Mechanism of accessory cell requirement in inducing IL 2 responsiveness by human T4 lymphocytes that generate colonies under PHA stimulation

PHA-driven monoclonal colony formation by low concentrations of resting T4 lymphocytes in agar culture requires the presence of interleukin 2 (IL 2) and accessory cells. Using recombinant IL 2 and anti-Tac monoclonal antibody as a probe for the IL 2 receptor, we demonstrate that the requirement of accessory cells (here an irradiated B cell line) in inducing IL 2 responsiveness relies on their enhancing effect in functional IL 2 receptor expression by the T colony progenitors. Furthermore, it is shown that cell to cell interaction between accessory cells and colony progenitors results in IL 2 response, i.e., colony formation, when the IL 2 receptor density reaches a critical threshold. The asynchronism in IL 2 responsiveness expression by the T colony progenitors upon activation and the short-lived T cell-accessory cell interaction, due to accessory cell death, determine the 10% colony efficiency of the culture system. In addition, we demonstrate that the accessory function in IL 2 receptor and IL 2 responsiveness expression by the T colony progenitors can be supported by irradiated T lymphocytes as well as B cells. The absence of lineage restriction of the signal delivered by accessory cells, and the requirement of physical interaction between T colony progenitors and accessory cells, emphasize the necessity of cross-linking the activation-signal receptors in inducing IL 2 responsiveness by resting T4 cells.     

Histamine acts directly on human T cells to inhibit interleukin-2 and interferon-gamma production

Histamine acts directly on human T cells to inhibit lymphokine production without the involvement of accessory cells. Histamine inhibits the production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by purified human peripheral T cells activated in the presence of either intact monocytes or metabolically inactive fixed Raji and U698 cells as accessory cells. Purified T cells do not respond more than marginally to staphylococcal enterotoxin A (SEA) or phytohemagglutinin (PHA) in the absence of accessory cells. However, activation by the phorbol ester PMA in conjunction with either PHA or the calcium ionophore A23187 induces large amounts of IFN-gamma and IL-2. Histamine suppresses the lymphokine production in these pure T-cell cultures to a similar extent as in monocyte-containing cultures. Histamine is also shown to suppress DNA synthesis by purified T cells cultivated at a low cell density, eliminating any possible involvement of small numbers of contaminating accessory cells. In vitro preactivated T cells are shown to retain their capacity to respond to histamine when stimulated by PMA and A23187 or by mitogen in the presence of Raji cells. The conclusion that histamine acts directly on T cells and does not require accessory cells to induce suppression is further confirmed by the demonstration that IL-2 production by the human T-cell leukemia line Jurkat was significantly suppressed by histamine in a H-2 receptor-restricted manner.     

Endogenous production of prothymocyte differentiating activity by phytohemagglutinin-stimulated T-cell-depleted human marrow

The PHA responsiveness of marrow T-cell precursors remains a matter of controversy. We have investigated the capacity of human marrow to proliferate under phytohemagglutinin (PHA) stimulation following extensive removal of mature T cells by complement-dependent cytotoxicity with MBG6 and RFT8 monoclonal antibodies. PHA-induced thymidine uptake by marrow cells occurred with a peak on Days 6-8 of incubation instead of Day 3 for PBL. This peak was observed 48 hr earlier in the presence of PHA-stimulated T-depleted marrow cell supernatants. These supernatants can also promote the growth of mature T-cell colonies from MBG6-, RFT8-, T11-, T3- marrow. However, full colony development requires exogenous interleukin 2 (IL-2). IL-2 could be detected in marrow supernatants but only at very low levels and beyond Days 3 and 4. In contrast Days 1-6 marrow supernatants were equally effective in promoting MBG6-RFT8- marrow cell responsiveness to PHA. We conclude that marrow T-cell precursors are not PHA responsive and that PHA induces the production by marrow non-T cells of a prothymocyte-differentiating activity (PTDA); PTDA can differentiate marrow T-cell progenitors into PHA-responsive T cells; following activation by PHA, these cells undergo limited proliferation induced by IL-2 endogenously released from de novo differentiated T cells. It is suggested that this mechanism may account for extrathymic differentiation of the T-cell lineage in heavily irradiated marrow transplantation recipients.     

Enhancement by transforming growth factor-beta 1 (TGF-beta 1) of the proliferation of leukemic blast progenitors stimulated with IL-3.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti-IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF-beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells.     

