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BRE (Brain and Reproductive Organ-Expressed) is an anti-apoptotic protein and a core component of DNA-repair BRCA1-A complex. Microarray-detected high BRE gene expression has been found to be associated with better patient survival in AML (acute myeloid leukemia) with MLL-AF9 translocation, and radiotherapy-treated non-familial breast cancer. A recent finding suggests that the high BRE gene expression in MLL-AF9 AML could be attributed to the additional expression of a transcript variant encoding a novel C-terminal BRE isoform. Using THP-1 as the MLL-AF9 AML cell model, we found that ectopic expression of the C-terminal BRE, which could not form an intact BRCA1-A complex, indeed increased cellular sensitivity to chemotherapeutic drugs and inhibited cell proliferation, while the complete opposite was achieved by the ectopic expression of full-length BRE. Our findings suggest that the C-terminal BRE-encoding transcript could be responsible for better patient survival and may have therapeutic potential for cancer.

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P

BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-alpha-induced activation of NF-kappaB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-alpha, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-alpha, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.

Epstein-Barr virus (EBV) is a human γ-herpesvirus causally linked with several different human diseases including nasopharyngeal carcinoma (NPC), Hodgkin's lymphoma, Burkitt's lymphoma, and infectious mononucleosis (15, 31). The incidence of NPC in southern China, including Guangdong province and Hong Kong, is among the highest in the world (28). A clear understanding of the molecular mechanisms underlying EBV-associated pathogenesis is of paramount importance in formulating an effective therapy. EBV can readily transform primary resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) (15, 31). EBV-encoded latent membrane protein 1 (LMP1) is indispensable for establishment of lymphoblastoid cell lines by EBV (23). Among several latent viral genes expressed in NPC and EBV-positive Hodgkin's disease, LMP1 is the only one with oncogenic properties (31). When introduced into rodent fibroblasts, LMP1 causes oncogenic transformation (43). LMP1 also promotes a higher incidence of lymphoma in transgenic mice when specifically introduced into lymphocytes (27). These results establish a critical role for LMP1 in EBV-associated malignances. LMP1 is a membrane protein of 386 amino acids containing six transmembrane domains. Both the amino (amino acids [aa] 1 to 24) and carboxyl (aa 186 to 386) termini of LMP1 are located in the cytoplasm (Fig. ​(Fig.1A).1A). While the short amino terminus of LMP1 is implicated in anchoring LMP1 on the plasma membrane, its carboxyl terminus is implicated in both cellular transformation and activation of intracellular signaling pathways. Two subregions in the carboxyl tail of LMP1 are critical in cell transformation and signaling: carboxyl-terminal activating region 1 (CTAR1, aa 194 to 231) and CTAR2 (aa 351 to 386) (Fig. ​(Fig.1A).1A). CTAR1 is capable of binding several tumor necrosis factor (TNF) receptor-associated factors (TRAFs) and activating the NF-κB pathway, while CTAR2 was shown to bind TNF receptor-associated death domain protein (TRADD) and receptor-interacting protein (RIP) and activate both the NF-κB and c-Jun N-terminal kinase (JNK) pathways (15, 31). Although CTAR1 can independently activate the NF-κB pathway, it is CTAR2 that is mainly responsible for activating both the NF-κB (accounting for ∼70% of total NF-κB activity induced by LMP1) and JNK (100% of LMP1-mediated JNK activation) pathways (15, 31). FIG. 1. LMP1(G335) and LMP1(D335) activate JNK equally well. (A) Schematic representation of LMP1. Two horizontal straight lines represent the plasma membrane. The six grey bars represent transmembrane domains, and the two black bars represent CTAR1 and CTAR2. ... LMP1 has generally been thought to functionally mimic members of the TNF receptor (TNFR) superfamily in signaling, as it was shown to constitutively oligomerize on the plasma membrane, interact with TRADD, RIP and several TRAFs, including TRAF2, and activate both the JNK and NF-κB pathways in host cells (15, 31). As both TRADD and TRAF2 are known to be involved in the TNF-α-mediated JNK and NF-κB pathways (5, 30, 32), they are also widely thought to be involved in LMP1-mediated JNK and NF-κB activation (15, 31). Overexpression of truncated TRADD and TRAF2 mutants were employed by earlier studies, which generated conflicting results (12, 20, 24). In order to clarify the confusion mainly arising from the use of various dominant-negative mutants and further elucidate the LMP1-mediated signaling pathways, we resorted to mouse embryonic fibroblasts (MEFs) derived from different knockout mice whenever possible. In cases where no knockout MEFs were available, the small interfering (siRNA) technique was used to silence the expression of endogenous genes in order to determine their involvement in LMP1-mediated signaling. In contrast to the prevailing paradigm, we demonstrate in this report that TRADD, TRAF2, RIP, TAB2, myeloid differentiation factor 88 (MyD88), and interleukin-1 (IL-1) receptor-associated kinases 1 and 4 are not essential for LMP1-mediated JNK activation. Instead, LMP1 activates JNK through sequential activation of TRAF6, TAK1/TAB1, and c-Jun N-terminal kinase kinases 1 and 2 (JNKK1/2).

