Characterization of a novel H2A(-)E+ transgenic model susceptible to heterologous but not self thyroglobulin in autoimmune thyroiditis: thyroiditis transfer with Vbeta8+ T cells.

Recently we reported on a novel H2E transgenic, IA-negative model of experimental autoimmune thyroiditis (EAT) that excludes reactivity to self in its susceptibility pattern to heterologous thyroglobulin (Tg). In conventional, susceptible mouse strains, EAT is inducible with both homologous and heterologous Tg; e.g., human (h)Tg shares conserved thyroiditogenic epitopes with mouse (m)Tg. However, when an H2Ea(k) transgene is introduced into class II-negative B10.Ab(0) mice, which express neither surface IA (mutant Abeta-chain) nor surface IE (nonfunctional Ea gene), the resultant H2E(b) molecules are permissive for EAT induction by hTg, but not self mTg. Also, the hTg-primed cells do not cross-react with mTg. To explore this unique capacity of E+B10.Ab(0) mice to distinguish self from nonself Tg, we have developed T cell lines to examine the T cell receptor repertoire and observed a consistent Vbeta8+ component after repeated hTg stimulation. Enrichment and activation of Vbeta8+ T cells by either superantigen staphylococcal entertoxin B or anti-Vbeta8 in vitro enabled thyroiditis transfer to untreated A-E+ recipients, similar to hTg activation. Vbeta8+ T cells isolated by FACS from hTg-immunized mice also proliferated to hTg in vitro. These studies support the contribution of Vbeta8 genes to the pathogenicity of hTg in this H2A-E+ transgenic model.     

Autoantibodies to thyroglobulin are encoded by diverse V-gene segments and recognize restricted epitopes.

Murine experimental autoimmune thyroiditis has been used as a model for human autoimmune thyroiditis. Experimental autoimmune thyroiditis is induced in mice by immunization with mouse thyroglobulin (Tg) in CFA. To characterize the antibodies to this autoantigen, we have studied the binding specificities and determined the nucleotide sequences of monoclonal anti-Tg antibodies. The specificities of the mAb for determinants on Tg varied extensively. Seven of 16 mAb showed reactivity to only mTg, 4 reacted to Tg from more than one species and four reacted to a variety of Ag. Many of the mAb were competitively inhibited by thyroid hormones, suggesting that they recognize the hormonogenic sites on the Tg molecule. The mAb could be divided into at least seven reactivity patterns based on reciprocal competitive inhibition studies, indicating that mTg contains at least seven antigenic regions. DNA sequence analysis of the mAb showed that a large number of V region gene segments encoded the H and L chains. No evidence for preferential use of any V region family or gene segment was found. Gene segments from the VH 7183, Q52, J558, and VH10 families were used by heavy chains, and the V kappa 1, 4, 8, 9, 19, and 21 families were used by kappa-chains. The results indicate that the antigenic epitopes on mTg elicit a very diverse autoantibody response that is derived from a large number of V region gene segments. Many of these autoantibodies show specific reactivity with mTg indicating they recognize species specific epitopes. The results suggest that clonal deletion of autoreactive Ab to certain self-epitopes may not occur.     

H2-E transgenic class II-negative mice can distinguish self from nonself in susceptibility to heterologous thyroglobulins in autoimmune thyroiditis

 Susceptibility to experimental autoimmune thyroiditis (EAT) is linked to H2-A class II genes;k and s haplotypes are susceptible, while b and f are resistant. EAT is inducible with thyroglobulins (Tgs) from several mammalian species which share portions of identical sequences. But cross-activation and cross-tolerance studies with mouse (m), human (h), and porcine (p) Tg have indicated mTg-unique T-cell epitope(s), in addition to conserved, in EAT induction. The recent introduction of the HLA-DRB1*0301 (DR3) transgene rendered major histocompatibility complex (MHC) class II-negative (Ab⁰) mice susceptible to EAT induction by both hTg and mTg, suggesting usage of conserved epitopes. Here, we introduced the H2-Ea ^k transgene into resistant B10 (H2 ^b ) or Ab⁰ mice with a defective Ea gene to provide functional surface H2E (b haplotype) expression. Surprisingly, both transgenic strains showed severe inflammation only after hTg, but not mTg, immunization, although the moderating influence of the A ^b gene in B10 was evident. In proliferative assays, hTg-primed cells did not respond to mTg, nor to conserved 12mer peptides from three primary hormonogenic sites, two of which can activate T cells for thyroiditis transfer and cytotoxicity. The vigorous response to hTg stimulation was reduced only by Eβ^b-specific monoclonal antibody. EAT induction with bovine and pTg showed responses similar to hTg, suggesting thyroiditogenic epitopes shared with hTg, but not mTg. This is the first demonstration of: (1) nonpermissiveness for EAT induction with mTg, normally the most thyroiditogenic Tg and the one with unique epitopes for susceptible mice, and (2) the separation of hTg from mTg in EAT induction in H2-E-transgenic mice. Received: 15 January 1999 / Revised: 23 April 1999     

