0192-0561/92 $5.00 + .00 Pergamon Press plc. International Society for lmmunopharmacology.

lnt. J. lmmunopharmac., Vol. 14, No. 3, pp. 377-382, 1992. Printed in Great Britain.

I M M U N E M O D I F I C A T I O N D U E TO C H E M I C A L I N T E R F E R E N C E W I T H T R A N S M E M B R A N E SIGNALLING: A P P L I C A T I O N TO P O L Y C Y C L I C AROMATIC HYDROCARBONS MARC PALLARDY,*t* ZOHAIR MISHAL,§ HERVI~ LEBREC*t and CLAUDE BOHUON*+ *Laboratoire de Toxicologie, Facult6 de Pharmacie Paris XI, rue JB. C16ment, 92296 Chatenay-Malabry; tUnit6 de Biochimie B, Institut Gustave Roussy, rue C. Desmoulins, 94805 Villejuif Cedex; and 3. ~Laboratoire de Cytom6trie, CNRS, IRSC, 7 rue Guy Mocquet, 94800 Villejuif, France

Triggering of the T-cell antigen receptor complex and some other surface molecules is coupled to the phosphodiesterase (phospholipase C)-mediated hydrolysis of membrane phosphoinositides, in particular, phosphatidylinositol-4,5-biphosphate (PIP2). PiP2 hydrolysis generates two products, inositol 1,4,5triphosphate and diacylglycerol, which act in concert as second messengers to increase the free intracellular calcium concentration and activate protein kinase C, respectively, thereby stimulating subsequent events leading to cellular activation and proliferation. Transmembrane signalling in T-lymphocytes represents a potential target for designated drugs as well as immunotoxicants. Immunotoxic effects of polycyclic aromatic hydrocarbons are discussed in the view of interaction with transmembrane signalling in the T-lymphocyte.

Abstract --

TRANSMEMBRANE SIGNALLING T-LYMPHOCYTE

IN

The binding of ligands to the "receptors of activation" initiates a series of events that results in T-cell activation. The major molecules of activation are: T-cell receptor/CD3 complex, CD2, CD5, CD28, CD4... Occupation of the T-cell receptor (TCR) by antigen and major histocompatibility complex (MHC) stabilized by interactions between adhesion molecules is accompanied by transmission of an activation signal across the T-cell plasma membrane. The clonotypic TCR may relay that signal via an invariant CD3 complex, composed of y, d, e, ~ and r] chains. One of the earliest events in T-cell activation is the phosphorylation of various components of the CD3 complex. In man, y and d chains of CD3 became phosphorylated on serine while the ~ chain is phosphorylated on tyrosine residues (Altman, Coggeshall & Mustelin, 1990). Phosphorylation on the serine seems to be mediated by protein kinase C (PKC), while the tyrosine protein kinase encoded by the lck protooncogene, designated p56 ~k has emerged as a strong candidate for tyrosine phosphorylation

(Alexander & Cantrell, 1989; Marth, Lewis, Wilson, Gearn, Krobs & Perlmutter, 1987). Lck activity is modified by CD45, a haematopoietic cell-specific glycoprotein, with tyrosine phosphatase activity in its cytoplasmic domain, suggesting that CD45 may have an essential role in the control of ~ chain phosphorylation, which in turn regulates the ability of the TCR to activate the phosphatidyl inositol pathway in response to stimuli (Mustelin, Coggeshall & Altman, 1989; Keretzky, Picus, Thomas & Weiss, 1990). Activation through the TCR/ CD 3 complex leads to the activation of a membrane associated phospholipase C (PLC) which is able to hydrolyse membrane phosphatidylinositol-4,5-biphosphate (PIP2) producing inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG) (Altman et al., 1990). IP3 is a polar product which diffuses in the cytosol where it binds to a putative IP3 receptor located on the endoplasmic reticulum membrane, leading to the increase in intracellular calcium. The mitochondrial pool of calcium is not concerned by this set of events (Carofoli, 1987). DAG is a neutral lipid which stays into the plasmic membrane and activates the PKC (Nishizuka, 1986). Activation of PKC is associated