Mouse Bre, an evolutionarily conserved stress-modulating gene, like its human counterpart, is expressed in multiple alternative transcripts. The main transcript, which is ubiquitously expressed, encodes a protein that binds tumor necrosis factor receptor 1 (TNF-R1) and downregulates TNF-induced activation of NF-kappaB. Alternative splicing of mouse Bre occurs only at the 5' region of the gene, generating either nonfunctional transcripts or transcripts that can encode putative protein isoforms differ at the N-terminal sequence. In contrast, alternative splicing of human BRE occurs at either or both ends of the gene; only the 3' alternative splicing can generate functional transcripts that encode putative protein isoforms differ at the C-terminus, occurrence of the 5' alternative splicing only results in forming nonfunctional transcripts. Unlike the human BRE alternative transcripts which are coexpressed at considerable levels with the main transcript, the mouse counterparts are expressed in a restricted pattern and generally in low abundance except in the heart. Both species, however, share a type of Bre alternative transcripts generated by cryptic splicing at a nonstandard, noncanonical acceptor site. Thus, a highly conserved gene in two species can generate alternative transcripts different in both of the sequence structure and expression pattern, as well as a similar class of transcripts resulting from unconventional transcript processing.

A panel of monoclonal antibodies specific to Hong Kong Chinese nasopharyngeal carcinoma (NPC)-associated Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) variants has been generated. These monoclonal antibodies not only differentiate the Hong Kong Chinese NPC-associated LMP1 variants from the prototype B95-8 LMP1, derived from Caucasian infectious mononucleosis, but also differentiate the 2 highly homologous LMP1 deletion variants commonly found in Hong Kong primary NPC. The predominant deletion type variant, DV-Asp335, is characterized by an aspartic acid at residue 335 located in the cytoplasmic C-terminal region, whereas the other minor deletion variant, DV-Gly335, has a glycine in the same residue position. 335D is hitherto found predominantly in LMP1 of the China 1 strain in association with NPC in the Chinese populations located in southern China and Malaysia. These antibodies, which are applicable in ELISA, immunofluorescence, immunoprecipitation, immunoblotting and immunohistochemistry on paraffin sections, are the first variant-specific anti-LMP1 monoclonal antibodies produced, and will be useful in investigating the functional significance of 335D in NPC. © 2002 Wiley-Liss, Inc.

Little is known about human anti-idiotypic antibodies. Phage display methodology was used to reconstruct these antibodies from lupus patients, which recognize a subset (T14(+)) of anti-DNA antibodies. Antigen-specific B cells were isolated from the blood using a peptide based on a complementarity determining region (V(H)CDR3) of the prototypic T14(+) antibody. cDNA fragments of the V(H) and V(L) genes prepared from the cells were expressed as phage displayed single chain Fv (scFv) fragments using the pCANTAB-5E phagemid vector. From a reactive clone obtained, the Ig genes used were identified to be V(H)3, D5-D3, J(H)4b, V(kappa)I and J(kappa)2. The heavy chain was highly mutated, especially in CDR3, which bears mutations mostly of the replacement type; this region is also unusual in being extremely long due to a D-D fusion. In contrast, a mouse hybridoma antibody, made to the same T14(+) peptide and transformed as a scFv fragment, uses a short V(H)CDR3 comprising five amino acids, three of which are tyrosines. Tyrosines may be important for antigen binding because two of these also exist in the human V(H)CDR3. The light chains of both antibodies may also contribute to the specificity of the protein, because their V(L) segments, including the CDRs, are highly homologous to each other.

