Human T-Lymphocyte Transformation with Human T-Cell Lymphotropic Virus Type 2

Human T-cell lymphotrophic virus type 2 (HTLV-2), a common infection of intravenous drug users and subpopulations of Native Americans, is uncommon in the general population. In contrast with the closely related HTLV-1, which is associated with both leukemia and neurologic disorders, HTLV-2 lacks a strong etiologic association with disease. HTLV-2 does shares many properties with HTLV-1, including in vitro lymphocyte transformation capability. To better assess the ability of HTLV-2 to transform lymphocytes, a limiting dilution assay was used to generate clonal, transformed lymphocyte lines. As with HTLV-1, the transformation efficiency of HTLV-2 producer cells was proportionately related to the number of lethally irradiated input cells and was comparable to HTLV-1-mediated transformation efficiency. HTLV-2-infected cells were reproducibly isolated and had markedly increased growth potential compared to uninfected cells; HTLV-2 transformants required the continued presence of exogenous interleukin 2 for growth for several months and were maintained for over 2 years in culture. All HTLV-2-transformed populations were CD2 and/or CD3 positive and B1 negative and were either CD4⁺ or CD8⁺ populations or a mixture of CD4⁺ and CD8⁺ lymphocytes. Clonality of the HTLV-2 transformants was confirmed by Southern blot analysis of T-cell receptor β chain rearrangement. Southern blot analysis revealed a range of integrated full-length genomes from one to multiple. In situ hybridization analysis of HTLV-2 integration revealed no obvious chromosomal integration pattern.     

Modulation of cell surface markers on NK-like T lymphocytes by using IL-2, IL-7 or IL-12 in vitro stimulation

Cytotoxicity and proliferation of NK-like T (CIK) cells are dependent on the continuous presence of exogenous cytokines, but it is not known which cytokine is optimal. Here, we compared the effect of exogenous interleukin 2 (IL-2), interleukin 7 (IL-7) or interleukin 12 (IL-12) on the generation of CIK cells in addition to IL-1, interferon-gamma and anti-CD3 antibodies. Cell surface markers important for cytotoxic activity and adhesion were defined and cytokines leading to their optimal expression were determined. The most important findings were: (a) IL-12 generates the most CD3/CD56-double-positive CIK cells, (b) the expression of LFA-1/CD11a which is important for cytotoxic activity is highest with IL-7, and (c) IL-7 also generates the most CD28-positive cells which may enhance T cell receptor co-stimulation. In summary, essential differences concerning antigen expression were found when generating CIK cells using IL-7 or IL-12 instead of IL-2. In particular, IL-12 may be of interest due to the high expansion of CD56 positive cells in CIK cell cultures and the important role of these cells in mediating cytotoxicity towards malignant tissues.     

Comparison of AAV/IL-7 autocrine (T cell) versus paracrine (DC) gene delivery for enhancing CTL stimulation and function

Adoptive transfer of antigen-specific cytotoxic T lymphocyte (CTL) into patients holds promise in treating cancer. Such anti-cancer CTL are stimulated by professional antigen-presenting dendritic cells (DC). We hypothesize the gene delivery of various Th1-response cytokines, such as interleukin 7 (IL-7), should further enhance CTL stimulation and activity. However, the issue as to which cell type, DC (paracrine) or the T cell (autocrine), should express a particular Th1 cytokine gene for optimal CTL stimulation has never been addressed. We used adeno-associated virus-2 (AAV) to compare delivery of IL-7 and IL-2 genes into DC or T cells and to exogenous commercial cytokines for generating robust carcinoembryonic antigen (CEA)-specific CTL. AAV/IL-7 transduction of T cells (autocrine delivery) generated CTL with the highest killing capability. Consistent with this, AAV/IL-7 delivery generated T cell populations with the highest proliferation, highest interferon γ expression, highest CD8(+):CD4(+) ratio, highest CD8(+), CD69(+) levels, and lowest CD4(+), CD25(+) (Treg) levels. These data are consistent with higher killing by the AAV/IL-7-altered CTL. These data strongly suggest that IL-7 autocrine gene delivery is optimal for CTL generation. These data also suggest Th1 cytokine autocrine versus paracrine delivery is an important issue for immuno-gene therapy and uncovers new questions into cytokine mechanism of action.     

