity. It was concluded that inhibition of secondary mixed lymphocyte responses by CsA may be due in part to the inhibition of TCGF production rather than the inhibition of the effect of TCGF on mature cytotoxic T lymphocytes.     

Direct suppression of lymphocyte induction by the immunoregulatory human serum low density lipoprotein, LDL-In

Preparations of LDL-In, an immunosuppressive lipoprotein subfraction, were analyzed for the capacity to directly suppress the response of human lymphocytes to the representative stimulant PHA vis-a-vis indirect mechanisms mediated by soluble factors or cell:cell interactions. Serum lipoprotein subfraction enriched in LDL-In induced a suppressed state in lymphocytes during 18-hr induction cultures. These lymphocytes, whether partially or completely suppressed, when added to fresh responder lymphocytes in the presence of PHA did not suppress the response of the responder lymphocytes. In contrast, the major low density lipoprotein (LDL) did not suppress lymphocytes at equivalent concentration in the induction culture, nor did LDL-exposed lymphocytes suppress responder lymphocytes. The supernatant medium from LDL-In-suppressed lymphocytes did not contain a newly synthesized or released suppressive factor. Finally, LDL-In-suppressed lymphocytes were not rescued by normal lymphocytes. Each of these observations, and previous evidence that adherent cells do not mediate the biologic effects of LDL-In, support the hypothesis that the biologic manifestations of LDL-In suppression of lymphocyte function result from a direct effect on the lymphocyte that is exposed to this lipoprotein, possibly via the previously demonstrated LDL-In receptor.     

The proto-oncogene c-myc is a direct target gene of Epstein-Barr virus nuclear antigen 2

Epstein-Barr virus (EBV) infects and transforms primary B lymphocytes in vitro. Viral infection initiates the cell cycle entry of the resting B lymphocytes. The maintenance of proliferation in the infected cells is strictly dependent on functional EBNA2. We have recently developed a conditional immortalization system for EBV by rendering the function of EBNA2, and thus proliferation of the immortalized cells, dependent on estrogen. This cellular system was used to identify early events preceding induction of proliferation. We show that LMP1 and c-myc are directly activated by EBNA2, indicating that all cellular factors essential for induction of these genes by EBNA2 are present in the resting cells. In contrast, induction of the cell cycle regulators cyclin D2 and cdk4 are secondary events, which require de novo protein synthesis.     

T cell surface molecules regulating noncognate B lymphocyte activation. Role of CD2 and LFA-1.

A central event in humoral responses is the Ag-mediated interaction of Th cells and B cells. This interaction leads to the activation of both cell types and results in cytokine secretion by the T cells and proliferation and secretion of Ig by the B cells. The proliferative and differentiative responses of B cells are dependent on contact-mediated signals and cytokines provided by the activated Th cells. Although the role of cytokines in B cell activation and differentiation is understood, the nature of the signals delivered by the activated Th cells and the molecules involved in this process are not known. In this study we have examined Ag-mediated "cognate" T-B cell interactions as well as B cell activation induced by contact with preactivated and fixed Th lymphocytes. Our results indicate that both the T cell surface molecules lymphocyte function associated Ag-1 and CD2 are important in the activation of T cells by Ag presented by B lymphocytes. This indicates that B cells have similar characteristics as other APC. However, once the T cells are activated, contact-mediated stimulation of resting B lymphocytes (the noncognate phase) is dependent on CD2 but not lymphocyte function associated Ag-1. Two lines of evidence indicate this; first, it is inhibited by blocking of CD2 on the T cells and, second, such stimulation is not efficiently mediated by a CD2- Th cell line. Thus, CD2 plays an obligatory role at several discrete stages of T cell-mediated activation of resting B lymphocytes.     

