Scand. J. Immunol. 36, 453-462, 1992
Qualitative Shift of Lymphokine Production in Response to Stimulation, as a Consequence of Preactivation In Vivo or In Vitro S, C A R D E L L , B. SANDER & G, MOLLER Department of Immunology, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden
Cardeil S, Sander B, Moller G, Qualitative Shift of Lymphokine Produclion in Response to Slimtthition, as a Consequence of Preactivation In Vivo or In Vilro, Sciind J Immunol 1992;36:453-62 Lymphokine produetion, analysed at the single cell level, was compared in reslmg and primed T-cell populations. Cells were preactivated in vitro by repealed milogen stimulalions, or isolated as large, low density ceils naturally activated in vivo, from normal spleens of unimmunized animals, A similar qualitative shift in the patlern of lymphokines synthesized after reslimulation was found as a resull of in vivo and in vitro preactivation of cells. Repealed slimulations in vilro resulted in a qualitative shift in the lymphokines produced in response to activation, from a dominance of I L-2 during the ftrst and second culture, lo a dominance of 1 L-4 and IL-5 in the later slimulations. In vivo activation lead to a similar separation of lymphokine produclion as primarily IL-2 was made by small resting cells, while large cells preferentially produced IL-4 and IL-5, IFN--/was produced by bolh small and large eells. Preaetivaiion in vitro lead lo a more rapid appearance of lymphokines dunng reslimulalion. In contrast, the in vivo naturally aclivated cells responded with a slow onset of lymphokine production when stimulated in vitro. S. Cardetl. Department of Ininmnohf-y, Arrhenius Laboratories for Natural Sciences, Stockholm tJniversitv, S-I06 91 Stockholm, Sweden
CD4 * T cells in adult animals consist of preactivated cells as well as resting cells. The two populations can be distinguished by the differential expression of a number of surface markers (reviewed in Refs I and 2), Naive and preactivated cells are also functionally different (reviewed in Refs I and 2). Studies of freshly isolated CD4+ T cells from different species revealed a subpopulation of CD4* T cells that gave help to B cells [3-8]. different from those producing IL-2. or mediating GVH reactivity, and from eells inducing suppressor activity. The phenotype of ihc helper CD4* T cells suggested that B-cell help was performed by the memory, or preactivated. subset of CD4* Tcells. and in vitro priming of naive human CD4' T eells induced the memory phenotype as well as helper function [9]. Approximately 10% ofthe lymphocytes in normal adult mice are large. These cells are thought to represent cells that have been activated in vivo [5], and can be separated on the basis of their low
density. Large CD4' T cells are enriched for effector cells expressing helper function [5], Differences in function, like help to B cells, would partly be mediated by the secretion of specific sets oflymphokines (reviewed in Ref. 10), Acquisition ofthe ability to produce a newset of lymphokines may be a part ofthe T-cell differentiation during activation [11-22]. leading to functional maturation. Such functional maturation of CD4^ T cells, detected as a qualitative change in lymphokine production, may contribute to the more eHicieni and rapid character of a secondary response. The present studies were initiated to investigate differences in lymphokine production by resting Tcells.andceltspreactivatedin vivoorin vitro, at the single cell level. Frequencies of lymphokineproducing cells were determined using in situ hybridization for the analysis of mRNA, and an immunofluorescence method to detect intracellular cytokines [23], Earlier experiments had shown 453
454
S. Cardeil ct al.
that one restimulation of spleen cells with Con A and PMA, 5 days after a first activation with Con A. did not result in a qualitative shift of the lymphokincs produced, but rather in an increase of IL-2 as well as of IL-4 and IL-5 [22], In the experiments of the present report iymphokine production was therefore followed during repeated restimulations with mitogen in vitro. To study the consequences of preactivation in vivo, we separated cells according to density, and after stimulation in vitro compared lymphokine production in small resting T cells, or CD4 ' T cells, with that of large, in vivo activated, cells. The results demonstrate a similar qualitative change in the pattern of lymphokines produced in response to stimuli as a consequence of in vitro or in vivo prestimulation, and suggest a need for collaboration between naive and preactivated cells for efficient memory responses.
