Antigen specific and MHC nonrestricted cytotoxicity of T cell receptor alpha beta+ and gamma delta+ human T cell clones isolated in IL-4

IL-4 has been shown to act as a growth factor for human T cells. In addition, IL-4 can enhance CTL activity in MLC, but blocks IL-2 induced lymphokine activated killer cell activity in PBL. In our study, the cloning efficiencies, Ag-specific CTL activity and non-MHC-restricted cytotoxicity of CTL clones generated in IL-2 were compared to those generated in IL-4. In a first experiment, T cells were stimulated with the EBV-transformed B cell line JY and cloned 7 days later with feeder cells and either IL-2 or IL-4. In a second experiment, stimulation of the T cells was carried out in the presence of IL-2 plus anti-IL-4 antibodies or IL-4 plus anti-IL-2 antibodies in order to block the effects of IL-4 and IL-2, respectively, produced by the feeder cells. Although the cloning efficiencies in the second experiment were lower than those obtained in the first experiment, the cloning efficiencies obtained with IL-2 or IL-4 were similar in both experiments. The overall proportion of TCR alpha beta+ T cell clones cytotoxic for the stimulator cell JY established in IL-2 or IL-4 were comparable. A striking difference between the clones obtained in IL-2 or IL-4 was that a large proportion of the clones obtained in IL-4 expressed CD4 and CD8 simultaneously, whereas none of the clones isolated in IL-2 were double positive. Also gamma delta+ T cell clones could be established with IL-4 as a growth factor. TCR gamma delta+ T cell clones isolated in either IL-2 or IL-4 were CD4-CD8- or CD4-CD8+, but the proportion of CD4-CD8+ clones isolated in IL-4 was higher. Interestingly, one TCR gamma delta+ clone isolated in IL-2 was CD4+CD8-. Most of the TCR alpha beta+ and TCR gamma delta+ CTL-clones isolated in IL-2 lysed the NK cell sensitive target cell K562. In contrast, only a small proportion of the TCR alpha beta+ or TCR gamma delta+ CTL clones isolated in IL-4, lysed K562. One TCR gamma delta+ T cell clone (CD-124) isolated in IL-4 and subsequently incubated in IL-2 acquired lytic activity against K562.(ABSTRACT TRUNCATED AT 400 WORDS)     

IL-2 and IFN-gamma are two necessary lymphokines in the development of cytolytic T cells

A filler cell-free limiting-dilution microculture system has been developed for the expansion and differentiation of a high proportion of single CD4-CD8+ T cells into cytolytic T cell (CTL) clones. The stimulus used was PMA together with the calcium ionophore ionomycin. The growth and differentiation factors were rIL-2, together with either a Con A-stimulated spleen cell supernatant (CAS) or rIFN-gamma. CTL activity was monitored by an autoradiographic 111In-release assay. With CAS and rIL-2 present, 50% of all potential precursors (CTL-p) produced cytolytic clones. Substitution of rIFN-gamma for CAS gave a similar efficiency with up to 42% of CTL-p producing cytolytic clones. rIL-2 alone allowed only a small proportion (6%) of CD4-CD8+ T cells to become cytolytic clones. Addition of rIL-2 and rIFN-gamma at various stages of the culture demonstrated that IL-2 was required throughout, but exogenous IFN-gamma was required only during the early stages. It is concluded that for at least 40% of all CTL-p, the lymphokines IL-2 and IFN-gamma are essential and act in synergy to induce proliferation and differentiation into CTL.     

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12

We investigated the effects of IL-18 on the development of CD8+ effector T cells in DBA/2 anti-BDF1 whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8+ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN-gamma after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF1 MLC induced type I CD8+ effector T cells when purified CD8+ T cells were used as responder cells in primary MLC. Furthermore, CD4+ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN-gamma production by CD8+ T cells, suggesting that IL-18-induced type I effector CD8+ T cell development was CD4+ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN-gamma production and CTL activity by CD8+ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. These findings show that IL-18 plays an important role in the generation of type I CD8+ effector T cells, and further suggest that functional maturation of CD8+ T cells is differentially regulated by IL-18 and IL-12.     