Cytokine requirements for production of a novel anti-CD8-resistant CTL population.

A population of CD8+ CTL can be generated in vitro in the presence of anti-CD8 mAb. Due to their apparent high avidity characteristic, these anti-CD8-resistant CD8+ CTL may have important functional in vivo roles in graft rejection, and may be important in antiviral and antitumor responses. We have previously reported that this anti-CD8-resistant subset of CD8+ CTL demonstrates functional differences from anti-CD8-sensitive CD8+ CTL. One important difference between the subsets is the markedly greater dependence of anti-CD8-resistant CTL upon exogenous cytokines for their generation in vitro. In this study, we have investigated in detail the cytokine requirements for the generation of allospecific CD8+ CTL in vitro and have found that IL-4 can augment the generation of anti-CD8-sensitive but not anti-CD8-resistant CTL, whereas IL-2 or IL-12 can augment the generation of both anti-CD8-sensitive and anti-CD8-resistant CTL. However, anti-CD8-resistant CTL require at least 10-fold higher concentrations of IL-2 than do anti-CD8-sensitive CTL. This more stringent IL-2 requirement precludes the efficient generation of anti-CD8-resistant CTL in vitro in the absence of exogenous IL-2 because they cannot produce sufficient IL-2 to meet their needs, in contrast to anti-CD8-sensitive CTL. By providing exogenous cytokines to allospecific CTL generation cultures, we further demonstrate that anti-CD8-resistant CTL can be functionally skewed to the Tc1 subset, but differ from anti-CD8-sensitive conventional CTL in that they cannot be skewed to the Tc2 subset.     

Generation of cytokine-induced killer cells using exogenous interleukin-2, -7 or -12

Immunologic effector cells termed cytokine-induced killer (CIK) cells are generated in vitro from peripheral blood lymphocytes by addition of interferon-gamma, interleukin (IL)-2, IL-1 and an antibody against CD3. CIK cells have been shown to eradicate established tumors in a SCID mouse/human lymphoma model. CIK cells are dependent on exogenous cytokines such as IL-2, IL-7, or IL-12. We studied the effect of these cytokines in detail. Cellular proliferation was analyzed using an MTT proliferation assay, surface antigen expression via flow cytometry, cytotoxic activity using an LDH release assay, and apoptosis via flow cytometric analysis. IL-2, IL-7 and IL-12 led to significant growth of lymphocytes. Cells grown in IL-2 and IL-7 showed higher proliferation rates than cells grown in IL-12 according to the MTT assay. Concerning surface antigen expression, exogenous IL-7 led to a decrease in IL-7 receptor expression (4.8% from 60.4%) and exogenous IL-2 to a decrease in IL-2 receptor expression (61.2% from 73.2%). CD28 expression was higher in cells grown in IL-7 (77.3%) than in cells grown in IL-2 (62.5%). IL-12 led to a decrease in ICAM-1 adhesion molecule expression (57.7% from 76.7%) and an increase in CD56 expression compared with exogenous IL-7. IL-7 led to higher number of CD4-positive cells than IL-2 (53.0% vs 49.5%). No significant difference was found between IL-2, IL-7 and IL-12 in cytotoxic activity measured in an LDH release assay. Small amounts of apoptotic cells were found with all cytokines. However, the percentage of necrotic cells was higher with exogenous IL-12 than with IL-2 or IL-7. In summary, CIK cells can be generated using exogenous IL-2, IL-7 or IL-12. No difference in cytotoxic activity was found. However, significant differences were found in cell proliferation rates, antigen expression and percentage of necrotic cells. Received: 26 February 1998 / Accepted: 4 August 1998     

Growth-promoting activity of IL-1 alpha, IL-6, and tumor necrosis factor-alpha in combination with IL-2, IL-4, or IL-7 on murine thymocytes. Differential effects on CD4/CD8 subsets and on CD3+/CD3- double-negative thymocytes