*Author to whom correspondance should be addressed. 377

378

M. PALLARDY el al.

with a translocation of the enzyme from the cytoplasm to the plasma membrane (Isakov & Airman, 1987). There is a need to evaluate how the TCR/CD3 complex is coupled to the PLC and if a G protein is involved in the regulation of PLC activity. Recent experiments have shown that the TCR/CD3 complex does not couple to PLC via a classical G protein coupling mechanism (direct coupling) but indicated that a G protein may modulate the TCR/CD3 linkage to PLC (Graves & Cantrell, 1991). There also appears to be a role for tyrosine phosphorylation in TCR/CD3 coupling to PLC since tyrosine kinase inhibitors prevent PLC activation subsequent events after cellular stimulation (June et al., 1990). The ability of cAMP to inhibit phosphatidylinositols (PIs) breakdown in various cells raises the possibility that it might exert a negative regulatory influence on P I - P L C . Based on the ability of the G protein activator, A1F~ , to bypass cAMP-mediated inhibition of T-cell activation it was suggested that protein kinase A (PKA) interferes primarily with PIs hydrolysis and formation of the relevant second messengers (O'Shea, Urdahl, Luong, Chused, Samelson & Klausner, 1987). However, the ability of cAMP to suppress even phorbol myristate acetate (PMA) plus calcium ionophore-induced T-cell activation suggests additional downstream sites of inhibition (Isakov & Altman, 1985). In addition, cAMP interferes with IL-2-induced T-cell proliferation at the early G1 phase of the cell cycle Isakov & Altman, 1985).

I N T E R F E R E N C E OF D R U G S W I T H T H E T R A N S M E M B R A N E S I G N A L L I N G P A T H W A Y IN T-LYMPHOCYTES

Cyclosporin A (CSA) and FK506 are potent inhibitors of the TCR-mediated signal transduction pathway, as evidenced by their ability to inhibit the transcription of early T-cell activation genes (Toci et al., 1989). CSA and FK506 inhibit the binding of NF-AT to the IL-2 enhancer and inhibit transcriptional activation by NF-AT (Emmel, Verweij, Durand, Higgins, Lacy & Crabtree, 1989). CSA and FK506 also inhibit transcription mediated by AP3 and Oct-I and partially inhibit transcription mediated by NF-KB (Emmel et al., 1989). Recently, Troposchug and Hofmann have found similarity of FK binding protein (FKBP), which binds FK506, to an inhibitor of PKC from bovine brain (Troposchug & Hofmann, 1991). They speculate that FK506 may

Table 1. PBMC proliferation following in vitro DMBA exposure and PHA or OKT3 activation DMBA(~M) 5 20 40

PHA~

OKT3'

65.4 -+ 15.9u 44.1" _+7.2 15" -+ 6.7

68* -+ 5.8 50.7* _+5.4 25.6* _+ 13.4

' Results represent the mean of two independent experiments. ~' Results are expressed as percentage of control ~ S.E.M. * P < 0.05. act through FKBP (PKC I-2) by interfering with the activity of PKC. Corticosteroids, including dexamethasone (DEX), have multiple influences on the immune system, affecting the functions of macrophages, B-cells and T-cells (Cupps & Fauci, 1982). Luster and colleagues have shown that DEX inhibits anti-Ig mediated PI turnover in B-cells (Luster, Germolec, Clark, Wiegand & Rosenthal, 1988). In this case, it appears that DEX may arrest B-cell maturation at multiple stages but with a primary effect on early events associated with the PI-dependent messenger system. This effect of DEX on PI turnover has not yet been reproduced in T-cells.

EFFECTS

OF T H E POLYCYCLIC AROMATIC HYDROCARBON PROTOTYPE 7 , 1 2 - D I M E T H Y L B E N Z ( a ) A N T H R A C E N E (DMBA) ON L Y M P H O C Y T E T R A N S M E M B R A N E SIGNALLING