A novel H2A-E+ transgenic model susceptible to human but not mouse thyroglobulin-induced autoimmune thyroiditis: identification of mouse pathogenic epitopes

The A⁻E⁺ transgenic mouse is highly susceptible to human thyroglobulin (hTg)-induced thyroiditis, but strongly tolerant to a challenge by mouse thyroglobulin (mTg), in stark contrast to traditionally susceptible strains, wherein mTg induces stronger thyroiditis. To identify mouse thyroid epitopes recognized by destructive, hTg-primed T cells, we selected the three hTg epitopes known to be presented by H2E^b, as the basis for synthesizing potential mTg epitopes. One 15-mer peptide, mTg409, did prime T cells, elicit Ab, and induce thyroiditis. Moreover, cells primed with corresponding, pathogenic hTg410 cross-reacted with mTg409, and vice versa. mTg409 contained 4/4 anchor residues, similar to the corresponding hTg peptide. Based on this finding, a second mTg epitope, mTg179, was subsequently identified. These mTg autoepitopes, identified by using thyroiditogenic hTg epitopes, help to explain the severe thyroiditis seen in this novel A⁻E⁺ transgenic model.     

H2E-derived Ealpha52-68 peptide presented by H2Ab interferes with clonal deletion of autoreactive T cells in autoimmune thyroiditis

Susceptibility and resistance to experimental autoimmune thyroiditis is encoded by MHC H2A genes. We reported that traditionally resistant B10 (H2(b)) mice permit thyroiditis induction with mouse thyroglobulin (mTg) after depleting regulatory T cells (Tregs), supporting A(b) presentation to thyroiditogenic T cells. Yet, Ea(k) transgenic mice, expressing A(b) and normally absent E(b) molecules (E(+)B10 mice), are susceptible to thyroiditis induction without Treg depletion. To explore the effect of E(b) expression on mTg presentation by A(b), seven putative A(b)-binding, 15-16-mer peptides were synthesized. Five were immunogenic for both B10 and E(+)B10 mice. The effect of E(b) expression was tested by competition with an Ealpha52-68 peptide, because Ealpha52-68 occupies approximately 15% of A(b) molecules in E(+)B10 mice, binding with high affinity. Ealpha52-68 competitively reduced the proliferative response to mTg, mTg1677, and mTg2342 of lymph node cells primed to each Ag. Moreover, mTg1677 induced mild thyroiditis in Treg-depleted B10 mice, and in E(+)B10 mice without the need for Treg depletion. Ealpha52-68 competition with mTg-derived peptides may impede clonal deletion of pathogenic, mTg-specific T cells in the thymus.     

High frequency of natural autoantibodies in normal newborn mice

Spleen cells from 6-day-old nonimmunized BALB/c and BALB.B10 mice were fused with the nonsecreting hybridoma cell line Sp2/0. Three hundred and eighty-four immunoglobulin-secreting hybrids were screened for antibody activity against mouse actin, tubulin, and myosin, and against TNP, peroxidase, renin, DNA, and neurofilaments. At least 24 hybridomas in the collection (6.25%) exhibited antibody activity against this panel of antigens. Ten of these hybrids were cloned, were propagated, and the corresponding monoclonal IgM protein was isolated from ascitic fluids and was further characterized. At least four groups of antibody specificities were identified: 1) one clone reacting with TNP only; 2) one clone reacting with both actin and tubulin; 3) two clones which bound to both TNP and actin; and 4) a fourth group, comprising the six other clones, which all exhibited widespread reactivity and bound to actin, tubulin, myosin, and TNP. These results indicate: 1) B cell clones directed against self antigens are activated in the internal environment and are recovered consequently by somatic cell hybridization; 2) the widespread antibody specificities found for these newborn mouse antibodies are very similar to those previously characterized with human natural antibodies and human monoclonal Ig; and 3) the frequency of B cells binding to cytoskeletal proteins and TNP is very high (at least 6.25%).     