Progressive differentiation and commitment of CD8+ T cells to a poorly cytolytic CD8low phenotype in the presence of IL-4

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4-CD8- cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.     

IL-10: a novel cytotoxic T cell differentiation factor

A previous report concluded that a new cytokine, designated IL-10, is a growth cofactor for thymocytes, spleen, and lymph node cells. In this report, we have focused on the effects of IL-10 on CD8+ spleen T cells. We first observed that IL-10 enhances the growth of CD8+ T cells to IL-2. We then investigated the effect of murine rIL-10 on the induction of murine effector CTL from CTL precursors (CTL-p) using both bulk and filler cell-free limiting-dilution cultures. IL-10 alone could not induce Con A-activated FACS-sorted CD8+ T cells either to proliferate or to generate effector CTL. In combination with IL-2, however, IL-10 augmented the cytolytic activity of effector CTL generated from Con A-activated spleen CD8+ T cells in bulk cultures incubated for 5 days. In limiting-dilution cultures (using solid-phase anti-CD3 mAb as stimulus), IL-10, in combination with IL-2, substantially increased the CTL-p frequency and augmented the cytolytic activity per clone expanded from one CD8+ T cell when compared with cells cultured in IL-2 alone. Kinetic studies showed that IL-10 is required at both early and late culture stages for optimal generation of effector CTL. The potentiating effects of IL-10 on CTL function were neutralized by an anti-IL-10 mAb. These results indicate that IL-10 has direct effects on mature T cells, and suggest that IL-10 also functions as a cytotoxic T cell differentiation factor, which promotes a higher number of IL-2-activated CTL-p to proliferate and differentiate into effector CTL. In contrast, IL-10 did not enhance significantly the lymphokine-activated killer cell activity of IL-2-grown CD8+ cytotoxic T cells.     

IL-2 in vivo activities and antitumor efficacy enhanced by an anti-IL-2 mAb

IL-2 is a potent immunostimulant and has been tested for clinical use, including in immunotherapy for cancers and HIV infection. Here we show that a widely used neutralizing anti-murine IL-2 mAb (S4B6) exhibits unexpected activities that enhance the treatment effects of IL-2 in vivo. Coinjection of the anti-IL-2 mAb with a plasmid carrying murine IL-2 cDNA significantly increased the serum IL-2 levels and induced a substantial increase in the division of CD8+ T and NK1.1(high) cells in vivo. Injection of the mAb premixed with recombinant murine IL-2 showed the same enhanced effect. A 5-day treatment with the anti-IL-2 mAb alone gradually increased the CD44(high)CD8+ population, and the increased population was maintained for >300 days, suggesting that the mAb can gradually maintain and potentially enhance the bioactivity of endogenous IL-2 for extended periods. Furthermore, combined treatment with the anti-IL-2 mAb plus the IL-2 plasmid markedly enhanced Ag-specific CTL activity in vivo and partially protected mice from tumor metastasis to the lungs, compared with the anti-IL-2 mAb or IL-2 plasmid alone. These results demonstrated IL-2-enhancing effects of the anti-IL-2 mAb in vivo and suggest that combining a neutralizing anti-IL-2 Ab with IL-2 gene delivery might be used effectively to enhance IL-2 functions in clinical applications.     

Treatment of fetal thymic organ culture with IL-1 leads to accelerated differentiation of subsets of CD4-CD8- cells.

Using fetal thymic organ culture (FTOC), we describe the effects of IL-1 on T cell differentiation, particularly within the CD4-CD8- subset. While treatment of FTOC with IL-1 led to a modest reduction in total thymocyte yield, it induced an increase in the percentage of CD4-CD8- cells that express IL-2R early in culture and a decrease in the number of their precursors (CD44+IL-2R- cells). The increase in the percentage of cells expressing IL-2R was not accompanied by an increase in the number of these cells. At later time points these IL-2R+ cells (and their precursors) were reduced relative to controls. The total number of CD4-CD8-CD3- precursor cells in IL-1-treated cultures was reduced to approximately half that in controls at Day 12 of culture. However, only minor inhibition of total cell number was observed, which, taken together with the greater frequency of IL-2R+ precursors, suggests that this depletion of the pool of precursors may have been due to the induction of premature differentiation rather than to its inhibition.     