Interferon-beta 2/interleukin 6 is a co-stimulant for human T lymphocytes

IFN-beta 2/IL-6 promotes the proliferation of human peripheral blood T cells in the presence of either PHA or Con A. This effect is observed with highly purified T cells and is masked by the addition of as few as 2% monocytes, suggesting that accessory cells are not required and that IFN-beta 2/IL-6 acts directly on the T cells. Both T4-positive and T8-positive cells respond to IFN-beta 2/IL-6 plus PHA. Studies of the time requirements of IFN-beta 2/IL-6 and of PHA in the response of T cells show that optimal co-stimulation occurs when both IFN-beta 2/IL-6 and PHA are added together at the outset of culture, suggesting that IFN-beta 2/IL-6 acts predominantly on resting T cells. Unlike T cell proliferation induced by IL-2, T cell proliferation induced by IFN-beta 2/IL-6 is not blocked by antibodies to the IL-2 R, suggesting that IFN-beta 2/IL-6 does not act by stimulating IL-2 production. Thus, in addition to its previously reported properties, IFN-beta 2/IL-6 stimulates T cells in the presence of mitogen, and may prove to be of considerable importance in the physiologic activation of T cells.     

T4 cell activation by immobilized phytohemagglutinin: differential capacity to induce IL-2 responsiveness and IL-2 production

Soluble mitogens, such as PHA induce accessory cell (AC)-dependent T cell proliferation. One function of the AC is to create a stimulatory matrix. Therefore, experiments were carried out to determine whether PHA immobilized onto microtiter plates could stimulate T cells in the absence of AC. Peripheral blood T4 cells were cultured under limiting dilution conditions with either soluble or immobilized PHA with or without rIL-1 beta, rIL-2, r-TNF-alpha, an anti-CD28 mAb (9.3), or irradiated EBV-transformed B cells as AC. The frequency of proliferating T4 cells was assessed by examining wells microscopically, and the frequency of T4 cells producing IL-2 was assessed by examining the ability of supernatants to support CTLL-2 proliferation. The percentage of T4 cells growing and producing IL-2 was determined by a maximum likelihood procedure. Immobilized, but not soluble, PHA induced a mean of 20.0 +/- 2.6% of T4 cells to grow in the complete absence of AC in medium supplemented with rIL-2. Whereas rIL-1 beta, rTNF-alpha, and 9.3 were unable to support T4 cell growth in the absence of rIL-2, each enhanced the percentage of T4 cells responding to immobilized PHA in the presence of rIL-2. In contrast, both soluble and immobilized PHA were unable to induce T4 cell IL-2 production in the absence of AC, even when cultures were supplemented with rIL-1 beta or 9.3. In the presence of AC, a small percentage of T4 cells (5.4 to 11.7%) was stimulated to produce detectable amounts of IL-2 by either immobilized or soluble PHA. Moreover, in the presence of AC, a very small population (approximately 1%) of PHA-stimulated T4 cells proliferated without supplemental rIL-2. The data indicate that a matrix of immobilized PHA is sufficient for some T4 cells to be activated to respond to IL-2, whereas others require additional signals provided by rIL-1 beta, rTNF alpha, 9.3, or AC. In contrast, neither soluble nor immobilized PHA is sufficient to induce T cell IL-2 production. This response requires signals provided by intact AC.     

Accessory cell-derived helper signals in human T-cell activation with phytohemagglutinin: induction of interleukin 2-responsiveness by interleukin 6, and production of interleukin 2 by interleukin 1 [corrected

