Melanoma cells can be eliminated by sialylated CD43 × CD3 bispecific T cell engager formats in vitro and in vivo.

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells.

Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Antibodies specific for the RSV prefusion F protein have guided RSV vaccine research, and in human serum, these antibodies contribute to >90% of the neutralization response; however, detailed insight into the composition of the human B cell repertoire against RSV is still largely unknown. In order to study the B cell repertoire of three healthy donors for specificity against RSV, CD27+ memory B cells were isolated and immortalized using BCL6 and Bcl-xL. Of the circulating memory B cells, 0.35% recognized RSV-A2-infected cells, of which 59% were IgA-expressing cells and 41% were IgG-expressing cells. When we generated monoclonal B cells selected for high binding to RSV-infected cells, 44.5% of IgG-expressing B cells and 56% of IgA-expressing B cells reacted to the F protein, while, unexpectedly, 41.5% of IgG-expressing B cells and 44% of IgA expressing B cells reacted to the G protein. Analysis of the G-specific antibodies revealed that 4 different domains on the G protein were recognized. These epitopes predicted cross-reactivity between RSV strain A (RSV-A) and RSV-B and matched the potency of antibodies to neutralize RSV in HEp-2 cells and in primary epithelial cell cultures. G-specific antibodies were also able to induce antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of RSV-A2-infected cells. However, these processes did not seem to depend on a specific epitope. In conclusion, healthy adults harbor a diverse repertoire of RSV glycoprotein-specific antibodies with a broad range of effector functions that likely play an important role in antiviral immunity.IMPORTANCE Human RSV remains the most common cause of severe lower respiratory tract disease in premature babies, young infants, the elderly, and immunocompromised patients and plays an important role in asthma exacerbations. In developing countries, RSV lower respiratory tract disease has a high mortality. Without an effective vaccine, only passive immunization with palivizumab is approved for prophylactic treatment. However, highly potent RSV-specific monoclonal antibodies could potentially serve as a therapeutic treatment and contribute to disease control and mortality reduction. In addition, these antibodies could guide further vaccine development. In this study, we isolated and characterized several novel antibodies directed at the RSV G protein. This information can add to our understanding and treatment of RSV disease.

Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis.

UNLABELLED: The family Picornaviridae is a large and diverse group of positive-sense RNA viruses, including human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral, and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity make HPeVs a good target for development of therapeutic human monoclonal antibodies (MAbs). In this study, we generated two different human MAbs specific for HPeV by screening culture supernatants of Ab-producing human B cell cultures for direct neutralization of HPeV1. Both MAbs showed HPeV1-specific neutralization as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, -5, and -6 and coxsackievirus A9 (CV-A9). VP1 capsid protein-specific assays confirmed that AM18 bound VP1 of HPeV1, -2, and -4 with high affinity (11.5 pM). In contrast, the HPeV1-specific MAb AM28, which neutralized HPeV1 even more efficiently than did AM18, showed no cross-reactivity with HPeV3 to -6 or other EVs and did not bind any of the capsid proteins, suggesting that AM28 is specific for a conformation-dependent, nonlinear epitope on the virus. The discovery of MAbs that are cross-reactive between HPeVs may help development of HPeV treatment options with antibodies and vaccine design based on epitopes recognized by these antibodies. IMPORTANCE: HPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given that the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with MAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human MAbs. Both MAbs show HPeV1-specific neutralization and cross-neutralized HPeV2. One MAb also cross-neutralized other HPeVs. Surprisingly, this MAb also neutralized CV-A9. These MAbs provide a unique tool for further research and for the diagnosis (antigen detection) and possible treatment of HPeV infections.

Id2 and Id3 inhibit development of CD34(+) stem cells into predendritic cell (pre-DC)2 but not into pre-DC1. Evidence for a lymphoid origin of pre-DC2.