Human peripheral lymphocyte growth regulation and response to phorbol esters is linked to synthesis and phosphorylation of the cytosolic protein, prosolin

Prosolin is a major cytosolic protein (Mr 18400, isoelectric point 5.9) first reported in HL-60 promyelocytic leukemia cells. It is rapidly phosphorylated (15 to 30 min) in response to TPA treatment as an early event in a sequence that leads to cessation of cell proliferation and to differentiation of promyelocytes into monocytes. In our study we examined the expression of prosolin in human peripheral lymphocytes and investigated the effects of TPA treatment on prosolin phosphorylation and on lymphocyte proliferation. Prosolin was not expressed in resting PBL but was induced after 24 to 36 h of PHA stimulation, simultaneously with induction of DNA synthesis. In rapidly proliferating (IL-2 dependent) PBL prosolin was a major cytosolic component, comprising 0.5% of total cytosolic protein, of which approximately 28% was phosphorylated. Expression of prosolin decreased again when either mitogen-induced or IL-2-dependent proliferation diminished during extended periods in culture. Thus, expression of prosolin is correlated with periods when PBL are cycling through S-phase. TPA treatment of IL-2-dependent PBL at the peak of their growth caused phosphorylation of about two-thirds of preexisting unphosphorylated prosolin within 1 h. This was accompanied by cessation of cell proliferation, as indicated by measurements of TdR incorporation. Although TPA has well known mitogenic effects in lymphocytes during initial activation, this result shows that it exerts an antiproliferative effect in rapidly dividing PBL. It is suggested that increased phosphorylation of prosolin may be an initiating event in the antiproliferative response to TPA, which would occur only in proliferating lymphocytes expressing prosolin.     

Antigen receptor-mediated anergy in resting T lymphocytes and T cell clones. Correlation with lymphokine secretion patterns.

The goal of these studies was to define the stimuli and factors that control the induction of anergy in unimmunized resting T lymphocytes. Initial experiments, aimed at establishing the system, showed that exposure of Th1 but not Th2 clones to immobilized anti-CD3 leads to a block in autocrine growth factor production and proliferation upon subsequent restimulation with Ag+APC. Anergy is not prevented by accessory cells, suggesting that this model of T cell tolerance may be due to receptor-mediated inhibitory signals, independent of costimulatory molecules. Culture of small (resting) unimmunized T lymphocytes with anti-CD3 +/- IL-2 induces unresponsiveness to restimulation with anti-CD3, but culture with anti-CD3+IL-4, which stimulates the differentiation of resting cells into IL-4 producers, does not induce anergy. Thus, IL-4-producing clones and bulk populations of IL-4-producing T cells are resistant to Ag receptor-mediated inhibitory stimuli. These results provide experimental models for studying the mechanisms of anergy in normal, unselected, mature T cells, and demonstrate fundamental similarities between cloned cell lines and unimmunized T lymphocytes in the induction of anergy.     

Endothelial cell adhesiveness for human T lymphocytes is inhibited by transforming growth factor-beta 1.

Recombinant human transforming growth factor-beta (TGF-beta) was found to inhibit the adhesive phenotype of human umbilical vein endothelial cells for human PBL, purified T lymphocytes, and PHA-activated lymphoblasts. TGF-beta inhibited lymphocyte attachment to resting human umbilical vein endothelial cells and also to endothelial monolayers stimulated with the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Our investigations also show that the ability of endothelial cells to respond to TGF-beta by altering their adhesiveness is lost with prolonged culture of the cells. However, this loss is selective as TGF-beta inhibits cell proliferation in both early and late passage endothelial cells. These results suggest that in vivo TGF-beta may inhibit the adhesive phenotype of endothelial cells and also may limit the immunologic response occurring at the endothelial cell barrier.     

Lymphocyte transformation induced by autologous cells. III. Lymphoblast-induced lymphocyte to stimulation does not correlate with EB viral antigen expression or immunity.