Many cytokines (including IL-1, IL-2, IL-4, IL-6, and TNF-alpha) have been shown to induce thymocyte proliferation in the presence of PHA. In this report, we demonstrate that certain cytokine combinations induce thymocyte proliferation in the absence of artificial comitogens. IL-1 alpha, IL-6, and TNF-alpha enhanced the proliferation of whole unseparated thymocytes in the presence of IL-2, whereas none of them induced thymocyte proliferation alone. In contrast, of these three enhancing cytokines, only IL-6 enhanced IL-4-induced proliferation. We also separated thymocytes into four groups based on their expression of CD4 and CD8, and investigated their responses to various cytokines. The results indicate that each cytokine combination affects different thymocyte subsets; thus, IL-1 alpha enhanced the proliferation of CD4-CD8- double negative (DN) thymocytes more efficiently than IL-6 in the presence of IL-2, whereas IL-6 enhanced the responses of CD4+CD8- and CD4-CD8+ single positive (SP) thymocytes to IL-2 or IL-4 better than IL-1 alpha. TNF-alpha enhanced the proliferation of both DN and both SP subsets in the presence of IL-2 and/or IL-7. None of these combinations induced the proliferation of CD4+CD8+ double positive thymocytes. Finally, DN were separated into CD3+ and CD3- populations and their responsiveness was investigated, because recent reports strongly suggest that CD3+ DN thymocytes are a mature subset of different lineage rather than precursors of SP thymocytes. CD3+ DN proliferated in response to IL-7, TNF-alpha + IL-2, and IL-1 + IL-2. CD3- DN did not respond to IL-7 or to IL-1 + IL-2, but did respond to TNF-alpha + IL-2. Finally, we detected TNF-alpha production by a cloned line of thymic macrophages, as well as by DN adult thymocytes. These results suggest that cytokines alone are capable of potent growth stimuli for thymocytes, and indicate that different combinations of these molecules act selectively on thymocytes at different developmental stages.     

Activation requirements of newborn thymic gamma delta T cells.

We have analyzed the requirements for the induction of proliferative responses by thymic CD4-CD8- gamma delta T cells. Enriched populations of CD4-CD8- thymocytes from newborn mice, purified by negative selection with anti-CD4, anti-CD8, and anti-TCR alpha beta mAbs were found to contain approximately 20% gamma delta T cells that were p55IL-2R-. When these cells were cultured with a panel of lymphokines (IL-1, -2, -4, and -7), a small response was observed to some of the cytokines tested individually; however, combinations of certain lymphokines (IL-1 + 2, IL-1 + 7, and IL-2 + 7) were found to induce significant proliferation and the selective outgrowth (75-90%) of gamma delta T cells. These cells were IL-2R+, remained CD4-, yet expressed variable levels of CD8. A limited analysis with specific anti-V gamma and V delta mAb suggested that there had not been a selective expansion of preexisting V gamma 2, V gamma 3, or V delta 4 populations in response to the stimulatory lymphokine combinations. Thymic CD4-CD8- gamma delta T cells were unresponsive to stimulation with immobilized anti-pan gamma delta mAb alone. However, in the presence of immobilized anti-pan gamma delta mAb and IL-1, IL-2, or IL-7, but not IL-4, a vigorous proliferative response was observed. Phenotypic analysis showed that 80 to 95% of the proliferating cells were polyclonally expanded gamma delta T cells, expressed the p55IL-2R, and the majority remained CD4-CD8-. Blocking studies with anti-IL-2R mAb showed that stimulation with anti-pan gamma delta + IL-1, but not anti-pan gamma delta + IL-7 was dependent on endogenously produced IL-2. Collectively, these studies suggest that the activation requirements of newborn thymic gamma delta T cells differ markedly from alpha beta T cells in that gamma delta T cells 1) respond to combinations of cytokines in the absence of TCR cross-linking, 2) can respond to TCR cross-linking in the presence of exogenous cytokines, 3) but are unable to activate endogenous cytokine production solely in the presence of TCR cross-linking.     