We found previously that Id3, which inhibits transcriptional activities of many basic helix-loop-helix transcription factors, blocked T and B cell development but stimulated natural killer (NK) cell development. Here we report that ectopic expression of Id3 and another Id protein, Id2, strongly inhibited the development of primitive CD34(+)CD38(-) progenitor cells into CD123(high) dendritic cell (DC)2 precursors. In contrast, development of CD34(+)CD38(-) cells into CD4(+)CD14(+) DC1 precursors and mature DC1 was not affected by ectopic Id2 or Id3 expression. These observations support the notion of a common origin of DC2 precursors, T and B cells. As Id proteins did not block development of NK cells, a model presents itself in which these proteins drive common lymphoid precursors to develop into NK cells by inhibiting their options to develop into T cells, B cells, and pre-DC2.

NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human T cells.

We have developed a new method that allows detection and isolation of viable, antigen-specific, human T cells from a heterogeneous pool of T cells. We have engineered a self-inactivating retroviral vector containing multiple (3 or 6) nuclear factor of activated T-cell (NFAT)-binding sites, followed by the minimal IL2 promoter and the reporter gene GFP. Jurkat cells, primary T-cell blasts, and T-cell clones were transduced with high efficiency (20%-40%). Stimulation of the transduced cells with phorbol myristate acetate (PMA) and ionomycin resulted in a high level expression of GFP that was maximal after 12 to 14 hours and remained stable for another 12 hours. Activation of T cells carrying the construct containing 6 NFAT-binding sites resulted in the highest mean fluorescence intensity. Cyclosporin-A and FK506 were able to block the activation-dependent GFP expression. Activation of transduced T-cell blasts with the superantigen staphylococcal enterotoxin B or of transduced antigen-specific T-cell clones with cognate antigen resulted in GFP expression. After an overnight stimulation of a heterogeneous T-cell bulk culture with an HLA mismatched stimulator cell (JY), the GFP expressing cells were cloned. As expected, the cloning frequency of the antigen-specific GFP(+) cells was considerably higher than that of the total T-cell population. Most of the T-cell clones were either cytolytic, or proliferative toward JY stimulator cells. Interestingly, we also isolated T-cell clones that were noncytolytic and nonproliferative toward JY cells, but specifically up-regulated GFP after an overnight stimulation with JY. (Blood. 2000;96:459-466)

Genetic modification of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3.

Transgenic and gene targeted mice have contributed greatly to our understanding of the mechanisms underlying B-cell development. We describe here a model system that allows us to apply molecular genetic techniques to the analysis of human B-cell development. We constructed a retroviral vector with a multiple cloning site connected to a gene encoding green fluorescent protein by an internal ribosomal entry site. Human CD34(+)CD38(-) fetal liver cells, cultured overnight in a combination of stem cell factor and interleukin-7 (IL-7), could be transduced with 30% efficiency. We ligated the gene encoding the dominant negative helix loop helix (HLH) factor Id3 that inhibits many enhancing basic HLH transcription factors into this vector. CD34(+)CD38(-) FL cells were transduced with Id3-IRES-GFP and cultured with the murine stromal cell line S17. In addition, we cultured the transduced cells in a reaggregate culture system with an SV-transformed human fibroblast cell line (SV19). It was observed that overexpression of Id3 inhibited development of B cells in both culture systems. B-cell development was arrested at a stage before expression of the IL-7Ralpha. The development of CD34(+)CD38(-) cells into CD14(+) myeloid cells in the S17 system was not inhibited by overexpression of Id3. Moreover, Id3(+) cells, although inhibited in their B-cell development, were still able to develop into natural killer (NK) cells when cultured in a combination of Flt-3L, IL-7, and IL-15. These findings confirm the essential role of bHLH factors in B-cell development and demonstrate the feasibility of retrovirus-mediated gene transfer as a tool to genetically modify human B-cell development.

HGF/SF and its receptor c-MET play a minor role in the dissemination of human B-lymphoma cells in SCID mice.