Interleukins (IL-) 1 and 6 have been shown to represent accessory signals for T-cell activation. In the present study, we further examined the effects of both cytokines on accessory cell-depleted human T cells stimulated with phytohemagglutinin (PHA). The addition of IL-6 to the cultures resulted in T-cell proliferation; however, IL-1 was unable to support PHA-induced T-cell growth. The addition of IL-1 consistently induced a low level of IL-2 production and strongly enhanced T-cell proliferation in the presence of IL-6. Thus, the effect of IL-1 on T-cell growth becomes apparent only in the presence of IL-6. Blocking the IL-2-receptor (IL-2R) with the monoclonal antibodies anti-Tac and MikBêta 1 (directed to the alpha and bêta chains of the IL-2R, respectively) had no effect on PHA/IL-6-supported proliferation, but completely eliminated the growth-enhancing effect of IL-1. On the other hand, a neutralizing anti-IL-4-antiserum did not affect PHA/IL-6- or PHA/IL-6/IL-1-induced proliferation. Further experiments showed that IL-6 enhances T-cell responsiveness to IL-2, as evidenced by enhanced IL-2-induced proliferation. However, we could not find an effect of IL-6 on the expression of IL-2R as measured by staining with anti-Tac and with MikBêta 1 or by binding of (125I)-IL-2 to T cells. It can be concluded from these studies that IL-1 and IL-6 have different helper effects on PHA-induced T-cell activation. In the presence of PHA, IL-6 induces limited IL-2/IL-4-independent growth, and more importantly it renders T cells responsive to IL-2. IL-1 provides a signal leading to IL-2 production. The combination of IL-1 and IL-6 represents a synergistic helper signal, leading to an IL-2-dependent pathway of proliferation.     

IL-10 inhibits human T cell proliferation and IL-2 production.

Human IL-10 has been reported previously to inhibit the secretion of IFN-gamma in PBMC. In this study, we have found that human IL-10 inhibits T cell proliferation to either mitogen or anti-CD3 mAb in the presence of accessory cells. Inhibited T cell growth by IL-10 was associated with reduced production of IFN-gamma and IL-2. Studies of T cell subset inhibition by human IL-10 showed that CD4+, CD8+, CD45RA high, and CD45RA low cells are all growth inhibited to a similar degree. Dose response experiments demonstrated that IL-10 inhibits secretion of IFN-gamma more readily than T cell proliferation to mitogen. In addition, IL-2 and IL-4 added exogenously to IL-10 suppressed T cell cultures reversed completely the inhibition of T cell proliferation, but had little or no effect on inhibition of IFN-gamma production. Thus, in addition to its previously reported biologic properties, IL-10 inhibits human T cell proliferation and IL-2 production in response to mitogen. Inhibition of IFN-gamma production by IL-10 appears to be independent of the cytokine effect of IL-2 production.     

IL-1 serves as a secondary signal for IL-9 expression.

IL-9 is produced in vitro by activated CD4+ T cell lines of the Th2 subtype and by naive CD4+ T cells. Here we show that T cell lines stimulated with Con A in the presence of accessory cells (AC) such as irradiated spleen cells or bone marrow-derived macrophages produced substantially more IL-9 than T cells stimulated with Con A alone. These data suggest that AC influence the production of IL-9 through accessory signals that result in an at least 10-fold increase of IL-9 secretion by the respective T cells. Addition of IL-1 to T cells activated by Con A, PHA, or anti-CD3 antibodies revealed that this monokine was responsible for the potentiation of IL-9 production. This finding was confirmed by applying anti-IL-1 antibodies. The production of other lymphokines, namely, IL-3, IL-4, and IL-6, by activated T cells was not or only marginally enhanced in the presence of AC or IL-1, thus indicating that the synthesis of IL-9 is regulated differently from that of other Th2-derived lymphokines. Furthermore, it was demonstrated by Northern blot analysis that IL-1 increases IL-9 expression at the pretranslational level. Because IL-1 alone failed to induce the production of IL-9 by T cells, this monokine acts as a costimulator in combination with a T cell receptor-mediated signal.     

Identification of the B cell derived T cell colony promoting activity to soluble CD23.

We previously reported that supernatants of phytohemagglutinin (PHA)-stimulated normal human B cells (NBCsup) contain a T cell colony promoting activity. NBCsup were able to (a) increase the number of secondary colonies generated under PHA and interleukin-2 (IL-2) stimulation by peripheral blood-derived primary T colony cells, (b) enhance the ability of CD4+ but not CD8+ peripheral blood T cells to form agar colonies in the presence of PHA and IL-2 and (c) support in vitro differentiation of CD2-3-4-8- prothymocytes into CD2+3+4+ T cells. This activity was therefore refered to as Prothymocyte Differentiating Activity (PTDA). Subsequent studies pointed to striking biochemical and cell source homologies between this B cell derived factor and the 25-kDa soluble CD23 (sCD23). sCD23 has been recently found to display prothymocyte differentiating activity.     