Role of CD47 in the induction of human naive T cell anergy

We recently reported that CD47 ligation inhibited IL-2 release by umbilical cord blood mononuclear cells activated in the presence of IL-12, but not IL-4, preventing the induction of IL-12Rbeta(2) expression and the acquisition of Th1, but not the Th2 phenotype. Here we show that in the absence of exogenous cytokine at priming, CD47 ligation of umbilical cord blood mononuclear cells promotes the development of hyporesponsive T cells. Naive cells were treated with CD47 mAb for 3 days, expanded in IL-2 for 9-12 days, and restimulated by CD3 and CD28 coengagement. Effector T cells generated under these conditions were considered to be anergic because they produced a reduced amount of IL-2 at the single-cell level and displayed an impaired capacity 1) to proliferate, 2) to secrete Th1/Th2 cytokines, and 3) to respond to IL-2, IL-4, or IL-12. Moreover, CD47 mAb strongly suppressed IL-2 production and IL-2Ralpha expression in primary cultures and IL-2 response of activated naive T cells. Induction of anergy by CD47 mAb was IL-10 independent, whereas inclusion of IL-2 and IL-4, but not IL-7, at priming fully restored T cell activation. Furthermore, CD28 costimulation prevented induction of anergy. Thus, CD47 may represent a potential target to induce anergy and prevent undesired Th0/Th1 responses such as graft vs host diseases, allograft rejection, or autoimmune diseases.     

Multiple cytokine secretion by IL-7-stimulated human T cells.

The induction of cytokine secretion by human peripheral blood (PB) T cells was examined. Highly purified T cells stimulated with interleukin 7 (IL-7), in the absence of co-mitogen, secreted IL-2, IL-4, IL-6 and interferon gamma (IFN-gamma) upon restimulation with phorbol ester and ionomycin. In contrast, induction of T-cell cultures initiated with IL-2 or IL-4 yielded only low levels of IL-6 and virtually undetectable levels of IL-4 or IFN-gamma, while IL-2 secretion was reduced. No difference was seen in the ability of CD4+ and CD8+ subpopulations, grown in IL-7, to produce cytokines. In contrast, subdivision of T cells into memory and naive populations using the CD45RO monoclonal antibody (mAb) UCHL1, revealed that almost all of the potential to secrete IL-4 and IL-6 in response to IL-7 resided in the CD45RO+ memory population. Stimulation of cytokine-secreting cells appeared to be a direct effect of IL-7 as neutralizing antibodies directed against IL-2 and IL-4 had no effect on the levels of cytokines produced. The differences observed in the ability of IL-2, IL-4, and IL-7 to potentiate cytokine production was supported by measurement of cytokine mRNA levels by PCR. The elevated levels of cytokine secretion seen in cells cultured with IL-7 was not due simply to increased viability in these cultures compared with those containing IL-2 or IL-4, as these populations showed comparable cloning frequencies in phytohemagglutinin (PHA) + IL-2. These results demonstrate that IL-7, in the absence of co-mitogen, is a potent initial stimulus for multiple cytokine production by human T cells upon restimulation.     

In vitro CD4+ lymphocyte transformation and infection in a rabbit model with a molecular clone of human T-cell lymphotrophic virus type 1.

We transfected human and rabbit peripheral blood mononuclear cells (PBMC) with the ACH molecular clone of human T-cell lymphotropic virus type 1 (HTLV-1) to study its in vitro and in vivo properties. PBMC transfected with ACH were shown to transfer infection to naive PBMC. ACH transformed rabbit PBMC, as indicated by interleukin-2-independent proliferation of a transfectant culture. This transformant culture was shown by flow cytometric analysis to be a CD4+ CD25+ T-lymphocyte population containing, as determined by Southern blot analysis, at least three integrated HTLV-1 proviral copies. HTLV-1 infection was produced in rabbits inoculated with ACH-transfected, irradiated PBMC. Inoculated rabbits seroconverted to positivity for antibodies against HTLV-1 and had steady or rising HTLV-1 enzyme-linked immunosorbent assay antibody titers. Western blot (immunoblot) analysis revealed sustained seroconversion of rabbits to positivity for antibodies against all major viral antigenic determinants. Infection of rabbits was further demonstrated by antigen capture assay of p24 in PBMC and lymph node cultures and PCR amplification of proviral sequences from PBMC. These data suggest that ACH, like wild-type HTLV-1, infects and transforms primary CD4+ T lymphocytes and is infectious in vivo. This clone will facilitate investigations into the role of viral genes on biological properties of HTLV-1 in vitro and in vivo.     

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines

Background

Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.

Methodology/Principal Findings

Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.

Conclusions/Significance

The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.     