Age-associated rapid and Stat6-independent IL-4 production by NK1-CD4+8- thymus T lymphocytes.

The source of IL-4 required for priming naive T cells into IL-4-secreting effectors has not been clearly identified. Here we show that upon TCR stimulation, thymus NK1-CD4+8- T cells produced IL-4, the magnitude of which was inversely correlated with age. This IL-4 production response by Th2-prone BALB/c mice was approximately 9-fold that of Th1-prone C57BL/10 mice. More than 90% of activated NK1-CD4+8- thymocytes did not use the invariant V alpha 14-J alpha 281 chain characteristic of typical CD1-restricted NK1+CD4+ T cells. Stat6-null NK1-CD4+8- thymocytes produced bioactive IL-4, with induction of IL-4 mRNA expression within 1 h of stimulation. Our results support the possibility that TCR repertoire-diverse conventional NK1-CD4+ T cells are a potential IL-4 source for directing naive T cells toward Th2/type 2 CD8+ T cell (Tc2) effector development.     

Regulation of human cytotoxic T lymphocyte development by IL-7

The effects of IL-7 on the generation of human CTL in alloantigen-, virus-, and lectin-stimulated systems were examined. Addition of IL-7 at the onset of cultures resulted in marked (up to 80-fold) augmentation of cytotoxicity accompanied by smaller (1.5- to 4-fold) increases in total lymphocyte number. Studies of CTL development in purified lectin-stimulated CD8+ T cell populations demonstrated that IL-7 could act directly on the CD8+ lymphocyte subset to augment cytotoxicity. In MLC, the IL-7-induced enhancement of cytotoxicity was found to be mediated primarily by the CD8+ subpopulation of lymphocytes. Late addition of IL-7 (day 5 of 7) resulted in an increase in cytolytic activity that was associated with little or no increase in total or activated CD8+ lymphocyte number indicating that IL-7 may act as a differentiation factor for human CTL. A role for endogenous IL-7 in CTL development was suggested by the observation that addition of neutralizing antiserum to IL-7 to MLC at initiation (or 5 days thereafter) resulted in decreased levels of cytotoxicity. These results indicate that IL-7 can exert major up-regulatory effects on human CTL development and suggest that these effects are both proliferative and differentiative.     

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.     

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.     

Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. I. Differential helper cell requirement for the generation of cytotoxic effector cells from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Previous studies have shown that both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells capable of developing into alloreactive CTL. In the current study, we have examined the mechanisms involved in the generation of CTL effector cells from these two CD8+ subsets. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic non-T cells, either alone or in the presence of CD4+ Th cells. Although the generation of CTL from CD8+CD45RA- precursor cells consistently required the presence of CD4+ Th cells, cytotoxic effector cells could be generated from CD8+CD45RA+ precursor cells in the absence of CD4+ cells. Several lines of evidence indicated that the helper cell-independent generation of cytotoxic effector cells from CD8+CD45RA+ precursors resulted from the unique ability of this subset to produce and use IL-2 in an autocrine fashion: 1) exogenous IL-2 could replace the effects of CD4+ helper cells for either CD8+ subset; 2) the helper cell-independent functional maturation of CD8+CD45RA+ cells could be blocked by anti-CD25 or anti-IL-2 antibodies; and 3) CD8+CD45RA+ cells produced IL-2 after activation with allogeneic cells. The finding that precursors for helper cell-independent CTL generation are restricted to the CD8+CD45RA+ subset suggests that this capability may vary as a function of the maturation of CD8+ cells.     