M A T E R I A L S AND METHODS Mice and reagents. (CBA x C57BL/6)FI hybrid or BALB/c mice were maintained in our own animal colony and used at the age of 6-12 weeks. Con A was purchased from Pharmacia (Uppsala. Sweden) and phorbol 12-myristate 13-acetate (PMA) from Sigma Chemical Co, (Si Louis, MO), Supernatant from the transfecied X63-Ag8,653 variant cell line producing IL2 ([24], a gift from Dr F, Melchers. Basel Inslitute of Immunology. Basel. Switzerland) was used as a source of recombinanl IL-2 (rIL-2) al a concentration ihat corresponded to approximately 20 U/ml of IL-2 (titrated in the CTLL assay [25] as described [26]), Preparation of cells. Spleen eells were prepared and washed in Earle's BSS (Gibeo. Paisley. UK), T cells were enriched for by depicting spleen cells of Ig+ cells by cell affinity chromatography as described previously [27], The recovered cells contained Xt) 85''4, T eells. and less than 1% Ig' eells, C D 4 ' cells were purified by depleting spleen cells of CDH ' cells using an anli-CD8 antibody (3,155. [28]) and low-iox rabbit complement (Buxted Rabbii Complement Ltd. UK), before the antiIg eell affinity chromatography. The remaining population contained 65 70% CD4^ cells, while CDS* cells were undetectable. For FCM analysis Ihe ceils were stained with PE-conjugated anli-CD4 and FITC-coupled anti-CD8 (Becton-Dickinson. Mountain View. CA. USA) and analy,sed on a FACScan flow eylofluorometer {Beeton-Dickinson), Cells were separated according to density on a discontinuous Percoll gradient (Pharmacia. Uppsala. Sweden) centrifuged at 1000,? for 25 mm as described [5. 29], Spleen cells and T cells were separated into three ditTerent interphases. The intermediate density fraction of cells was discarded, while the high (spleen eells: p > 1,091 g/ml. T cells;
p > 1,085 g/ml) and low (1,060 < ^ x 1,070 g/ml) density fractions were used for experiments, CD4 eells were separated into two fractions only (high density p> 1,078. and low density cells \.i}59
Qualitative Shift of Lymphokine Production in Response to Stimulation, as a Consequence of Preactivation In Vivo or In Vitro S, C A R D E L L , B. SANDER & G, MOLLER Department of Immunology, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden
Cardeil S, Sander B, Moller G, Qualitative Shift of Lymphokine Produclion in Response to Slimtthition, as a Consequence of Preactivation In Vivo or In Vilro, Sciind J Immunol 1992;36:453-62 Lymphokine produetion, analysed at the single cell level, was compared in reslmg and primed T-cell populations. Cells were preactivated in vitro by repealed milogen stimulalions, or isolated as large, low density ceils naturally activated in vivo, from normal spleens of unimmunized animals, A similar qualitative shift in the patlern of lymphokines synthesized after reslimulation was found as a resull of in vivo and in vitro preactivation of cells. Repealed slimulations in vilro resulted in a qualitative shift in the lymphokines produced in response to activation, from a dominance of I L-2 during the ftrst and second culture, lo a dominance of 1 L-4 and IL-5 in the later slimulations. In vivo activation lead to a similar separation of lymphokine produclion as primarily IL-2 was made by small resting cells, while large cells preferentially produced IL-4 and IL-5, IFN--/was produced by bolh small and large eells. Preaetivaiion in vitro lead lo a more rapid appearance of lymphokines dunng reslimulalion. In contrast, the in vivo naturally aclivated cells responded with a slow onset of lymphokine production when stimulated in vitro. S. Cardetl. Department of Ininmnohf-y, Arrhenius Laboratories for Natural Sciences, Stockholm tJniversitv, S-I06 91 Stockholm, Sweden
CD4 * T cells in adult animals consist of preactivated cells as well as resting cells. The two populations can be distinguished by the differential expression of a number of surface markers (reviewed in Refs I and 2), Naive and preactivated cells are also functionally different (reviewed in Refs I and 2). Studies of freshly isolated CD4+ T cells from different species revealed a subpopulation of CD4* T cells that gave help to B cells [3-8]. different from those producing IL-2. or mediating GVH reactivity, and from eells inducing suppressor activity. The phenotype of ihc helper CD4* T cells suggested that B-cell help was performed by the memory, or preactivated. subset of CD4* Tcells. and in vitro priming of naive human CD4' T eells induced the memory phenotype as well as helper function [9]. Approximately 10% ofthe lymphocytes in normal adult mice are large. These cells are thought to represent cells that have been activated in vivo [5], and can be separated on the basis of their low
