Feb 5, 1997 - Michael Croft,2 Sean B. and Kent. T. Miner. Tolerance is thought to occur when Ag is presented to T cells in the absence of costimulatory ...
Partial Activation of Naive CD4 T cells and Tolerance Induction in Response to Peptide Presented by Resting 6 Cells' Michael Croft,2 Sean B.
and Kent
T. Miner
Tolerance is thought to occur when Ag is presented to T cells in the absence of costimulatory interactions from APC accessory molecules. O f the professional APC, the resting B cell may bethe main tolerizing cell in vivo. We have analyzed several aspects of activation of naive transgenic CD4 cells stimulated with resting or activated B cells presenting peptide Ag. Similar results were obtained with stimulation from peptide presenting fibroblast APC lacking or expressing B7-1 with intracellular adhesion molecule-l. TCR ligation with littleor no accessory molecule coreceptor engagement induced efficient blastogenesis; up-regulation of CD25, CD44, CD69,CD95 and CD71; and down-regulation of CD62L over a 48-h period. Accessory molecule help enhanced the expression of CD25, CD44, CD69, and CD71, but tovery modest degrees. Only twomolecules, CD40 ligand and IL-2, were found to be extremely dependent on accessory molecule help, with little or no expression evident with peptide presented on resting B cells or class It-positive fibroblasts. T cells induced on resting B cells expanded minimally over 3 days, and this was followed by extensive cell death and hyporesponsiveness of the resulting cells.These studies suggest that under tolerizing conditions, such as Ag presentation by resting B cells, much of the naive CD4 response is induced efficiently. Partial activation, however, may be the overall result due to the lack of CD40 ligand expression, which may regulate costimulatory activity in APC and, in turn, may contribute to limiting the production of IL-2 required for T cell expansion and survival. The Journal of Immunology, 1997,159: 3257-3265.
A
lthough a majority of self-reactive CD4 T cells are eliminated in the thymus. it is clear that a number of such cells exist in the periphery, and these are potentially capable of inducing autoimmune reactions. To prevent this self-reactivity, a number of mechanisms appear to exist to maintain a state of tolerance, although generally these are not fully understood. One such mechanism is the sequestration of the appropriate self Ag so that the specific T cell may never encounter the Ag. These T cells pose no threat unless tissue damage occurs, resulting in release of the antigenic determinants. The most favorable mechanism, however, involves the two-signal hypothesis for T cell activation, which suggests that encounter with an Ag-presenting APC is only productive if signals brought about by engagement of the TCR are coupled with signals received after interaction of APC-derived accessory molecules with T cell coreceptors (1-8). Ligation of CD28 by B7-2KD86 has been the focus of most studies of T cell activation. although it i s now clear that many ligand
La Jolla Institute ior Allergy and Immunology, Division of Immunochemistry,San Diego, CA 92121
Received forpuhlication J 1 , 1997.
February 5 , 1997.AcceptedforpublicationJuly
The costs of publication of this article were defrayed in part by the payment of i page charges. This article must therefore be hereby marked advertisement n accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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This work was supported by National Institutes of Health Grant A136259 and an investigator award from the Arthritis Foundation (both to M.C.). This manuscript IS publication 172 from the La lolla Institute for Allergy and Immunology.
pairs, such as ICAM4-LFA-I,LFA-3-CD2, and CD7O-CD27, can also be costimulatory (reviewed in Ref. 9). Thus, T cell recognition of self Ag presented on an MHC-expressing cell that lacks or possesses insufficient levels of these accessory molecules is thought to lead to a state of tolerance whereby a noneffective T cell response is elicited. Tolerance may involve peripheral deletion or a phenomenon known as anergy, in which the T cells survive but are hyporesponsive to further stimulation (2, 6, IO). Several hypotheses have been put forward to explain the development of either state, including defective signaling to the T cell and inefficient signaling through the TCR leading largely to a null event. Alternatively, efficient signaling through the TCR may occur, but partial activation may be the result due to lack of the essential late phases of the response, such as IL-2 gene transcription, which must be brought about by accessory molecule costimulation. Several cell types are capable of expressing class 11 MHC, but generally Ag presentation to CD4T cells is thought to occur on the professional APC, dendritic cells, macrophages, and B cells that constitutively express these molecules. Unactivated B cells are usually regarded as tolerizing and are the most likely candidates for inducing tolerance in the CD4 subset due to their abundance compared with other APC ( 1 1-13). However, all resting APC may be able to induce tolerance if little or no accessory molecule expression is evident, and recently studies in B cell-deficient mice have shown that tolerance can indeed be brought about in the absence of B cells (14, 15). Although several reports in different systems have shown that interaction with resting B cells leads to little or no T cell proliferation or IL-2 production ( I I , 12, 16-24),