Enhanced cytotoxic T cell activity in IL-4-deficient mice

CD8+ effectors are critical components of type 1 responses against viral infections as well as for antiviral vaccines. IL-4 plays a clear role as an inhibitor of CD4+ Th1 cells; however, its role in CD8+ T cell regulation appears to be more complex. Thus, IL-4 may augment CD8+ T cell growth, but also limit effector function. Moreover, abundant IL-4 is inhibitory for viral clearance, but the lack of IL-4 appears not to affect CTL-mediated immunity. This report investigates these disparate roles of IL-4 in CD8+ T lymphocyte regulation by comparing T cell responses specific for a single HIV-IIIIB gp120-derived epitope in BALB/c mice deficient in IL-4 to those in wild-type controls. CTL activation was monitored during the acute and memory phases following immunization with recombinant vaccinia virus. Similar frequencies of gp120-specific CTL precursors in splenocytes from both groups indicated that IL-4 plays no significant role in either CTL priming or the establishment of memory. However, cytolytic activity in cultures derived from IL-4-deficient mice developed earlier and was strikingly enhanced following in vitro restimulation, an effect exhibited by both primary and memory T cells. Secretion of IL-2 and IFN-gamma by CD8+ T cells from IL-4-deficient mice was also elevated, reflecting their enhanced activation. Thus, IL-4 appears to limit the activation, expansion, and differentiation of CD8+ T cells with high cytolytic potential.     

Expression of murine IL-2 receptor beta-chain on thymic and splenic lymphocyte subpopulations as revealed by the IL-2-induced proliferative response in human IL-2 receptor alpha-chain transgenic mice

Lymphocytes from the human (h) IL-2R alpha chain transgenic mice (TGM) constitutively express high affinity binding sites for hIL-2, consisting of transgenic h-IL-2R alpha and endogenous murine IL-2R beta, and therefore easily proliferate in vitro in response to hIL-2. Our study was undertaken to clarify the hIL-2-responsive lymphocyte subsets in the TGM, which should most likely reflect the normal distribution of m IL-2R beta expression. In both thymus and spleen, the majority of expanded cells by hIL-2 was CD3+CD4-CD8+ TCR alpha beta+ cells. The proliferation of CD4+ cells was not observed at all from either organ despite the expression of transgenic hIL-2R alpha. Potent cellular proliferation was also observed from the thymocytes that had been depleted of CD8+ cells, the expanded cells consisting of CD3- (15-40%) and CD3+ populations (60-85%). Among CD3+ cells, approximately the half portion expressed TCR alpha beta, whereas the other half was suggested to express TCR gamma delta. A variable portion (5-20%) of the CD3+ cells expressed CD8 (Lyt-2) in the absence of Lyt-3, and the CD3+CD8+ cells were confined preferentially to the TCR alpha beta- (TCR gamma delta+) population. In the culture of splenocytes depleted of CD8+ cells, however, the proliferated cells were mostly CD3-CD4-CD8-TCR-Mac1-, whereas a minor portion (10-30%) was CD3+CD4-CD8-TCR alpha beta- (TCR gamma delta+. Analysis of TCR genes at both DNA and mRNA levels confirmed the phenotypical observations. These results strongly suggested that IL-2R beta was constitutively and selectively expressed on the primary murine thymocytes and splenic T and NK cells, except for CD4+ cells in both organs.     

Activation of CD 4-, CD 8- thymocytes with IL 4 vs IL 1 + IL 2

Thymocytes from C57BL/6 mice were highly purified to obtain the CD 4-, CD 8- subpopulation which constitutes only 5% of all thymocytes. Substantial proliferation was induced in vitro with either IL-1 + IL-2 or with IL-4 in the presence of PMA. IL-1 and IL-2 synergized in inducing proliferation of these purified CD 4-, CD 8- thymocytes whereas neither synergized with IL-4. In order to determine whether stimulation with IL-1 + IL-2 acted via IL-4 or vice versa, cultures were treated reciprocally with affinity-purified anti-IL-2 or anti-IL-4 antibodies. Cultures with IL-4 were inhibited by anti-IL-4 but were unaffected by anti-IL-2. The CD 4-, CD 8- thymocytes cultured with IL-1 + IL-2 + anti-IL-2 were inhibited to baseline IL-1 stimulation. At low concentrations of IL-1 (1 U/ml) and IL-2 (100 U/ml), anti-IL-4 had no effect, whereas at higher levels of IL-1 (2 U/ml IL-1), and 100 or 200 U/ml IL-2, anti-IL-4 significantly reduced DNA synthesis. This result suggests that at higher concentrations the combination of IL-1 + IL-2 can induce cells to produce IL-4 which then contributes to overall proliferation. When CD 4-, CD 8- thymocytes were cultured with the low doses of IL-1 + IL-2 for 72 h, 62% expressed cell surface T3 complex (vs 11% at initiation) and 27% were F23.1+ (vs 5% at initiation). In contrast, culture with IL-4 led to no increase in numbers of T3+ cells and none were F23.1+; however, there was coexpression of Thy1 and 6B2 on 20% of cells at the end of culture (vs 4% at initiation). Thus, IL-1 + IL-2 causes expansion of a CD 4-, CD 8- thymocyte population expressing the alpha, beta-T cell receptor, whereas IL-4 induces cells to express a phenotype present in small numbers in the periphery of normal mice and in larger numbers in mice bearing the lpr gene.     