The MET protooncogene, c-MET, encodes a cell surface tyrosine kinase receptor. The ligand for c-MET is hepatocyte growth factor (HGF), also known as scatter factor (SF), which is known to affect proliferation and motility of primarily epithelial cells. Recently, HGF/SF was also shown to affect haemopoiesis. Studies with epithelial and transfected NIH3T3 cells indicated that the HGF/SF-c-MET interaction promotes invasion in vitro and in vivo. We previously demonstrated that HGF/SF induces adhesion of c-MET-positive B-lymphoma cells to extracellular matrix molecules, and promoted migration and invasion in in vitro assays. Here, the effect of HGF/SF on tumorigenicity of c-MET-positive and c-MET-negative human B-lymphoma cell lines was studied in C.B-17 scid/scid (severe combined immune deficient) mice. Intravenously (i.v.) injected c-MET-positive (BJAB) as well as c-MET-negative (Daudi and Ramos cells) B-lymphoma cells formed tumours in SCID mice. The B-lymphoma cells invaded different organs, such as liver, kidney, lymph nodes, lung, gonads and the central nervous system. We assessed the effect of human HGF/SF on the dissemination of the B-lymphoma cells and found that administration of 5 microg HGF/SF to mice, injected (i.v.) with c-MET-positive lymphoma cells, significantly (P = 0.018) increased the number of metastases in lung, liver and lymph nodes. In addition, HGF/SF did not significantly influence dissemination of c-MET-negative lymphoma cells (P = 0.350 with Daudi cells and P= 0.353 with Ramos cells). Thus the effect of administration of HGF/SF on invasion of lymphoma cells is not an indirect one, e.g. via an effect on endothelial cells. Finally, we investigated the effect of HGF/SF on dissemination of c-MET-transduced Ramos cells. In response to HGF/SF, c-MET-transduced Ramos cells showed an increased migration through Matrigel in Boyden chambers compared to wild-type and control-transduced Ramos cells. The dissemination pattern of c-MET-transduced cells did not differ from control cells in in vivo experiments using SCID mice. Also no effect of HGF/SF administration could be documented, in contrast to the in vitro experiments. From our experiments can be concluded that the HGF/SF-c-MET interaction only plays a minor role in the dissemination of human B-lymphoma cells.

Enrichment of an antigen-specific T cell response by retrovirally transduced human dendritic cells.

The superior ability of dendritic cells (DC) in triggering antigen-specific T cell responses makes these cells attractive tools for the generation of antitumor or antiviral immunity. We report here an efficient retroviral transduction system for the introduction of antigens into DC. A retroviral vector encoding several CTL epitopes in a string-of-beads fashion in combination with the marker gene green fluorescence protein (GFP) was generated. Polyepitope transduced EBV-LCL could be isolated on the basis of GFP expression and were found to be sensitive to lysis by antigen-specific cytotoxic T cells, demonstrating that antigens encoded by the retroviral construct were stably expressed, processed, and presented in the context of HLA class I molecules. CD34(+) cells isolated from G-CSF mobilized peripheral blood were transduced with high efficiency (40-60%) with this retroviral construct. These cells could be considerably expanded in vitro and differentiated into mature DC without loss of the transduced antigen. DC transduced with the polyepitope constructs were able to mount a CTL response against an influenza epitope in the context of HLA-A2, demonstrating the antigen-specific CTL priming capacity of retrovirally transduced DC. Staining of the T cells with tetramers of HLA-A2 and the influenza virus peptide demonstrated a marked antigen-specific CTL enrichment after 2 in vitro stimulations using DC transduced with the polyepitope. However, additional in vitro stimulations of the T cells with transduced DC did not result in a further enrichment of tetramer staining cells.

Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors.

Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint.

TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation.

Recent studies have identified several populations of progenitor cells in the human thymus. The hematopoietic precursor activity of these populations has been determined. The most primitive human thymocytes express high levels of CD34 and lack CD1a. These cells acquire CD1a and differentiate into CD4(+)CD8(+) through CD3(-)CD4(+)CD8(-) and CD3(-)CD4(+) CD8alpha+beta- intermediate populations. The status of gene rearrangements in the various TCR loci, in particular of TCRdelta and TCRgamma, has not been analyzed in detail. In the present study we have determined the status of TCR gene rearrangements of early human postnatal thymocyte subpopulations by Southern blot analysis. Our results indicate that TCRdelta rearrangements initiate in CD34(+)CD1a- cells preceding those in the TCRgamma and TCRbeta loci that commence in CD34(+)CD1a+ cells. Furthermore, we have examined at which cellular stage TCRbeta selection occurs in humans. We analyzed expression of cytoplasmic TCRbeta and cell-surface CD3 on thymocytes that lack a mature TCRalphabeta. In addition, we overexpressed a constitutive-active mutant of p56(lckF505) by retrovirus-mediated gene transfer in sequential stages of T-cell development and analyzed the effect in a fetal thymic organ culture system. Evidence is presented that TCRbeta selection in humans is initiated at the transition of the CD3(-)CD4(+)CD8(-) into the CD4(+)CD8alpha+beta- stage.