PAHs are ubiquitous environmental contaminants generated as by-products of the incomplete combustion of fossil fuels, wood and other organic materials and many are carcinogenic (Zadek, 1980). Previous studies with the prototype carcinogenic PAH, DMBA, have shown a suppression of both humoral and cell-mediated immunity (Ward, Murray, Lauer, House, Irons & Dean, 1984; Dean et al., 1988). Inhibition of IL-2 production and IL-2 high-affinity receptor expression have been demonstrated in DMBA-treated mouse splenocytes (Pallardy, House & Dean, 1989). Stimulation of cytotoxicity by direct ligation of the CD3 subunit of the TCR was also suppressed following in vitro DMBA exposure while antigen non-specific cytolysis including LAK cells and polyclonal cytotoxic T-lymphocyte (CTL) activation was unaffected (House, Pallardy & Dean, 1989). Taken together, these results suggest transmembrane signalling or events immediately prior to this as the molecular target for DMBA action. Recently Burchiel,

Immune Modification due to Chemical Interference

379

(167.8)

DMBA40uM

f

DMBA40uM

D M B A 20 u M

z

160

m

z

DMBA20uM

z 140

m

o DMSO

d

o

z

d z

12o

DMSO I

I

I

I

2

4

6

8

TIME

(minutes)

Fig. 1. Intracellular calcium level in PBMC following DMBA exposure and P H A activation. Results of a typical experiment. Values in parentheses correspond to the maximum value of the mean fluorescence intensity of each curve.

I

I

I

I

I

1

2

3

4

5

TIME

(minutes)

Fig. 2. Calcium release from intracellular stores in PBMC following DMBA exposure and P H A activation. EGTA was added 2 min before P H A stimulation. Results of a typical experiment.

Table 2. DMBA needs to be present at the initiation of cell activation to induce immunosuppression DMBA(taM) 0 5 20 40

Non-washed cells ,.b

Washed cells a.b

56,403 + 5590 ~ 45,939 + 4439* (81.4 %)d 30,725 +_ 2262* (51.3 %) 13,016 _+ 1801" (21.7 070)

64,689 _+ 4182 55,878 + 4870 (86.3 070) 69,366 _+ 2139 (107.2 %) 58,312 _+ 4451 (90.1%)

Results of a typical experiment. b Washed cells: DMBA was removed from the media before the addition of PHA; non-washed cells: DMBA was not removed before addition of PHA. Results are expressed as mean _+ S.E.M. in counts/min. '~ Percentage of control. * P < 0.05.

Table 3. Extracellular calcium influx in PBMC following DMBA exposure and A23147 addition Time (rain)

DMSO"

DMBA (5 / a M )

DMBA (20/aM)

DMBA (40 taM)

0 1 2

43.4 b 119.2 121.5

57.8 131 131.8

71.3 132.2 130.5

86 136.8 138.2

3

115.4

129.2

4 5

111 107.8

125 123.3

131.8 (-22%) 130.3 129.9

139.5 (-31°70) 139.1 137.6

(-5O7o) ~

Results of a typical experiment. b Mean fluorescence intensity. Percentage of control.

380

M. PALLARDY et al. Table 4. PBMC proliferation following DMBA exposure and A23147 and PMA activation A23147 ~

PMA'

A23147 + PMA"

40,232 +_ 9552 N.D. N.D.

65,416 _+ 16,104 68,462 _+ 6779 74,033 _+ 10,647 (108%) ~ 65,396 _+ 6873 (95.5%) 73,348 _+ 4738 (107.1%)

Control DMSO DMBA 5 ~M

3684 + 3 5 0 N.D. N.D.

DMBA 20/~M

N.D.

N.D.

DMBA 40 ~M

N.D.

N.D.

b

~' Results of a typical experiment. b Results are expressed as mean _+ S.E.M. in counts/rain. Percentage of control. N.D.: not done.