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.     

CD1d-independent developmental acquisition of prompt IL-4 gene inducibility in thymus CD161(NK1)-CD44lowCD4+CD8- T cells is associated with complementarity determining region 3-diverse and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2 T cell receptor usage

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.     

IL-12 drives IFN-gamma-dependent autoimmune kidney disease in MRL-Fas(lpr) mice

IL-12 is secreted by kidney tubular epithelial cells in autoimmune MRL-Fas(lpr) mice before renal injury and increases with advancing disease. Because IL-12 is a potent inducer of IFN-gamma, the purpose of this study was to determine whether local provision of IL-12 elicits IFN-gamma-secreting T cells within the kidney, which, in turn, incites injury in MRL-Fas(lpr) mice. We used an ex vivo retroviral gene transfer strategy to construct IL-12-secreting MRL-Fas(lpr) tubular epithelial cells (IL-12 "carrier cells"), which were implanted under the kidney capsule of MRL-Fas(lpr) mice before renal disease for a sustained period (28 days). IL-12 "carrier cells" generated intrarenal and systemic IL-12. IL-12 fostered a marked, well-demarcated accumulation of CD4, CD8, and double negative (CD4-CD8- B220+) T cells adjacent to the implant site. We detected more IFN-gamma-producing T cells (CD4 > CD8 > CD4-CD8- B220+) at 28 days (73 +/- 14%) as compared with 7 days (20 +/- 8%) after implanting the IL-12 "carrier cells;" the majority of these cells were proliferating (60-70%). By comparison, an increase in systemic IL-12 resulted in a diffuse acceleration of pathology in the contralateral (unimplanted) kidney. IFN-gamma was required for IL-12-incited renal injury, because IL-12 "carrier cells" failed to elicit injury in MRL-Fas(lpr) kidneys genetically deficient in IFN-gamma receptors. Furthermore, IFN-gamma "carrier cells" elicited kidney injury in wild-type MRL-Fas(lpr) mice. Taken together, IL-12 elicits autoimmune injury by fostering the accumulation of IFN-gamma-secreting CD4, CD8, and CD4-CD8- B220+ T cells within the kidney, which, in turn, promote a cascade of events culminating in autoimmune kidney disease in MRL-Fas(lpr) mice.     

CD8+ T cells can be primed in vitro to produce IL-4.

IL-4 production by T lymphocytes from naive mice in response to stimulation by plate-bound anti-CD3 is concentrated among CD4+ T cells. In vitro stimulation of lymph node T cells with anti-CD3 plus IL-2 and IL-4 strikingly increases the frequency of cells that produce IL-4 in response to subsequent stimulation with anti-CD3 plus IL-2. Separation of these primed cell populations into CD4+ and CD8+ T cell by cell sorting reveals that the frequency of IL-4-producing cells in both population is similar. Verification that CD8+ T cells produce IL-4 is provided by the capacity of anti-IL-4 mAb to inhibit the response of the indicator cell line to the growth factor produced by the primed cells and by detection of IL-4 by an IL-4-specific ELISA. The in vitro "priming" of CD8+ T cells to produce IL-4 is not dependent on the presence of CD4+ T cells because highly purified CD8+ T cells can be stimulated to develop into cells capable of producing IL-4 by culture with plate-bound anti-CD3 plus IL-2 and IL-4.