Modulating the differentiation status of ex vivo-cultured anti-tumor T cells using cytokine cocktails

The genetic modification of CD8+ T cells using anti-tumor T-cell receptors (TCR) or chimeric antigen receptors is a promising approach for the adoptive cell therapy of patients with cancer. We previously developed a simplified method for the clinical-scale generation of central memory-like (Tcm) CD8+ T cells following transduction with lentivirus encoding anti-tumor TCR and culture in the presence of IL-2. In this study, we compared different cytokines or combinations of IL-2, IL-7, IL-12, IL-15, and IL-21 to expand genetically engineered CD8+ T cells. We demonstrated that specific cytokine combinations IL-12 plus IL-7 or IL-21 for 3 days followed by withdrawal of IL-12 yielded the phenotype of CD62L^highCD28^high CD127^highCD27^highCCR7^high, which is associated with less-differentiated T cells. Genes associated with stem cells (SOX2, NANOG, OCT4, and LIN28A), were also up-regulated by this cytokine cocktail. Moreover, the use of IL-12 plus IL-7 or IL-21 yielded CD8 T cells showing enhanced persistence in the NOD/SCID/γc?/? mouse model. This defined cytokine combination could also alter highly differentiated TIL from melanoma patients into cells with a less-differentiated phenotype. The methodology that we developed for generating a less-differentiated anti-tumor CD8+ T cells ex vivo may be ideal for the adoptive immunotherapy of cancer.     

IL-7 induces human lymphokine-activated killer cell activity and is regulated by IL-4.

Induction of lymphokine-activated killer (LAK) activity by IL-2 has been described and characterized as broadly cytolytic activity against both fresh and cultured tumors. rIL-7 in the absence of IL-2 also induces LAK activity in human cells. This activity is unique for IL-7, because it is not shared by other cytokines including IL-1, IL-4, IL-6, and TNF-alpha. IL-7 also induces either de novo or increased expression of the surface markers CD25 (Tac, IL-2R alpha-chain), CD54 (ICAM-1), Mic beta 1 (IL-2R beta-chain) and CD69 (early T cell activation Ag). IL-7-induced LAK activity is independent of IL-2 secretion, because it is not abrogated by IL-2 antisera. The LAK precursor responding to IL-7 stimulation is enriched in the null cell fraction as has been demonstrated for IL-2-induced LAK cells. TGF-beta and IL-4 interfere with generation of LAK activity by IL-7. Anti-IL-4 antiserum enhances IL-7-induced LAK activity and augments induction of surface marker expression by IL-7. This may be indirect evidence that IL-7 stimulation leads to induction of IL-4 activity. Our results describe the activation of mature lymphoid cells by IL-7. This and the previously described role of IL-7 in lymphohemopoiesis makes it a cytokine of potential therapeutic value for treatment of immunodeficiency states and possibly the immunotherapy of cancer.     

IL-33, a potent inducer of adaptive immunity to intestinal nematodes

IL-33 (IL-1F11) binds ST2 (IL-1R4), both of which are associated with optimal CD4(+) Th2 polarization. Exogenous IL-33 drives induction of Th2-associated cytokines and associated pathological changes within the gut mucosa. Th2 polarization is also a prerequisite to expulsion of the intestinal-dwelling nematode Trichuris muris. In this study, we demonstrate that IL-33 mRNA is expressed early during parasite infection and susceptible mice can be induced to expel the parasite by a regime of exogenous IL-33 administration. IL-33 prevents an inappropriate parasite-specific Th1-polarized response and induces IL-4, IL-9, and IL-13. This redirection requires the presence of T cells and must occur at the initiation of the response to the pathogen. Interestingly, exogenous IL-33 also induced thymic stromal lymphopoietin mRNA within the infected caecum, an epithelial cell-restricted cytokine essential for the generation of Th2-driven parasite immunity. IL-33 also acts independently of T cells, altering intestinal pathology in chronically infected SCID mice, leading to an increased crypt length and intestinal epithelial cell proliferation, but reducing goblet cell hyperplasia. Thus, the ability of IL-33 to induce Th2 responses has functional relevance in the context of intestinal helminth infection, particularly during the initiation of the response.     

Induction of cytokines in mice with parainfluenza pneumonia.