Effective Elicitation of Human Effector CD8(+) T Cells in HLA-B*51:01 Transgenic Humanized Mice after Infection with HIV-1

Humanized mice are expected to be useful as small animal models for in vivo studies on the pathogenesis of infectious diseases. However, it is well known that human CD8(+) T cells cannot differentiate into effector cells in immunodeficient mice transplanted with only human CD34(+) hematopoietic stem cells (HSCs), because human T cells are not educated by HLA in the mouse thymus. We here established HLA-B*51:01 transgenic humanized mice by transplanting human CD34(+) HSCs into HLA-B*51:01 transgenic NOD/SCID/Jak3(-/-) mice (hNOK/B51Tg mice) and investigated whether human effector CD8(+) T cells would be elicited in the mice or in those infected with HIV-1 NL4-3. There were no differences in the frequency of late effector memory and effector subsets (CD27(low)CD28(-)CD45RA(+/-)CCR7(-) and CD27(-)CD28(-)CD45RA(+/-)CCR7(-), respectively) among human CD8(+) T cells and in that of human CD8(+) T cells expressing CX3CR1 and/or CXCR1 between hNOK/B51Tg and hNOK mice. In contrast, the frequency of late effector memory and effector CD8(+) T cell subsets and of those expressing CX3CR1 and/or CXCR1 was significantly higher in HIV-1-infected hNOK/B51Tg mice than in uninfected ones, whereas there was no difference in that of these subsets between HIV-1-infected and uninfected hNOK mice. These results suggest that hNOK/B51Tg mice had CD8(+) T cells that were capable of differentiating into effector T cells after viral antigen stimulation and had a greater ability to elicit effector CD8(+) T cells than hNOK ones.     

Induction of diabetes in nonobese diabetic mice by Th2 T cell clones from a TCR transgenic mouse

We have produced a panel of cloned T cell lines from the BDC-2.5 TCR transgenic (Tg) mouse that exhibit a Th2 cytokine phenotype in vitro but are highly diabetogenic in vivo. Unlike an earlier report in which T cells obtained from the Tg mouse were cultured for 1 wk under Th2-promoting conditions and were found to induce disease only in NOD.scid recipients, we found that long-term T cell clones with a fixed Th2 cytokine profile can transfer disease only to young nonobese diabetic (NOD) mice and never to NOD.scid recipients. Furthermore, the mechanism by which diabetes is transferred by a Tg Th2 T cell clone differs from that of the original CD4+ Th1 BDC-2.5 T cell clone made in this laboratory. Whereas the BDC-2.5 clone rapidly causes disease in NOD.scid recipients less than 2 wk old, the Tg Th2 T cell clones can do so only when cotransferred with other diabetogenic T cells, suggesting that the Th2 T cell requires the presence of host T cells for initiation of disease.     

Isolation of cDNAs encoding the CD19 antigen of human and mouse B lymphocytes. A new member of the immunoglobulin superfamily

The CD19 (B4) molecule is a m.w. 95,000 cell-surface protein of human B lymphocytes that is expressed before Ig and persists throughout differentiation. In this report, cDNA clones that encode the CD19 molecule were isolated and the amino acid sequence of CD19 was determined. A cDNA clone that selectively hybridized to RNA from CD19+ cell lines was selected from a human tonsilar cDNA library using differential hybridization. This cDNA was used to isolate additional cDNA clones. Four of the five longest cDNA clones isolated were sequenced and found to contain unique sequences presumed to be introns. One clone, pB4-19, was near full length (2.1 kb) and did not contain these putative introns. pB4-19 contained an 1685 bp open reading frame that could encode a protein of about 60 kDa. COS cells that were transfected with pB4-19 expressed a nascent cell surface structure reactive with the anti-B4 antibody. Immunoprecipitation of this structure from surface-iodinated COS cells with the anti-B4 antibody revealed a m.w. 85,000 protein. Northern blot analysis indicated that pB4-19 hybridized with a predominant mRNA species of 2.4 kb and a minor species of 1.5 kb, found in only CD19+ cells. The pre-B cell line, PB-697, also expressed four larger RNA species that hybridized with pB4-19. cDNA clones that encode the putative cytoplasmic portion (247 amino acids) of the mouse CD19 molecule were also isolated and found to be highly homologous (79 and 75%) with the human CD19 nucleotide and amino acid sequences. The deduced amino acid sequence of the CD19 cytoplasmic tail shared no significant homology with other known proteins but the putative extracellular region contained two Ig-like domains indicating that CD19 is a new member of the Ig superfamily.     