BRE is a multifunctional adapter protein involved in DNA repair, cell survival and stress response. To date, most studies of this protein have been focused in the tumor model. The role of BRE in stem cell biology has never been investigated. Therefore, we have used HUCPV progenitor cells to elucidate the function of BRE. HUCPV cells are multipotent fetal progenitor cells which possess the ability to differentiate into a multitude of mesenchymal cell lineages when chemically induced and can be more easily amplified in culture. In this study, we have established that BRE expression was normally expressed in HUCPV cells but become down-regulated when the cells were induced to differentiate. In addition, silencing BRE expression, using BRE-siRNAs, in HUCPV cells could accelerate induced chondrogenic and osteogenic differentiation. Hence, we postulated that BRE played an important role in maintaining the stemness of HUCPV cells. We used microarray analysis to examine the transcriptome of BRE-silenced cells. BRE-silencing negatively regulated OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and components of the TGF-β/BMP and FGF signaling pathways which are crucially involved in maintaining stem cell self-renewal. Comparative proteomic profiling also revealed that BRE-silencing resulted in decreased expressions of actin-binding proteins. In sum, we propose that BRE acts like an adaptor protein that promotes stemness and at the same time inhibits the differentiation of HUCPV cells.

Objective. To investigate whether antibodies to a T14 anti-DNA antibody can be found in patients with systemic lupus erythematosus (SLE). Methods. Seventy-six serum samples (37 from patients with SLE) were randomly selected from among sera submitted for routine antinuclear antibody testing. Short, overlapping peptides based on the partial VH (variable region of the heavy chain) sequence of the T14 antibody were synthesized on multipins and screened for reactivity with SLE sera. In addition, selected peptides from T14 and related proteins were synthesized in bulk and screened for reactivity with both SLE and control sera. A monoclonal antibody was generated to determine the prevalence of the T14 idiotype (T14+ Id) in the different study populations. Results. Antibodies were detected by a peptide based on the third complementarity-determining region (CDR3) of the T14 protein in 15 (41%) of 37 patients with SLE or 15 (54%) of 28 who had anti-DNA antibodies, in 3 (9%) of 34 patients without anti-DNA antibodies (9 of whom had SLE), and in 6 (10%) of 57 healthy controls. In SLE sera, the antiidiotypic (anti-Id) responses (IgM and IgG) correlated well with the anti-DNA responses (IgG), and both responses correlated well with the T14+ Id activity in SLE sera. Control peptides based on the 18/2 (16/6+ Id) and S107 proteins detected low antibody activities in SLE sera, attributable to cross-reactivity with the T14 peptide. A peptide based on an unrelated human antibody was not reactive with these sera. Conclusion. Anti-Id antibodies directed to T14 VHCDR3 were found commonly in the sera of patients with SLE, and they appeared to be induced by the anti-DNA antibodies present in the sera. Based on these findings, these secondary antibodies may be pathogenic in SLE.

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8 months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.

Stress-responsive genes play critical roles in many biological functions that includes apoptosis, survival, differentiation and regeneration. We have identified a novel stress-responsive gene called BRE which interacts with TNF-receptor-1 and blocks the apoptotic effect of TNF-alpha. BRE enhances tumor growth in vivo and is up-regulated in hepatocellular and esophageal carcinomas. BRE also regulates the ubiquitination of the DNA repair complex BRCC, and the synthesis of steroid hormones. Here, we examined BRE-mRNA in cells after treatments with UV and ionizing radiation (IR). UV and IR treatment alone suppressed BRE-mRNA levels by more than 90% at 24 h, while hydroxyurea, fluorodeoxyuridine, aphidicolin, known inhibitors of S-phase DNA synthesis, had no significant effect. BRE protein expression was unaltered in cells treated with TNF-alpha, Interleukin-1 and Dexamethasone, while a threefold increase was observed following chorionic gonadotropin exposure. Although BRE plays a regulatory role in many different pathways, yet its expression is apparently under very stringent control.

Cyclooxoygenase (COX)-2 overexpression is involved in gastric carcinogenesis. While high-salt intake is a known risk factor for gastric cancer development, we determined the effects of high salt on gastric chemical carcinogenesis in COX-2 transgenic (TG) mice. COX-2 TG mice were developed in C57/BL6 strain using the full-length human cox-2 complementary DNA construct. Six-week-old COX-2 TG and wild-type (WT) littermates were randomly allocated to receive alternate week of N-methyl-N-nitrosourea (MNU, 240 p.p.m.) in drinking water or control for 10 weeks. Two groups of mice were further treated with 10% NaCl during the initial 10 weeks. All mice were killed at the end of week 50. Both forced COX-2 overexpression and high-salt intake significantly increased the frequency of gastric cancer development in mice as compared with WT littermates treated with MNU alone. However, no additive effect was observed on the combination of high salt and COX-2 expression. We further showed that MNU and high-salt treatment increased chronic inflammatory infiltrates and induced prostaglandin E(2) (PGE(2)) production in the non-cancerous stomach. Whereas high-salt treatment markedly increased the expression of inflammatory cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-1 beta and IL-6) in the gastric mucosa, COX-2 overexpression significantly altered the cell kinetics in the MNU-induced gastric cancer model. In conclusion, both high salt and COX-2 overexpression promote chemical-induced gastric carcinogenesis, possibly related to chronic inflammation, induction of PGE(2), disruption of cell kinetics and induction of inflammatory cytokines.

The Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3a locus encodes a 274 a.a. novel protein, and its expression has been confirmed in SARS patients. To study functional roles of 3a, we established a transgenic fly model for the SARS-CoV 3a gene. Misexpression of 3a in Drosophila caused a dominant rough eye phenotype. Using a specific monoclonal antibody, we demonstrated that the 3a protein displayed a punctate cytoplasmic localization in Drosophila as in SARS-CoV-infected cells. We provide genetic evidence to support that 3a is functionally related to clathrin-mediated endocytosis. We further found that 3a misexpression induces apoptosis, which could be modulated by cellular cytochrome c levels and caspase activity. From a forward genetic screen, 78 dominant 3a modifying loci were recovered and the identity of these modifiers revealed that the severity of the 3a-induced rough eye phenotype depends on multiple cellular processes including gene transcriptional regulation.

Objective To investigate why the serum of a pediatric patient with systemic lupus erythematosus was persistently (>30 months) and strongly positive for antibodies to double-stranded DNA (dsDNA) as revealed by enzyme-linked immunosorbent assay (ELISA), but yielded negative results on the antinuclear antibody test (HEp-2 immunofluorescence [IF]). Methods The patient's antibodies were isolated on dsDNA and single-stranded DNA (ssDNA) supports, which were then examined by dsDNA ELISA and HEp-2 IF. Tests included the use of various inhibitors to determine the fine specificity of the antibodies. Other tests performed included immunoblotting, immunoprecipitation, Crithidia luciliae IF, and neutrophil IF. Results The antibodies isolated from the dsDNA and ssDNA supports were similar, in that they were of the IgG type, bound well in the dsDNA ELISA, and recognized a normally hidden nucleolar RNA antigen in HEp-2 cells. With both the dsDNA ELISA and nucleolar antigens, inhibition studies revealed that the epitope recognized was guanosine 5′-triphosphate (GTP). Binding of the antibodies was better to GTP than to guanosine 5′-monophosphate or cytidylyl (3′-5′) guanosine, and, in turn, was better than to guanosine, while N7-methylated GTP was unreactive. The antibodies did not bind to dsDNA present in solution or in HEp-2 or Crithidia cells, but bound transfer RNA well and recognized a cytoplasmic RNA antigen in neutrophils. Conclusion A new problem in dsDNA ELISA is revealed in the occurrence of a hitherto-unknown and unusual buckling of the insolubilized DNA molecule, which, absent in dsDNA found in solution or in whole cells, presumably creates gaps of single-strandedness in the molecule. A new antibody specific for GTP is described in this patient, which may be clinically important.

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), a common cancer in Hong Kong. The EBV-encoded LMP1 protein is believed to play an important role in cell transformation. We have previously identified a prevalent LMP1 variant (2117-LMP1) that is expressed in 86% of primary NPC in Hong Kong. In this study, the biologic phenotypes induced by 2117-LMP1 were compared with those of the prototypic B95.8-LMP1 in an immortalized nasopharyngeal epithelial cell line, NP69. The 2117-LMP1 could induce cell proliferation and resistance to apoptosis induced by growth factor deprivation. Expression of 2117-LMP1 also suppressed expression of p16, p21 and Bax but induced expression of CDK2 and A20. Compared with B95.8-LMP1, 2117-LMP1 could induce a higher migration ability in NP69 cells but was less efficient in inducing morphologic changes, anchorage-independent growth and cell invasion. Relatively weaker ability of 2117-LMP1 than B95.8-LMP1 in upregulation of vimentin, VEGF and MMP9 as well as in downregulation of E-cadherin was observed. 2117-LMP1 could activate higher level of NF-kappaB activity in HEK 293 cells than B95.8-LMP1. The present study supports a role of 2117-LMP1 in NPC development by enhancing cell proliferation, cell death inhibition and migration in premalignant nasopharyngeal epithelial cells. Furthermore, our study reveals significant functional differences between 2117-LMP1 and the prototypic B95.8-LMP1. Our results provide insights into the pathologic significance of this prevalent LMP1 variant, 2117-LMP1, in the development of NPC in the Hong Kong population.