The possible involvement of cytokines in the acute viral pneumonia induced by the murine parainfluenza type 1 virus, Sendai virus, was studied. Cytokine profiles for both the respiratory tract and the draining mediastinal lymph node (MLN) of virus-infected C57BL/6J mice were quantified by using the single-cell cytokine (ELISPOT) assay with freshly isolated cell populations and enzyme-linked immunosorbent assay for lung lavage fluids and culture supernatants. Maximal levels of interleukin 2 (IL-2), gamma interferon (IFN-gamma), tumor necrosis factor, IL-6, and IL-10 were detected at the inflammatory site 7 to 10 days after infection, about the time that virus is cleared from the lung. The frequencies of cells producing IL-2, IL-4, IL-6, IL-10, IFN-gamma, and tumor necrosis factor were much higher for the bronchoalveolar lavage (BAL) cell population than for the MLN cell population. Cytokine production after in vitro restimulation of MLN cells was dominated by IL-2 and IFN-gamma, with low levels of IL-10 and IL-6 also being present. Most of the cytokine was produced by the CD4+ cells, although the CD8+ subset was also involved. No IL-4 was found in the BAL fluid or in culture supernatants from restimulated BAL or MLN cells, although a high frequency of IL-4-producing cells was demonstrated in the BAL population by ELISPOT analysis.     

Effect of IL-7 on the growth of fetal thymocytes in culture

The effects of IL-7 on the in vitro growth and differentiation of day 12 to 14 murine fetal thymocytes were examined in three culture systems. In single cell suspension cultures, IL-7 and IL-2 induced a DNA synthetic response in a short term (1 day) assay, but neither cytokine supported continued cell growth. In conventional fetal thymus organ cultures, the addition of exogenous IL-7 resulted in a twofold increase in cell number over that which normally develops in unsupplemented fetal thymus organ cultures during a 7-day period. The most striking effects of IL-7 were noted in lobe submersion cultures (LSC), a system in which thymocyte growth was totally dependent on the addition of exogenous cytokine. Cells proliferated for a period of approximately 2 wk in IL-7, and cell viability could be maintained even longer. A high percentage of cells recovered after 7 to 14 days from IL-7-supplemented LSC resembled the earliest detectable fetal thymocytes with regard to cell surface markers: they expressed Pgp-1, lacked CD4, CD8, and CD3 and many expressed the IL-2R. These results suggest that IL-7 promotes the growth of cells that occur early in the T cell lineage. Cell populations recovered from LSC supplemented with IL-7 and IL-2 exhibited differential expression of some surface markers, particularly CD3 and NK1.1. In addition, cells from LSC supplemented with IL-7 were found to proliferate upon subsequent exposure to IL-2, but cells from LSC containing exogenous IL-2 were no longer responsive to IL-7. These results imply that IL-7 and IL-2 may act at different stages of thymocyte differentiation. Together with previous observations of IL-7-specific mRNA expression in the thymus, this study provides evidence highly suggestive of a pivotal role for IL-7 in T cell development.     

Enhanced proliferation of murine T cell lines following interaction of poly(Glu60,Phe40) (GPhe) and antigen-presenting accessory cells. II. The role of accessory cells and cytokines in the activity of GPhe on antigen-independent proliferation.