Green fluorescent protein as a selectable marker of fibronectin-facilitated retroviral gene transfer in primary human T lymphocytes.

The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of gene transfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes to mammalian cells and improvement of transduction protocols remains dependent on markers that can be rapidly monitored and used for efficient selection of transduced cells. The enhanced green fluorescent protein (EGFP) is a suitable reporter molecule for gene expression because of its lack of cytotoxicity and stable fluorescence signal that can be readily detected by flow cytometry. However, attempts to adapt the GFP system to stable transduction of human lymphocytes have not been satisfactory. In this article, transductions of primary human T lymphocytes were performed using cell-free supernatants from a PG13 packaging cell line in which a retroviral vector expressing EGFP was pseudotyped with the gibbon ape leukemia virus (GALV) envelope. Using this system combined with a fibronectin-facilitated protocol, primary lymphocytes were transduced with a mean gene transfer efficiency of 27.5% following a 2-day stimulation with either PHA or anti-CD3/CD28 antibodies. Conditions that increased the entry of lymphocytes into cell cycle did not consistently correlate with enhanced gene transfer, indicating that factors other than proliferation are important for optimal retroviral gene transfer. These results demonstrate the utility of EGFP as a marker for human T cell transduction and will enable further optimization of T cell gene therapy protocols.

Functional analysis of Ran/TC4 as a protein regulating T-cell costimulation.

Antigen (Ag)-triggered activation of T cells requires engagement of both the T-cell Ag receptor and a costimulatory receptor, for which CD28 can function as a prototypical example. CD80 and CD86 represent ligands for this receptor, and although they are present on professional Ag-presenting cells, these molecules are absent from most tumors. Yet some tumors are still able to costimulate a T-cell response, while others cannot. Therefore, a key question concerns the molecular basis for the costimulation of T cells by those tumor cells not expressing the CD28 ligands CD80 and CD86. Upon screening a cDNA library of such a tumor cell line in a transient COS cell transfection assay for costimulatory activity, we identified Ran/TC4 as a protein whose overexpression results in costimulatory activity. Ran/TC4 is a ubiquitously expressed member of the Ras gene superfamily of small guanosine triphosphate-binding proteins and is involved in nuclear transport; Ran/TC4 cDNA-transfected COS cells specifically costimulate CD8 T cells and not CD4 T cells. Transfection of Ran/TC4 into the costimulation-deficient murine RMA lymphoma cell line introduced costimulatory capacity for CD8 T cells and resulted in markedly elevated levels of nuclear Ran/TC4 protein expression. In addition, in vivo priming of mice with Ran/TC4-transfected RMA cells induced protection against wild-type (wt) RMA tumor cells. Ran/TC4-transfected RMA cells and wt RMA tumor cells exhibit comparable in vivo growth rates in mice lacking T and B cells, and Ran/TC4-mediated tumor rejection thus involves B and/or T cells. This possibility is substantiated by the observation that T cells from normal mice challenged with Ran/TC4-transfected RMA cells can mount a cytotoxic T-cell response not only against the Ran/TC4-transfected tumor cells but also against wt RMA tumor cells. Based on these results, we conclude that gene transfer-mediated elevations in Ran/TC4 can confer costimulatory function for CD8 T cells to tumor cells. This finding suggests a novel application of Ran/TC4 as a protein capable of regulating costimulation in tumor cells.

Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3.

Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

Expression of tyrosine kinase gene in mouse thymic stromal cells.