' Address correspondence and reprint
requests to Dr. Michael Croft, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diegu, CA 92121. E-mail address: mick-croft8liai.org
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Present address: Graduate Program for Molecular and Cellular Life Sciences, Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095. Copyright 0 19'37 by The American Association of Immunologists
Abbreviations used in this paper: ICAM,Intracellularadhesionmolecule; low level; FSc, forward scatter; PCC, pigeon cytclCD40L, CD40 ligand: chrome c. 0022-1 767/'17/$02.00
3258 the early events following Ag presentation have received little attention; in particular, it is not clear whether IL-2 secretion is the major or the only defect in the T cell response. In this report we have used a simplified, yet highly informative, in vitro system to assess other facets of naive CD4 activation. Our studies show that peptide presentation by resting B cells is also insufficient for the induction of high levels of CD40L, suggesting that additional aspects of the T cell response may be lacking as well as the ability to produce IL-2. Because CD4OL expression appears tobe controlled by the magnitude of signals generated through the TCR (25), these results initially indicated that inefficient or defective signaling may occur through the TCR during interaction with resting B cells. However, additional analyses conclusively show that many events associated with T cell activation, including blastogenesis and up- or down-regulation of several other cell surface molecules, are induced successfully by resting B cells and other APC that have few or no accessory molecules. In particular, resting B cells are shown to be equivalent in their capacity to activate many phases of the T cell response compared with a highly costimulatory APC. Thus, much of the response of naive T cells does not rely on high level accessory molecule expression, showing that ligation of Ag/MHC on APCs that provide little accessory molecule help is neither inefficient nor defective for most events associated with stimulation. These studies suggest that tolerance induction by resting APC may simply involve partial T cell activation and that responses are only limited in certain key restriction points that include CD40L induction and IL-2 production.
Materials and Methods Mice AND TCR transgenic mice expressing the Vp3/VcuI I TCR were bred on a B I0.BR background (H2k) as previously described (20, 25). Nontransgenic B I0.BR mice were either purchased from The Jackson Laboratory (Bar Harbor, ME) or also bred in the animal facilities.
T cells CD4+ T cells were purified from the spleens of TCR transgenic mice as previously described (20, 25) by nylon wool depletion followed by complement treatment with Abs to CDX (3.155), heat-stable Ag (JIID), and class I1 MHC (MY1 14 and CA-4.A12), cross-linked with mouse anti-rat K Ab (MAR 18.5). Any residual APC and any in vivo-activated T cells were removed by isolating high density cells spun through a Percoll gradient (45, 53, 62. and 80%). The resultant cells were resting (low FSc, CD69T. CD95Y, CD71-. CD40L-, C D 2 Y ) and >95% CD4+; >95% of these cells possessed a phenotype associated with naive CD4 cells (CD45RB+, CD62L+, CD44'"") along with expression of the VP3IVul 1 TCR (20.25).
Ag-presenting cells Either fibroblast cells transfected with IEk (originally generated by Dr. R. Germain, National Institutes of Health, Bethesda, MD) or B cells purified from B I 0.BR mice were used as APC. Two fibroblast lines were used as previously described (25) that expressed or lacked B7-1 andICAM-I (DCEKJCAM, referred to as ICAM+B7+; DCEK-", referred to as ICAM- B7T). These cells do not express vascular cell adhesion molecule-I, very late Ag-4, B7-2, OX-~OL, 4-1BBL, LFA-1, heat-stable Ag, and CD48 as judged by negative staining with FACS analysis. Resting B cells were purified as previously described (26). Briefly, high density total splenocytes were isolated on a four-layer Percoll gradient (45, 53, 62, and 80%) followed by depleting T cells and other APC by complement treatment with anti-Thy 1.2 (F7D5 and H0.13.4), antiLCD4 (RL172.4). antiCD8 (3.155). anti-Mac-I (M1/70). and anti-dendritic cells (33DI). The remaining cells were again spun through a four-layer Percoll gradient; high density cells were removed and incubated on plastic for 2h. B cells purified in this way were >99% B220+, with low FSc. Activated B cells were derived from resting B cells by stimulation for 2 days with a 25 pgiml mixture of LPS (Sigma Chemical Co., St. Louis, MO) and dextran sulfate (Sigma Chemical Co.) at 5 X 1OS/ml.APC populations were treated with mitomycin C (75-100 pg/ml; Sigma Chemical Co.) for 30 minat 37°C before use.