Human mitogen-induced and cell line B lymphoblasts stimulate the proliferation of allogeneic and autologous lymphocytes in culture. The role in thes reaction of EB viral determinants on the stimulating cells and immunity of the lymphocyte donor to the EB virus has been studied. The stimulatory capacity of cultured cell line lymphoblasts is not inhibited by incubating lymphoblasts with antisera to EB viral determinants. Cultured cell line B lymphoblasts stimulate as much thymidine incorporation by lymphocytes from donors with or without immunity to the EB virus. Further, a B lymphoblast cell line (U-698) which lacks the EB viral genome stimulated as much lymphocyte proliferation as did B lymphoblasts with the EB genome. Cultured T lymphoblast cell lines do not stimulate allogeneic lymphocyte proliferation. These cells appear to lack the determinants which stimulate lymphocyte transformation. No evidence was found that cultured cell line T lymphoblasts suppressed allogeneic lymphocyte proliferation. Mitogeninduced lymphoblasts from EB-immune and non-immune subjects stimulated the proliferation of autologous lymphocytes comparably. It is concluded that neither immunity to the EB virus nor expression of EB viral antigens on mitogen-induced on cell line lymphoblasts is necessary for the stimulation of lymphocyte proliferation.     

Regulation of apoptosis and replicative senescence in CD8+ T cells from patients with viral infections

Activated T lymphocytes are generated during an immune response. The induction of T lymphocyte proliferation is one way in which cell numbers can be controlled. However, once generated, the increased numbers of cells must be removed in order to re-establish cellular homoeostasis within the immune system. In this paper we describe how the numbers of activated T cells can be regulated by two distinct mechanisms, namely apoptosis and replicative senescence. In addition, we suggest that the regulation of cell clearance, as opposed to cell persistence, after an immune response is intimately involved in the generation of immune memory.     

A comparison of serum-free media for the support of in vitro mitogen-induced blastogenic expansion of cytolytic lymphocytes

The high cost and potential dangers of including human AB serum in incubation media used to expand lymphocyte populations in vitro for adoptive immunotherapy have stimulated efforts to develop defined media which can support both the expansion and induction of lymphocytes with tumor cytolytic activity in the absence of serum. Lymphocyte proliferation following exposure to either PHA or the combination of phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore, ionomycin, was evaluated. Although the media tested, X-Vivo 10, HB-104, AIM V, and HL-1, supported the generation of comparable levels of LAK activity after 3–5 days incubation with 10³ U human recombinant interleukin-2 (rIL-2)/ml, there were striking differences in the ability of each medium to support mitogenically stimulated lymphocytes in the absence of serum, with cells in AIM V and X-Vivo 10 showing the highest levels of DNA synthesis. In long-term cultures (17 days) of blood MNC stimulated by PDBu and ionomycin, X-Vivo 10 and HB-104 yielded the greatest numbers of cells. The addition of 2% AB serum greatly enhanced the ability of each medium to support cell proliferation to equivalent maximum levels. The results indicate that while all four serum-free media were suitable for lymphocyte culture and support the development of LAK activity, they differ in their capacity to support expansion of lymphocyte populations in response to polyclonal mitogenic activation. This latter characteristic should be considered before choosing a particular serum-free formulation as its constituents may affect mechanistic interpretations regarding signal transduction events.     

Suppression of T lymphocyte function by measles virus is due to cell cycle arrest in G1

Measles virus suppresses T lymphocyte functions in vitro. When measles virus-infected T lymphocytes are stimulated with PHA or 12-O-tetradecanoylphorbol-13-acetate, plus calcium ionophore, the cells secrete IL-2 and express the IL-2R or Tac Ag to a similar extent as uninfected cells, yet proliferation is reduced by 50 to 90%. Stimulated infected T cells also express the cell surface activation Ag 4F2, transferrin R, and HLA-DR. The secretion of IFN-gamma by infected T cells in response to PHA is not suppressed at 24 to 72 h after stimulation. Total RNA synthesis at 48 and 72 h after stimulation is reduced in infected T lymphocytes. Infectious measles virus progeny are produced during this interval. Thus infected T lymphocytes can become activated in response to mitogenic stimuli and the cells support efficient viral replication before the block in cell proliferation.