Amongst the most important signal transduction molecules involved in regulating growth and differentiation are the protein tyrosine kinases (PTK). Since T cell development is a consequence of interactions between thymic stromal cells (TSC) and thymocytes, identification of the PTK in both compartments is required to dissect the mechanisms that control this process. Here we report a search for PTK in mouse TSC, using RT-PCR to survey the repertoire of PTK mRNAs expressed in a freshly isolated TSC preparation. We identified 10 different PTK cDNAs among the 216 cDNAs sequenced, and demonstrate that transcripts of three of those (ufo, fyn and fer) are widely expressed among a large panel of immortalized thymic epithelial cell lines (TEC) and in primary cultures of TSC. Of the other seven, none were expressed in established TEC lines but, instead, displayed distinct expression patterns in cell types likely to have contaminated the fresh TSC preparation, i.e., macrophages, B cells, T cells and fibroblasts. Among the three PTK expressed in TEC lines, only one, ufo, exhibited expression exclusively in cells of non-hemopoietic origin. Although expression of ufo (also known as tyro 7, axl or ark) is not thymic-specific, in that it is also expressed in cell types of mesodermal origin in other tissues, its presence in TEC suggests a role for ufo in differentiation of the TSC compartment. Consistent with this notion, high-level expression of this receptor PTK at the protein level could be documented in every TEC line investigated, as well as in fresh thymus tissue sections. These data provide the first example of a receptor PTK in TSC and open new approaches to study the regulation of TSC differentiation.

Use of bicistronic retroviral vectors encoding the LacZ gene together with a gene of interest: a method to select producer cells and follow transduced target cells.

The coordinate expression of a marker gene and a therapeutic gene in one retroviral vector has considerable advantages. High-titer producer lines can potentially be selected on the basis of marker gene expression, and the expression of transduced genes in target cells can readily be followed. Moreover, target cells with stable high expression can be selected before use in therapeutic protocols or research questions. We used internal ribosomal entry site (IRES) sequences to express two genes in the same retroviral vector. We used the LacZ gene as the marker gene and the cytokine interleukin (IL)-7 or dominant negative (dn) forms of the T-cell tyrosine kinases ZAP-70 and lck as genes of interest. Amphotropic packaging cells transfected with MFG-IL-7-IRES-LacZ, MFG-dnZAP-70-IRES-LacZ, or MFG-dnlck-IRES-LacZ were sorted on the basis of beta-galactosidase expression. These LacZ-positive producer cells also expressed the gene of interest, produced high-titer retrovirus, and were capable of efficiently transducing Jurkat T cells and T-cell clones. When MFG-IL-7-IRES-LacZ-transduced Jurkat T cells were sorted on the basis of LacZ expression, a positive correlation with the amount of IL-7 produced by these cells was found. This demonstrates that selection of the LacZ marker gene also selects for cells that express the gene of interest at high levels. Moreover, T cells transduced with the dn tyrosine kinases and selected on the basis of LacZ expression showed functional alterations after T-cell receptor stimulation, demonstrating that retrovirally transduced signaling molecules can alter the function of T cells.

DNA sequence analysis of spontaneous mutations at a LacZ transgene integrated on the mouse X chromosome.

Transgenic mice with integrated shuttle vectors containing the LacZ mutational target gene were used to study spontaneous mutational events in vivo. The transgenic mouse strain used carries the LacZ transgene on the X chromosome and was previously found to be characterized by approximately 25-fold higher spontaneous mutation frequency in liver and brain compared with at least three other transgenic mouse strains. To determine the nature of in vivo spontaneous mutational events, 35 mutant LacZ genes isolated from liver and brain of mice from strain 35.5 were analyzed at the DNA sequence level. The results obtained indicate that single base-pair changes were predominant in both liver and brain. However, in liver the majority of mutations were transitions whereas in brain transversions were predominantly observed. Six mutants appeared to contain multiple dispersed mutations, separated by as much as 44 bp. Mutations were generally located within a 500 bp region encoding the active site of the beta-galactosidase protein. Our results indicate that spontaneous mutations at the LacZ transgene are tissue specific and dependent on the chromosomal position of the LacZ transgene.