PARTIAL ACTIVATION OF NAIVE T CELLS Cell cultures Cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA) wlth penicillin, streptomycin, glutamine, 2-ME, sodium pyruvate, and 7% FCS (HyClone Laboratories (Logan, UT) and Sigma Chemical Co.). Cultures were generally set upin1-ml volumes in48-we11 plates (Costar, Cambridge, MA) in replicates of four to six. Naive CD4 cells were plated at a concentration of 5 X 10S/ml with an equal number of APC prepulsed (2 X IOh/ml, 2 h, 37°C) with a saturating dose (20 p,M) of pigeon cytochrome c (PCC) peptide 88-104 (HPLC purified from the whole protein; Sigma Chemical Co.). In cases where B7-CD28 interactions and soluble IL-2 were blocked, 5 pg/ml of either CTLA4-lg (supernatant from the cell line provided by Dr. P. Linsley, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle,WA)or rat IgG2a anti-mouse IL-2(GenzymeCorp., Cambridge, MA) was added to the culture at 0 h. CD4 cells were harvested from replicate cultures, pooled at 24 and 48 h, and stained for various cell surface markers as described below. Pooled supernatants were collected at 40 to 44 h for IL-2 assay. For tolerance induction, 1 X 10' naive CD4 were stimulated as described above at 5 X 105/ml with an equal number of APC prepulsed with PCC. After 3 days, some T cells were restimulated in 0.2-ml volumes at 1 X 10'/ml with an equal number of [CAM+ B7+ or ICAM- B7- APC, and assayed for IL-2 secretion after 24h. Another 1 X 10' cells were recultured in fresh medium at 5 X 1@/ml without APCs or Ag for 2 more days. The resultant cells were again restimulated at I X IOs/ml (2 X lo4 cells) with an equal number of the two fibroblast APCs and assayed for IL-2. In cases where IL-2 secretion was adjusted to take into account cell recoveries, the IL-2 levels from 2 X IO4 cells were multiplied to reflect the total number of cells that were alive after the 3- or 5-day culture periods.
Fluorescence analysis CD40L expression was measured at 3 to 5 h as described in previous studies (25) using biotinylated MRI (hamster IgG) purified from supernatant obtained from the MRI cell line (provided by Dr. R. J. Noelle, Dartmouth Medical School, Lebanon, NH). Positive CD4 cells were vlsualized with phycoerythrin-streptavidin(PharMingen, San Diego, CA) and FITClabeled anti-CD4 (PharMingen).A biotinylated hamster IgG Ab (PharMingen) was used as the control. CD25, CD44, CD62L, CD45RB, CD69, CD95, and CD71 expressions were assessed using 7D4 (rat IgM, supernatant from the 7D4 cell line, American Type Culture Collection, (ATCC), Rockville, MD), Pgp-l (rat IgC2b. supematant from I M7.8.1 cell line, ATCC), Mel-14 (rat IgG2a, supernatant from Mel-14 cell line, ATCC), 23G2 (rat IgC2a, supernatant from the 2362 cell line, ATCC), FITC-labeled anti-CD69 (hamster IgG, PharMingen), anti-Fas (hamster IgG, PharMingen), and antiLCD7 I (rat IgGI, PharMingen). Unlabeled rat and hamster Abs were visualized using FITC-conjugated RG7/9. I (mouse anti-rat k, ATCC) and FITC-conjugated goat anti-hamster IgG (PharMingen), respectively. Controls were a mixture of purified rat IgGI, IgG2a, IgG2b. and IgM or hamster IgG followed by the appropriate secondary Abs. Background staining was identical for each control, whether used alone or in combination. CD4+ T cells were identified using phycoerythrin-conjugated anti-CD4 (PharMingen). FACS analyses were performed on a FACScan flow cytometer with CellQuest software (Becton Dickinson, Mountain View, CA). Data were gated for viable CD4+ Tcells and are single color histograms with relative cell number on the y-axis and log,,, fluorescence intensity on the x-axis. Changes in cell size were also examined and are presented on linear scales of FSc.
Cytokine secretion IL-2 production was assessed as previously described (20, 25) by titrating pooled culture supernatant in duplicate onto NK.3 cells in the presence of anti-IL-4 ( I IBI 1). Standard curves were constructed with purified IL-2 (supernatant from the X63.Ag.IL-2 cell line).
Results Studies from many sources have established that accessory molecules providing costimulatory signals are necessary to elicit maximum proliferation and IL-2 secretion from resting T cells. This has now been demonstrated unequivocably for the least differentiated of peripheral T cells, the naive cell, in both mitogen and Ag systems (23, 24, 27-31). For example, as shown in Table I, naive CD4 T cells derived from V/331val1 TCR transgenic mice secrete
3259
The Journal of Immunology
Table I. IL-2 secretion b y naive CD4 cells" IL-2 Secretion
(ngrnl) ICAM- 67- APC ICAM+ 67' APC
. Mer1 177:Y25. 39. Sloan-Lancahter. J.. A. S . Shaw. J. B. Rothbard, and P. M. Allen. 1994. Par~ialT Cell signaling: altered phospho-zeta and lack nf zap70 recruitnlent In APL-Induced T cell anergy. Cell 79:Y13.