T h o m p s o n & De A n n (1991) have d e m o n s t r a t e d using the J U R K A T cell line, a cloned T-cell leukaemia, t h a t D M B A induced a dose a n d timed e p e n d e n t i n h i b i t i o n o f C a 2+ m o b i l i z a t i o n following in vitro exposure. The decrease in Ca 2+ m o b i l i z a t i o n was correlated with a n increase in baseline levels o f cytoplasmic free Ca 2~ . These results have p r o m p t e d us to evaluate the interference of DMBA with t r a n s m e m b r a n e signalling using human peripheral blood m o n o n u c l e a r cells ( P B M C ) . Blood from healthy volunteers was collected o n h e p a r i n - c o a t e d tubes. P B M C were isolated o n a F i c o l l - H y p a q u e gradient a n d then cultured in tissue culture m e d i a with 5 % foetal calf serum (FCS) a n d 5 × 10 5 M 2-mercapt o e t h a n o l . The cells were always treated with D M B A for 1 h before the activation signal was added. P h y t o h a e m a g g l u t i n i n ( P H A - P ) or the m o n o c l o n a l a n t i b o d y OKT3, directed to the h u m a n CD3, were used to produce l y m p h o c y t e activation. In vitro exposure o f h u m a n P B M C to D M B A ( 0 - 4 0 / a M ) resulted in a decreased P H A - P a n d O K T 3 - i n d u c e d l y m p h o p r o l i f e r a t i o n after 48 h o f i n c u b a t i o n as assessed by 3H-thymidine i n c o r p o r a tion (Table 1). In a d d i t i o n , the presence o f D M B A was required at the time o f initial activation to be effective since r e m o v i n g D M B A before the a d d i t i o n of PHA-P resulted in n o r m a l lymphocyte proliferation. This result suggested t h a t D M B A may interfere with early cell p r o g r a m m i n g . T h e next step was to evaluate the intracellular calcium level since it has been s h o w n t h a t calcium plays an i m p o r t a n t role in cell activation a n d intracellular signalling ( A l t m a n et al., 1990). For this p u r p o s e we used P B M C loaded with indo-1, a highly fluorescent indicator o f free calcium, a n d then analysed the cells with a flow c y t o m e t e r

(Grynkiewicz, Poenie & Tsien, 1985; R a b i n o v i t c h , June, G r o s s m a n n & Ledbetter, 1986). A shift in the fluorescence emission of indo-1 f r o m blue to violet following calcium chelation has been f o u n d to be a sensitive indicator o f intracellular free calcium levels. A rapid increase in the m e a n v i o l e t / b l u e ratio occurred within seconds after exposure o f P B M C to P H A . The peak calcium response is reached a r o u n d 3 m i n and then declined slowly to reach a basal level after 15 m i n (data not shown). W h e n the cells are exposed to D M B A ( 0 - 4 0 ~M), we n o t e d t h a t baseline levels of calcium were increased in a dosed e p e n d e n t m a n n e r (data not shown). This result was in a c c o r d a n c e with Burchiel et al. (1991) w h o f o u n d a similar increase o f basal calcium level in J U R K A T cells exposed to D M B A . P H A - i n d u c e d calcium m o b i l i z a t i o n was inhibited at all D M B A concentrations tested (Fig. 1). In addition, the peak o f calcium response was delayed in a d o s e - d e p e n d e n t m a n n e r ( 3 m i n at 0 / a M ; 3 m i n 3 0 s at 2 0 ~ M ; 4 r a i n at 40/aM). W h e n P B M C are triggered by a lectin or an antigen in the abscence o f extracellular calcium there is a little a n d transient a u g m e n t a t i o n o f intracellular calcium. It is believed t h a t this a u g m e n t a t i o n is due to the release of calcium f r o m the e n d o p l a s m i c reticulum (endogenous calcium). In the presence o f extracellular calcium there is a n i m p o r t a n t and sustained elevation of intracellular calcium suggesting that the m a j o r part o f this a u g m e n t a t i o n is linked to calcium influx (exogenous calcium). To distinguish the effect o f D M B A exposure o n the different c o m p o n e n t s of the calcium increase we have p e r f o r m e d two types o f experiments: (1) measurements o f intracellular calcium levels following P H A activation in the presence of E G T A in the i n c u b a t i o n media; a n d (2) m e a s u r e m e n t s of

Immune Modification due to Chemical Interference intracellular calcium levels following addition of the calcium ionophore A23147. In the first set of experiments, we observed a rapid and transient augmentation of the intracellular calcium level which was completely abrogated when the cells were exposed to DMBA (Fig. 2). However, when we studied the extracellular calcium influx induced by A23147 we observed that DMBA induced only a partial inhibition of the influx (Table 3). These results suggested that DMBA was mainly effective on the release of calcium from intracellular stores. One approach to study the function of second messengers in cellular activation has been to use pharmacological agents that by-pass receptor triggering and mimic downstream events. The use of a combination of P M A plus calcium ionophore is able to induce interleukin-2 (IL-2) production and lymphocyte proliferation (Altman et al., 1990). Lymphoproliferation induced by calcium ionophore plus P M A was unaffected at all DMBA concentrations tested (Table 4). This result confirmed that the molecular target of DMBA-induced immunosuppression seems to be located before calcium release and PKC activation. Previous experiments using murine lymphocytes have demonstrated that DMBA was able to alter IL-2 responsiveness by either a direct effect on the receptor or an action on the second messengers cascade linked to the IL-2 receptor (IL-2R) (Pallardy et aL, 1989). Thus, it appears that DMBA is able to modify T-cell activation through the TCR as well as IL-2 responsiveness. In this regard, it is important to point out that DMBA, due to its high lipophilicity,