The IgG2a antibody response to thyroglobulin is linked to the Igh locus in mouse

The IgG-subclass usage by several strains of mice in the response to immunization with mouse thyroglobulin (mTg) was examined in the experimental autoimmune thyroiditis model. While the subclass usage by most mouse strains was similar, the Igh^b allotype-bearing mice consistently produced lower IgG2a levels to mTg. Using CBA-Igh ^b congenic and recombinant inbred strains of mice, the lower level of IgG2a in the Igh^b mouse was mapped to the Igh locus. The regulation of IgG2a appeared to be cis controlled, as the CBA x C57BL/6F₁ mouse also produced reduced IgG2a of the Igh^b (B6) allotype but not Igh^j (CBA) allotype.     

Assignment of the human gene for hexosaminidase B to chromosome 5

Mouse-human somatic cell hybrids between different mouse and human cells were studied for the expression of human hexosaminidases A and B activities. The expression of human hexosaminidase B in the hybrids was found to segregate concordantly with the presence of the human chromosome 5. Mouse-human hybrid clones containing either the human chromosomes 5 and 7 only or the human chromosome 7 only were also included in this study. Expression of human hexosaminidase B activity was detected only in those clones containing human chromosome 5. These results indicate that the gene(s) for human hexosaminidase B is located on chromosome 5. No hexosaminidase A activity was detected in clones which contained either human chromosomes 5 and 7 or chromosome 7.     

Tolerance induction in antigen-specific helper T cell clones and lines in vitro

The induction of T cell tolerance in vitro was investigated by using HGG-specific murine helper T cell (Th) clones and cell lines. It was found that exposure of Th to monomeric HGG (tolerogen) for 18 hr rendered the Th unable to reconstitute the PFC response of HGG-primed B cells. The tolerant state was not a result of Th cell death, as up to 100% of Th could be recovered after exposure to the monomer, and in addition, the recovered cells proliferated in response to IL2. B cells were shown not to be significantly affected by the presence of monomeric HGG in amounts calculated to be carried over from the tolerization cultures into the assay cultures. Consequently, it was concluded that interaction between Th and monomeric HGG induced unresponsiveness at the T cell level. A comparison of the tolerogenic potential of monomeric, soluble, and aggregated HGG revealed that only the monomer could induce tolerance in Th. Monomeric HGG was also shown to induce tolerance in an antigen-specific manner. Th reactive to HGG could be tolerized by monomeric HGG, but not Th reactive to FGG or OVA. Helper function of Th was also shown to be antigen specific in that HGG-reactive Th helped only HGG-primed B cells. Certain HGG-specific Th clones were found to be refractory to tolerization with monomeric HGG, whereas other clones derived from the same uncloned cell line were tolerizable.     

Preferential retention of the human chromosome C-7 in human-(thymidine kinase deficient) mouse hybrid cells

On karyotyping human—mouse hybrid cells derived from various parental crosses, we found that if the mouse parental cells were thymidine-kinase deficient, the hybrid clones retained not only the human chromosome E-17 containing the thymidinekinase gene, but a high proportion (82%) also contained the human C-7 chromosome. Other human chromosomes were also found in these clones.     

Somatic cell hybrids of mouse myeloma cells and B lymphocytes from a patient with agammaglobulinemia: failure to secrete human immunoglobulin.