Accessory function of human fibroblasts in mitogen-stimulated interferon-gamma production by T lymphocytes. Inhibition by interleukin 1 and tumor necrosis factor

Highly purified human T cells from peripheral blood fail to produce interferon (IFN)-gamma in the absence of accessory cells. The ability of T cells to produce IFN-gamma upon stimulation with phytohemagglutinin (PHA) or concanavalin A could be restored by the addition of cultured allogeneic human foreskin fibroblasts. Addition of antibodies specific for HLA-DR, DQ, and DP antigens failed to block this accessory function of the fibroblasts. In contrast, antibodies to HLA-DR and DQ antigens inhibited the accessory cell activity of autologous monocytes. Allogeneic fibroblasts failed to exert accessory activity when exogenous interleukin 2 (IL-2) was used as the stimulus for IFN-gamma production. In contrast, autologous monocytes were active as accessory cells for IL-2-stimulated T cells. Addition of recombinant human interleukin 1 alpha (IL-1 alpha) or IL-1 beta to PHA-stimulated T cells co-cultured with fibroblasts stimulated IFN-gamma production. In contrast, preincubation of fibroblasts with IL-1 alpha or IL-1 beta caused a dose-dependent suppression of the ability of fibroblasts to augment PHA- and concanavalin A-induced IFN-gamma production by T cells. Preincubation of fibroblasts with recombinant human tumor necrosis factor (TNF) also reduced their accessory activity. Incubation of fibroblasts with IFN-gamma produced some reduction in their accessory activity and the inhibitory effect of TNF was further enhanced in the presence of IFN-gamma. A 4- to 10-hr incubation of fibroblasts with IL-1 or TNF was sufficient to produce a maximal suppression of accessory activity. Fixation of fibroblasts with formaldehyde decreased their accessory activity, but fixation did not abolish the suppression of accessory function induced by earlier incubation with IL-1. Supernatants of IL-1-treated fibroblast cultures had less suppressive activity than the IL-1-treated fibroblasts per se, and no suppressive activity at all was detected in the supernatants of TNF-treated fibroblasts. Enhanced prostaglandin synthesis may play a role in the IL-1- and TNF-induced suppression of accessory cell function, but other factors are likely to be involved. Our results show that fibroblasts can have a marked effect on T cell function and that IL-1 and TNF can exert immunoregulatory activities indirectly by altering the interactions of fibroblasts with T cells.     

Synergistic effects of purified recombinant human and murine B cell growth factor-1/IL-4 on colony formation in vitro by hematopoietic progenitor cells. Multiple actions

Purified recombinant human B cell growth factor-1/IL-4 was evaluated, alone and in combination, with purified preparations of recombinant human (rhu) CSF or erythropoietin (Epo) for effects on colony formation by human bone marrow CFU-GM progenitor cells (GM) and burst forming unit-E progenitor cells. rhu IL-4 synergized with rhu G-CSF to enhance granulocyte colony formation, but had no effect on CFU-GM colony formation stimulated by rhu GM-CSF, rhu IL-3, or rhu CSF-1. Rhu IL-4 synergized with Epo to enhance BFU-E colony formation equal to that of Epo plus either rhu IL-3, rhu GM-CSF, or rhu G-CSF. Removal of adherent cells and T lymphocytes did not influence the synergistic activities of rhu IL-4. Rmu IL-4, synergized with rhu G-CSF, but not with rmu GM-CSF, rmu IL-3, or natural mu CSF-1, to enhance CFU-GM (mainly granulocyte) colony numbers by a greater than 90% pure preparation of murine CFU-GM. Also, rhu IL-4 at low concentrations enhanced release of CSF and at higher concentrations the release also of suppressor molecules from human monocytes and PHA-stimulated human T lymphocytes. Use of specific CSF antibodies suggested that rhu IL-4 was enhancing the release of G-CSF and CSF-1 from monocytes and the release of GM-CSF and possibly G-CSF from PHA-stimulated T lymphocytes. Use of antibodies for TNF-alpha, IFN-gamma, or TNF-beta as well as measurement of TNF and IFN titers suggested that the suppressor molecule(s) released from monocytes were acting with TNF-alpha and those released from PHA-stimulated T lymphocytes were acting with IFN-gamma. These results implicate B cell growth factor-1/IL-4 as a synergistic activity for hematopoietic progenitors and suggest that the actions can be on both progenitor and accessory cells.     

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.     