381

might interact at the membrane level and disrupt transduction signals (PI turnover, PLC, tyrosine kinase) and conformation of receptors (TCR/CD3, IL-2R...). Preliminary experiments performed in our laboratory have revealed that DMBA could decrease the membrane fluidity of resting human T-lymphocytes (data not shown). A candidate for a molecule involved in membrane fluidity changes after receptor cross-linkage and so, a potential target of DMBA action, is a guanine nucleotide-binding protein because sodium fluoride, which is an activator of guanine nucleotide-binding protein, causes increases in membrane fluidity (Mizuguchi, Utsunomiya, Nakanishi & Arata, 1988). In conclusion, understanding of T-cell signal transduction is still far from complete. Future works will uncover more receptor kinases and phosphatases that are involved in T-cell activation. This question is of more than academic interest, as the design of specific pharmacological agents to inhibit or stimulate lymphokine production is dependent on precise knowledge of the biochemical events relevant to lymphokine production. On the other hand, transduction of activation signals through the lymphocyte membrane seems to be a potential target for immunotoxicants, as it has been demonstrated with DMBA. Acknowledgement - - The authors wish to thank Dr Jack

Dean for helpful discussion concerning DMBA mechanism of action.

REFERENCES

ALEXANDER, D. R. & CANTRELL, D. A. (1989). Kinases and phosphatases in T cell activation. Immun. Today, 10, 200 - 205. ALTMAN,A., COGGESHALL,M. & MUSTELIN,T. (1990). Molecular events mediating T cell activation. Adv. Immun., 48, 227 - 360. BURCHIEL, S. W., THOMPSON, T. A. & DE ANN, D. (1991). Alterations in mitogen-induced calcium mobilization and intracellular free calcium produced by 7,12-dimethylbenz(a)anthracene in the Jurkat human T-cell line. Int. J. Immunopharmac., 13, 109- 115. CAROFOLI,E. (1987). Intracellular calcium homeostasis. A. Rev. Biochem., 56, 395-433. CUPPS, T. R. & FAUCI,A. S. 0982). Corticosteroid-mediated immunoregulation in man. Immun. Rev., 65, 133. DEAN, J. H., WARD,E. C., MURRAY,M. J., LAUER,L. D., HOUSE,R.V., STILEMAN,W. S., HAMILTON,T. A. & ADAMS, D. O. (1988). Immunosuppression following 7,12-dimethylbenz(a)anthracene exposure in B6C3F~mice. II -- Altered cell-mediated immunity and tumoral resistance. Int. J. lmmunopharmac., 8, 189- 198. EMMEL, E. A., VERWEIJ,C. L., DURAND,D. B., HIGGINS, K. M., LACY, E. & CRABTREE,G. R. (1989). Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science, 246, 1817. GRAVES, J. & CANTRELL,D. A. (1991). An analysis of the role of guanine nucleotide binding proteins in antigen receptor/ CD3 antigen coupling to phospholipase. C. J. lmmun., 146, 2102-2107.