Somatic cell hybrid clones were isolated from the fusion of RPC5.4 mouse myeloma cells and B lymphocytes from a patient with common varied agammaglobuinemia. The patient has B lymphocytes that synthesize immunoglobulin but fail to secrete immunoglobulin. The hybrid character of the six clones was established by examination of metaphase chromosome spreads. Most of the hybrid clones expressed mouse and human surface immunoglobulin. All of the clones synthesized immunoglobulin of mouse and human parental origin. Mouse parental immunoglobulin was secreted, whereas the human parental immunoglobulin was not secreted. Human light chain molecules were secreted as part of hybrid H2L2 molecules formed with mouse heavy chains. Human heavy chains had a reduced m.w. in comparison to the mouse heavy chains. Kinetic experiments indicated that human Ig was synthesized in amounts that were comparable to the mouse Ig. Pulse-chase experiments showed that that the intracellular human Ig was removed from the cytoplasm, probably by degradation. These experiments demonstrate that the hybrid cells are an in vitro model of naturally occurring failure of immunoglobulin secretion from agammaglobulinemia. The failure of fusion with mouse myeloma cells to complement the secretion defect suggests that these B cells produce an altered immunoglobulin molecule that is not programmed for secretion.     

Analysis of human monoclonal antibodies derived from lymphocytes of patients with cancer

Human lymphocytes from tumor-bearing patients and normal individuals have been fused with the NS-1 mouse myeloma line or the LICR -LON- HMY2 ( LICR -2) or SK0 -007 human cell lines. For a given number of lymphocytes, fusions with NS-1 produced 8 times more clones than fusions with LICR -2 and greater than 20 times more clones than fusions with SK0 -007. The percentage of clones that secrete human immunoglobulin (Ig) and the range of Ig production were comparable for clones derived from the three myeloma/lymphoblastoid lines. Clones derived from fusions with LICR -2 and SK0 -007 were found to secrete new species of light and heavy Ig chains in addition to those of the myeloma/lymphoblastoid lines, and clones derived from fusions with NS-1 secreted human Ig and contained both mouse and human chromosomes, which indicates that true hybrid cells were derived from fusions with each of the myeloma/lymphoblastoid lines under study. The stability of Ig production was similar for clones derived from fusions with NS-1, LICR -2, or SK0 -007; these results were comparable to those obtained with standard mouse/mouse hybrids. Stable clones producing human monoclonal antibodies that react with cell surface, cytoplasmic, cytoskeletal, nuclear, or nucleolar antigens have been isolated from tumor-bearing patients and normal individuals. A number of human monoclonal antibodies reactive with cytoskeletal antigens appear to be directed against components of the intermediate filament family. Techniques for the production of human monoclonal antibody appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer.     

Identification and immunological characterization of a novel 40-kDa protein linked to CD98 antigen.

Monoclonal antibodies (mAbs) were obtained from hybridoma clones established by cell fusion between mouse myeloma cells and spleen cells from a mouse immunized against an affinity-purified 40-kDa component of rat 125-kDa glycoprotein (GP125). Two mAbs designated as 3F2 and 6B4 detected a 40-kDa and a 125-kDa band under reducing and nonreducing conditions, respectively, in extracts prepared from rat, mouse and human tumor cells. Association of the 40-kDa protein with CD98 was revealed by sandwich-type enzyme-linked immunosorbent assay. The two mAbs were strongly reactive with various tumor cells and activated lymphocytes, but were only weakly reactive with resting lymphocytes. Confocal microscopy indicated colocalization of CD98 and the 40-kDa protein defined with 3F2 and 6B4 at the cell surface and perinuclear regions. On immunohistochemical analysis of frozen sections of rat tongue, the anti-rat CD98 mAb B3 selectively stained the basal layer and 3F2 stained the upper epithelial part in addition to the basal layer, indicating the existence of CD98-unlinked 40-kDa protein.     

Identification of a thyroiditogenic sequence within the thyroglobulin molecule.