A single-chain antibody fragment (scFv) was constructed from a hybridoma antibody that binds to phosphorylcholine (PC) only when this hapten is presented in the form of the immunizing antigen (derived from Trichinella ) but not when it is presented on other carriers (as found, for example, in pneumococcal capsules). The scFv derivative was found to lack this carrier specificity as it bound indiscriminately, but specifically, to the various PC-associated antigens, and exhibits a two-fold lower affinity (3.5×10 5 M −1 ) for nitrophenyl-PC than the native antibody. The findings suggest that the scFv combining site is different in fine structure from that of the native antibody.

BRE, a putative stress-modulating gene, found able to down-regulate TNF-alpha-induced NF-kappaB activation upon overexpression, is now shown in human cells expressed as multiple mRNA isoforms. A total of six isoforms are produced by alternative splicing predominantly at either end of the gene. Predicted from the cDNA sequences of these isoforms, three of them (alpha(a), alpha(b), and alpha(c)) code for BRE of different C-terminus, and the other three (beta(a), beta(b), and beta(c)) may possibly be the nonfunctional counterparts. All human cells examined coexpress all the predominant splice variants, albeit at different ratios. Comparing with normal cells, immortalized human cell lines uniformly express higher levels of BRE. Interestingly, peripheral blood monocytes responded to LPS by down-regulating the expression of all the BRE isoforms, which was however less obvious in the cell line counterpart, THP-1. Isoform alpha(a), which codes for the canonical BRE with a C-terminal peroxisomal targeting sequence, is the most abundant transcript. We propose that the function of BRE and its isoforms is to regulate peroxisomal activities.

IgA nephropathy (IgAN) is characterized by raised serum IgA1 and mesangial IgA1 deposits. We have previously shown increased T-cell activation in IgAN. Recently, transforming growth factor-beta (TGF-beta) has been shown to induce IgA isotype switch at a clonal level and interleukin 5 (IL5) promotes differentiation into IgA-bearing B cells. In the present study we have examined the TGF-beta and IL5 mRNA expression by mitogen-activated CD4-positive T cells from patients with IgAN (n = 25), patients with other primary nephritides (CGN) (n = 24), and healthy control subjects (n = 25). The cytokine genes were analysed by reverse transcription (RT)-polymerase chain reaction (PCR) and were semi-quantitated by normalizing the differences occurring during RT and PCR using a housekeeping gene, beta-actin. CD4-positive T cells from IgA nephritic patients expressed a higher level of IL5 mRNA than healthy controls (P < 0.01) and patients with CGN (P < 0.005). CD4-positive T cells from IgA nephritic patients expressed a higher level of TGF-beta mRNA than healthy controls (P < 0.01) but no difference was demonstrated on comparison with CGN patients. Elevated TGF-beta mRNA expression in patients with CGN probably reflects its other important function as a 'sclerogenic' factor involved in the glomerulosclerosis found in these nephritides. Our data suggest that there is increased expression of cytokine genes which induce the IgA isotype switch and differentiation; these immunological abnormalities may be important in the pathogenesis of IgAN.

SUMMARYCytokinesare important mediators involved in the development of effector cells and in the regulation of immune responses. The gene expression of these mediators in T cell subset has yet to be fully elucidated. Using sensitive reverse transcription-polymerasc chain reaction (RT-PCR). the kinetics of cytokine gene expression in human CD4+ and CD8+ T cells were examined. CD4+ T cells were more readily activated by phorbol myrislatc acetate (PMA) and phytohaemagglutinin (PHA) than CD8+ T cells in terms of the IL-2 receptor (IL-2R) mRNA expression. Quantitative differences in cytokine gene expression between CD4+ and CD8+ T cells were confirmed and higher levels of cytokine mRNAs were induced in CD4+ than in CD8+ T cells. Early induction of IL-2 mRNA was observed in both T cell subsets. The demonstration of diffcrenl kinetics of cytokine gene expression illustrates one of the examples of the complexity of immunoregulation. The differential response of cytokine gene expression in different T cell subsets should be taken into consideration when clinical studies in cytokine production by peripheral blood mononuclcar cells arc interpreted.