Our previous study (1) demonstrated the "cytokine-like" activity of poly(Glu60,Phe40)(GPhe) in augmenting the antigen-dependent proliferation of a variety of long-term murine T cell lines, particularly the bulk, BALB/c anti-poly (Glu36,Lys24,Ala40) (GLA), interleukin-4-producing, DCL-2 T cell line. GPhe was found to also augment the antigen-independent proliferation of DCL-2 in response to exogenous cytokines ([interleukin(IL)-2 +/- IL-1] in most experiments). Such exogenous cytokine-driven proliferative responses of DCL-2 were used to investigate further the role of accessory cells and of various soluble factors in the action of GPhe. GPhe did not act as a direct mitogen for T cells, rather it acted in a costimulatory fashion, requiring the presence of plastic-adherent accessory cells and a T cell growth factor (either IL-2 or IL-4). In the presence of accessory cells and exogenous IL-2, augmentation of antigen-independent DCL-2 proliferation by GPhe or by IL-1 depended upon the induction of autocrine IL-4 production. However, GPhe also augmented the response of these cells in the presence of exogenous IL-4 (+/- IL-2, +/- IL-1), and exogenous IL-4 added in combination with exogenous IL-2 (+/- IL-1) failed to mimic the GPhe effect, suggesting that another signal was involved in the mechanism of action of GPhe. The ability of allogeneic accessory cells to interact with GPhe to augment proliferative responses suggested that either a soluble factor or an unusual non-MHC-restricted cell-cell interaction provided this signal. In the presence of uv-irradiated accessory cells, DCL-2 proliferation was enhanced over that observed in the presence of non-uv-treated accessory cells, mimicking the GPhe effect, and interaction of GPhe with uv-irradiated accessory cells did not result in further enhancement of DCL-2 cytokine-driven proliferation. Using monoclonal antibodies which could block the function of IA or CD4 molecules, these cell-surface "adhesion" molecules were shown not to participate in the activity of GPhe. By the addition of recombinant cytokines, neutralizing antibodies, or indomethacin, the mechanism of action of GPhe was also shown not to be dependent upon IL-1, IL-6, IL-7, TNF alpha, or prostaglandin production by accessory cells. However, the presence, individually, of some of these factors (IL-1, IL-7, or prostaglandins) could influence to a variable degree the magnitude of the GPhe effect.     

Cytokine detection by multiplex technology useful for assessing antigen specific cytokine profiles and kinetics in whole blood cultured up to seven days

Cytokine profile assessment is important to characterize immune responses to pathogens. To identify optimal time points for determination of cytokine profiles, we diluted whole blood 1:10, to enable daily cytokine measurements during one week. Cultures for 10 blood donors were set up in the presence of phytohemagglutinin (PHA), cytomegalovirus (CMV) or Candida. Supernatant levels of interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, interferon-gamma (IFN-gamma), granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha), were determined by multiplex technique, and intracellular cytokine staining (ICS) was employed to detect IFN-gamma, IL-2, IL-4 and IL-13 in CD3+ cells. The multiplex analysis detected representative cytokine profiles for the majority of the cytokines on day 7 by identifying peak levels or good correlation with peak levels, with the exception of IL-2 and TNF-alpha in PHA and CMV cultures and IL-10 in PHA cultures. For these cytokines an extracellular measurement on day 2-3 would be appropriate. The intracellular cytokines showed distinct kinetics for IFN-gamma and IL-2, while IL-4 and IL-13 were not detected at all with ICS. In conclusion, the combination of whole blood cultures with multiplex analysis is a simple and powerful tool that can be used to identify detailed cytokine profiles of specific cell-mediated immune responses.     

Human acute lymphoblastic leukemia (ALL) blasts as accessory cells during T-cell activation: differences between patients in costimulatory capacity affect proliferative responsiveness and cytokine release by activated T cells

The ability of acute lymphoblastic leukemia (ALL) blasts to mediate costimulatory signals during T-lymphocyte activation was investigated in an experimental model in which monoclonal T-cell populations were stimulated with standardized activation signals (anti-CD3 and anti-CD28 monoclonal antibodies; phytohemagglutinin, PHA). Leukemia cells from 12 consecutive ALL patients with high peripheral blood blast counts were studied. Proliferative T-cell responses were detected for a majority of these patients when irradiated leukemia blasts were used as accessory cells during activation. T-cell cytokine release was also observed for most patients when using nonirradiated ALL accessory cells. Low or undetectable cytokine levels were usually observed for CD8+ clones, whereas the CD4+ clones often showed a broad cytokine response with release of interleukin-2 (IL-2), IL-4, IL-10, IL-13 and interferon gamma(IFN-gamma) in the presence of the ALL accessory cells. ALL blasts were also able to function as allostimulatory cells for normal peripheral blood mononuclear responder cells. However, both T-cell proliferation and cytokine release showed a wide variation between ALL patients. The accessory cell function of ALL blasts showed no correlation with the release of immunomodulatory mediators (IL-2, IL-10, IL-15) or the expression of any single adhesion/costimulatory membrane molecule (CD54, CD58, CD80, CD86) by the blasts. We conclude that for a majority of patients, native ALL blasts can mediate costimulatory signals needed for accessory cell-dependent T-cell activation, but differences in costimulatory capacity between ALL patients affects both the proliferative responsiveness and cytokine release by activated T cells.