Thyroglobulin (Tg)-specific T cells are important in the induction of experimental autoimmune thyroiditis (EAT), but the nature and the number of the Tg T cell epitopes involved in the disease process are unknown. Through the use of computerized algorithms that search for putative T cell epitopes, a 17-mer peptide (TgP1) was identified within the known portion of the rat Tg sequence (corresponding to amino acids 2495 to 2511 of the human Tg sequence) that induced strong mononuclear cell infiltration of the thyroid in classic EAT-susceptible murine strains such as SJL, C3H, and B10.BR and low or undetectable infiltration in EAT-resistant strains such as BALB/c and B10. TgP1 appears to be phylogenetically conserved since it is completely homologous to its bovine counterpart and differs at a single amino acid position from its human analogue. After priming with TgP1 in vivo, significant proliferative T cell responses to TgP1 in vitro were observed only with lymphocytes from susceptible (high responder) strains, thus correlating proliferative capacity with EAT induction. TgP1-primed T cells did not respond to intact mouse Tg (MTg) or rat Tg in vitro and, conversely, T cells primed in vivo with MTg or rat Tg did not respond to TgP1 in culture, suggesting that TgP1 is comprised of non-immunodominant T cell determinants. TgP1 was defined as a serologically nonimmunodominant epitope as well, since in vivo priming of all strains with MTg led to strong MTg-specific IgG responses but no TgP1-specific responses in ELISA assays. This was not due to lack of immunogenic B cell determinants on TgP1, however, because peptide challenge of EAT-susceptible strains elicited TgP1-specific IgG that also cross-reacted with MTg and rat, human, bovine, and porcine Tg. The data demonstrate that TgP1 delineates nonimmunodominant but highly immunogenic determinants at both the T and B cell level, which may play an important role in the development of autoimmune thyroiditis.     

Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-1-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-1-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Newborn and Wiskott-Aldrich patient B cells can be activated by TNP-Brucella abortus: evidence that TNP-Brucella abortus behaves as a T-independent type 1 antigen in humans

TNP-Brucella abortus (TNP-Ba) has been classified as a T-independent type 1 (TI-1) antigen in the mouse on the basis that it activates neonatal and CBA/N (X-linked immunodeficient) murine B cells in contrast to T-independent type 2 (TI-2) antigens. Therefore, it was of interest to determine whether human newborn and X-linked Wiskott-Aldrich syndrome B cells could be triggered by TNP-Ba. Previous studies had shown that human B cells from both these latter sources were relatively insensitive to stimulation with T-dependent and polysaccharide antigens (TI-2 in mouse). In this study, we show that TNP-Ba can trigger human cord blood B cells to differentiate into anti-TNP plaque-forming cells (PFC) in a hapten-specific and T-independent manner. The dose response and kinetics were similar to those previously seen with adult cells. The newborn responses, however, were lower than adult PFC responses. Precursor frequency and clone size analyses revealed that this lower response was not due to newborn cells containing fewer precursors but was the result of a reduced ability of these anti-TNP clones to expand. The ability of TNP-Ba to activate immature newborn B cells implies that this antigen can be used to assess B cell function in very young children. It also implies that TNP-Ba behaves as a TI-1 antigen in humans as well as in mice. This was supported by the finding that B cells from Wiskott-Aldrich patients, which were unreactive to polysaccharide antigens, were generally responsive to TNP-Ba. Therefore, it would appear that human newborn and Wiskott-Aldrich patients do possess a functionally competent B cell subset possibly equivalent to Lyb-5- immature murine B cells.     

The 5'-flanking region of the human CGL-1/granzyme B gene targets expression of a reporter gene to activated T-lymphocytes in transgenic mice.

The human CSP-B/CGL-1 gene is the homologue of the mouse granzyme B/CCPI gene and encodes a cytotoxic T-lymphocyte-specific serine protease. We have used regulatory sequences upstream from the CSP-B gene to drive human growth hormone gene expression in transgenic mice. Eleven founder mice were screened for transgene expression in activated T-cells. Expression was detected in 10 mice; levels of expression were integration site-dependent. The transgene was not expressed in resting lymphocytes but could be activated by treatment with concanavalin A or interleukin-2, indicating that CSP-B regulatory sequences are responsive to signals originating at either the T-cell receptor or the interleukin-2 receptor. Transgene expression was detected at the whole organ level only in lymph nodes and small intestine, where endogenous mouse CCPI mRNA was also present. The time course of transgene activation in T-lymphocytes was similar to that of the mouse CCPI gene. No differences in levels of expression of the transgene were observed in activated lymphocyte populations that had been depleted of either CD4+ or CD8+ cells; in contrast, the mouse CCPI gene was expressed primarily in CD8+ cells. Six CD4+ T-cell clones with Th0, Th1, or Th2 phenotypes were generated from a transgenic animal. All clones expressed moderate to high levels of the transgene, but only three clones expressed mouse CCPI, indicating that the transgene is disregulated in CD4+ T-cell subsets. The CSP-B regulatory unit represents a novel reagent for targeting gene expression to activated T-lymphocytes.