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GRYNKIEWlCZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca -'+ indicators with greatly improved fluorescence properties. J. biol. Chem., 260, 3440. HousE, R. V., PALLARDY,M. J. & DEAN, J. H. (1989). Suppression of murine cytotoxic T-lymphocyte induction following exposure to 7,12-dimethylbenz(a)anthracene: dysfunction of antigen recognition. Int. J. Immunopharmac., 11, 207-219. 1SAKOV,N. & ALTMAN, A. (1985). Tumor promoters in conjunction with calcium ionophores mimic antigenic stimulation by reactivation of alloantigen-primed murine T lymphocytes. J. l m m u n . , 135, 3674. ISAKOV, N. & ALTMAN, A. (1987). Human T lymphocyte activation by tumor promoters: role of protein kinase C. J. I m m u n . , 138, 3100. JUNE, C. H., FLETCHER, M. C., LEDBETTER, J. A., SCHIEVFN, G. L., SIEGEL, J. N., PHII.IPPS, A. F. & SAMELSON,L. E. (1990). Inhibition of tyrosine phosphorylation prevents T cell receptor-mediated signal transduction. Proc. natn. Acad. Sci. U.S.A., 87, 7722-7726. KERETZKY, G. A., PtcUS, J., THOMAS, M. L. & WEISS, A. (1990). Tyrosine phosphatase CD45 is essential for coupling T cell antigen receptor to the phosphatidyl inositol pathway. Nature, 346, 66-68. LUSTER, M. T., GERIVlOLEC, D. R., CLARK, G., WIEGAND,G. & ROSENTHAL, G. (1988). Selective effects of 2,3,7,8tetrachlorodibenzo-p-dioxin and corticosteroid on in vitro lymphocyte maturation. J. h n m u n . , 140, 928- 935. MARTH, J. D., LEWIS, D. B., WILSON, C. B., GEARN, M. E., KROBS, E. G. & PERLMUTTER, R. M. (1987). Regulation of pp56 ~k during T cell activation: functional implications for the src-likc protein tyrosine kinases. E M B O J., 6, 2727. MIZUGUCHI, J., UTSUNOIVnYA,N., NAKAN1SHI,M. & ARATA, Y. (1988). Phorbol myristate inhibits increases in membrane fluidity induced by anti-IgM in B cells. J. I m m u n . , 140, 2495- 2499. MUSTELIN, T., COGGESHALL,K. M. & ALTMAN, A. (1989). Rapid activation of the T cell tyrosine protein kinase pp56 j~kby the CD45 phosphotyrosine phosphatase. Proc. natn. ,4cad. Sci. U.S.A., 86, 6302-6306. NISHIZUKA, Y. (1986). Studies and perspectives of protein kinase C. Science, 233, 305- 312. O'SHEA, J. J., URDAHL, K. B., LUON~, H. T., CHUSED, T. M., SAMELSON,L. E. & KLAUSNER,R. D. (1987). Aluminium fluoride induces phosphatidyl inositol turnover, elevation of cytoplasmic free calcium and phosphorylation of the T cell antigen receptor in murine T cells. J. l m m u n . , 139, 3463. PALLARDY, M. J., HOUSE, R. V. & DEAN, J. H. (1989). Molecular mechanism of 7,12-dimethylbenz(a)anthracene-induced immunosuppression: evidence for action via the interleukin-2 pathway. Molec. Pharmac., 36, 128- 133. RABINOVITCH, P. S., JUNE, C. H., GROSSMAN, A. & LEDBETTER, J. A. (1986). Heterogenity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry. J. l m m u n . , 137, 952-961. Tocl, M. J., MATKOVITCH, D. A., COLLIER, K. A., KWOK, P., DUMONT, F., LIN, S., DEGUDICIBUS,S., SIEKERKA,J. J., CHIN,, J. & HUTCHINSON, N. I. (1989). The immunosuppressant FK 506 selectively inhibits expression of early T cell activation genes. J. I m m u n . , 143, 718. TROPOSCHUG,M. & HOFMANN,R. (1991). FK 506 and protein kinase C. Nature, 351, 195. WARD, E. C,, MURRAY,M. J., LAUER, L. D., HOUSE, R. V., IRONS, R. & DEAN, J. H. (1984). Immunosuppression following 7,12-dimethylbenz(a)anthracene exposure in B~C~F~ mice. I - - Effects on humoral immunity and host resistance. Toxic. appl. Pharmac., 74, 299-308. ZADEK, M. S. (1980). Polycyclic aromatic hydrocarbons: a review. J. Envir. Path. Toxic., 3, 357-367.

Immune modification due to chemical interference with transmembrane signalling: application to polycyclic aromatic hydrocarbons.

Triggering of the T-cell antigen receptor complex and some other surface molecules is coupled to the phosphodiesterase (phospholipase C)-mediated hydr...
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