Accessory cell function of human melanoma cells in mitogen-induced T cell proliferation

Six out of eight human melanoma cell lines were found to be able to function as accessory cells in PHA-induced proliferation of autologous and allogeneic T cells. The accessory cell function of the melanoma cell lines appears to be similar to that of monocytes, requires the presence of viable cells, and does not correlate with the cell surface binding sites for PHA and with the level of expression of HMW-MAA and of HLA Class I antigens. HLA Class II antigens do not appear to play a major role in these phenomena, since there is no relationship between level of expression of HLA Class II antigens and accessory cell function of melanoma cells. Furthermore, addition of anti-HLA Class II monoclonal antibodies does not affect proliferation of T cells stimulated with PHA in the presence of melanoma cells with accessory cell function. Although melanoma cells exert accessory cell function, functional and immunological assays did not detect IL-1 in the spent medium of the melanoma cell lines. Furthermore, Northern blotting analysis with IL-1 alpha and IL-1 beta probes did not detect IL-1-specific mRNA in melanoma cell lines. These results suggest that PHA-induced proliferation of T cells in the presence of melanoma cells can bypass the requirement for IL-1 or utilizes factors other than IL-1.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts; studies of normal polyclonal T cells and T lymphocyte clones derived early after allogeneic bone marrow transplantation

 T cell clones (CD4⁺CD8^–TCRαβ⁺γδ^–) derived from bone marrow transplant recipients were stimulated with phytohaemagglutinin (PHA) +interleukin-2 (IL-2) in the presence of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) derived from acute leukaemia patients(leukaemic PBMC containing more than 95% blast cells). Leukaemic PBMC could function as accessory cells during mitogenic T cell activation resulting in both T cell proliferation and a broad T cell cytokine response [IL-3, IL-4, IL-10, granulocyte/macrophage-colony-stimulating factor (GM-CSF) tumour necrosis factor α (TNFα) and interferon γ (IFNγ) secretion]. Blockade of IL-1 effects by adding IL-1 receptor antagonist together with PHA+IL-2+leukaemia blasts increased T cell proliferation, whereas IL-6-neutralizing antibodies did not alter T cell proliferation. A qualitatively similar T cell cytokine response and a similar cytokine profile (highest levels detected for GM-CSF and IFNγ) were detected when normal polyclonal T cell lines were stimulated with PHA in the presence of non-irradiated leukaemic PBMC. When leukaemic PBMC derived from 18 acute myelogenous leukaemia patients were cultured with PHA and cells from a polyclonal T cell line, increased concentrations of the T cell cytokines IFNγ and IL-4 were detected for all patients. We conclude that T cell activation resulting in proliferation and a broad cytokine response can take place in the presence of excess acute myelogenous leukaemia blasts. Received: 30 November 1995 / Accepted: 9 January 1996     

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.     

Comparative studies of the granulopoietic enhancing effects of biosynthetic human insulin-like growth factors I and II

The effect of biosynthetic human insulin-like growth factor I (IGF-I) and IGF-II on the in vitro growth of human marrow myeloid progenitors in the presence of recombinant human granulocyte colony stimulating factor (rhG-CSF), granulocyte-macrophage CSF (rhGM-CSF), or interleukin-3 (rhIL-3), was investigated. IGF-I and IGF-II similarly enhanced the growth of myeloid progenitors in cultures stimulated with any of the above hemopoietic regulators. Analysis of colony composition showed an increase in the numbers of granulocyte colonies, but no alteration in the numbers of macrophage or granulocyte/macrophage colonies. IGF-I induced an increase of 62 ± 16%, 84 ± 13%, and 107 ± 18% in granulocyte colony numbers in the presence of G-CSF, GM-CSF, or IL-3, respectively. The values for IGF-II were 66 ± 13%, 96 ± 12%, and 91 ± 12%. Similar enhancement of myeloid colony formation by both peptides was also detected in G-CSF and GM-CSF-stimulated cultures of marrow cells that had been depleted of accessory cells, while neither peptide exerted any effect in the presence of IL-3 in such cultures. The growth-promoting effects of IGF-I and IGF-II were completely abrogated by monoclonal antibodies directed against the IGF-I (Type I) membrane receptor. IGF-I and IGF-II thus appear to exert their effects on human marrow myeloid progenitors via a direct mechanism involving the Type I receptor. © 1993 Wiley-Liss, Inc.