density. Large CD4' T cells are enriched for effector cells expressing helper function [5], Differences in function, like help to B cells, would partly be mediated by the secretion of specific sets oflymphokines (reviewed in Ref. 10), Acquisition ofthe ability to produce a newset of lymphokines may be a part ofthe T-cell differentiation during activation [11-22]. leading to functional maturation. Such functional maturation of CD4^ T cells, detected as a qualitative change in lymphokine production, may contribute to the more eHicieni and rapid character of a secondary response. The present studies were initiated to investigate differences in lymphokine production by resting Tcells.andceltspreactivatedin vivoorin vitro, at the single cell level. Frequencies of lymphokineproducing cells were determined using in situ hybridization for the analysis of mRNA, and an immunofluorescence method to detect intracellular cytokines [23], Earlier experiments had shown 453
454
S. Cardeil ct al.
that one restimulation of spleen cells with Con A and PMA, 5 days after a first activation with Con A. did not result in a qualitative shift of the lymphokincs produced, but rather in an increase of IL-2 as well as of IL-4 and IL-5 [22], In the experiments of the present report iymphokine production was therefore followed during repeated restimulations with mitogen in vitro. To study the consequences of preactivation in vivo, we separated cells according to density, and after stimulation in vitro compared lymphokine production in small resting T cells, or CD4 ' T cells, with that of large, in vivo activated, cells. The results demonstrate a similar qualitative change in the pattern of lymphokines produced in response to stimuli as a consequence of in vitro or in vivo prestimulation, and suggest a need for collaboration between naive and preactivated cells for efficient memory responses.
M A T E R I A L S AND METHODS Mice and reagents. (CBA x C57BL/6)FI hybrid or BALB/c mice were maintained in our own animal colony and used at the age of 6-12 weeks. Con A was purchased from Pharmacia (Uppsala. Sweden) and phorbol 12-myristate 13-acetate (PMA) from Sigma Chemical Co, (Si Louis, MO), Supernatant from the transfecied X63-Ag8,653 variant cell line producing IL2 ([24], a gift from Dr F, Melchers. Basel Inslitute of Immunology. Basel. Switzerland) was used as a source of recombinanl IL-2 (rIL-2) al a concentration ihat corresponded to approximately 20 U/ml of IL-2 (titrated in the CTLL assay [25] as described [26]), Preparation of cells. Spleen eells were prepared and washed in Earle's BSS (Gibeo. Paisley. UK), T cells were enriched for by depicting spleen cells of Ig+ cells by cell affinity chromatography as described previously [27], The recovered cells contained Xt) 85''4, T eells. and less than 1% Ig' eells, C D 4 ' cells were purified by depleting spleen cells of CDH ' cells using an anli-CD8 antibody (3,155. [28]) and low-iox rabbit complement (Buxted Rabbii Complement Ltd. UK), before the antiIg eell affinity chromatography. The remaining population contained 65 70% CD4^ cells, while CDS* cells were undetectable. For FCM analysis Ihe ceils were stained with PE-conjugated anli-CD4 and FITC-coupled anti-CD8 (Becton-Dickinson. Mountain View. CA. USA) and analy,sed on a FACScan flow eylofluorometer {Beeton-Dickinson), Cells were separated according to density on a discontinuous Percoll gradient (Pharmacia. Uppsala. Sweden) centrifuged at 1000,? for 25 mm as described [5. 29], Spleen cells and T cells were separated into three ditTerent interphases. The intermediate density fraction of cells was discarded, while the high (spleen eells: p > 1,091 g/ml. T cells;
p > 1,085 g/ml) and low (1,060 < ^ x 1,070 g/ml) density fractions were used for experiments, CD4 eells were separated into two fractions only (high density p> 1,078. and low density cells \.i}59
