Retroviral Vector-mediated Gene Transfer Research Articles

Chronic Expression of Murine flt3 Ligand in Mice Results in Increased Circulating White Blood Cell Levels and Abnormal Cellular Infiltrates Associated With Splenic Fibrosis

The effect of chronic expression of flt3 ligand (FL) on in vivo hematopoiesis was studied. Retroviral vector-mediated gene transfer was used in a mouse model of bone marrow transplantation to enforce expression of mouse FL cDNA in hematopoietic tissues. As early as 2 weeks posttransplantation, peripheral blood white blood cell counts in FL-overexpressing recipients were significantly elevated compared with controls. With the exception of eosinophils, all nucleated cell lineages studied were similarly affected in these animals. Experimental animals also exhibited severe anemia and progressive loss of marrow-derived erythropoiesis. All of the FL-overexpressing animals, but none of the controls, died between 10 and 13 weeks posttransplantation. Upon histological examination, severe splenomegaly was noted, with progressive fibrosis and infiltration by abnormal lymphoreticular cells. Abnormal cell infiltration also occurred in other organ systems, including bone marrow and liver. In situ immunocytochemistry on liver sections showed that the cellular infiltrate was CD3+/NLDC145+/CD11c+, but B220− and F4/80−, suggestive of a mixed infiltrate of dendritic cells and activated T lymphocytes. Infiltration of splenic blood vessel perivascular spaces resulted in vascular compression and eventual occlusion, leading to splenic necrosis consistent with infarction. These results show that FL can affect both myeloid and lymphoid cell lineages in vivo and further demonstrate the potential toxicity of in vivo treatment with FL.

Chronic Expression of Murine flt3 Ligand in Mice Results in Increased Circulating White Blood Cell Levels and Abnormal Cellular Infiltrates Associated With Splenic Fibrosis

AbstractThe effect of chronic expression of flt3 ligand (FL) on in vivo hematopoiesis was studied. Retroviral vector-mediated gene transfer was used in a mouse model of bone marrow transplantation to enforce expression of mouse FL cDNA in hematopoietic tissues. As early as 2 weeks posttransplantation, peripheral blood white blood cell counts in FL-overexpressing recipients were significantly elevated compared with controls. With the exception of eosinophils, all nucleated cell lineages studied were similarly affected in these animals. Experimental animals also exhibited severe anemia and progressive loss of marrow-derived erythropoiesis. All of the FL-overexpressing animals, but none of the controls, died between 10 and 13 weeks posttransplantation. Upon histological examination, severe splenomegaly was noted, with progressive fibrosis and infiltration by abnormal lymphoreticular cells. Abnormal cell infiltration also occurred in other organ systems, including bone marrow and liver. In situ immunocytochemistry on liver sections showed that the cellular infiltrate was CD3+/NLDC145+/CD11c+, but B220− and F4/80−, suggestive of a mixed infiltrate of dendritic cells and activated T lymphocytes. Infiltration of splenic blood vessel perivascular spaces resulted in vascular compression and eventual occlusion, leading to splenic necrosis consistent with infarction. These results show that FL can affect both myeloid and lymphoid cell lineages in vivo and further demonstrate the potential toxicity of in vivo treatment with FL.

Involvement of Retinoic Acid Receptor-α–Mediated Signaling Pathway in Induction of CD38 Cell-Surface Antigen

Human leukocyte antigen CD38, a 45-kD single-chain, transmembrane glycoprotein, is a bifunctional ectoenzyme that participates in signal transduction pathways involved in the regulation of cell growth and differentiation. In this study, we demonstrate the nature of retinoid receptors involved in retinoic acid–induced expression of CD38 protein in the human myeloblastic leukemia cell line HL-60. We used a variant HL-60 cell line, HL-60R, in which retinoid receptor function has been abrogated by a trans-dominant negative mutation. We introduced the normal retinoic acid receptors (RAR)-α, -β, and -γ or retinoid X receptor (RXR)-α into HL-60R cells by retroviral vector-mediated gene transfer. Based on experiments using these cell lines and receptor-specific synthetic retinoids that preferentially bind to one of the RARs or RXRs, we conclude that RAR-α is involved in retinoid-induced CD38 expression in HL-60 cells. Further evidence included our demonstration that blocking of RAR-α with the antagonist Ro 41-5253 completely suppressed the retinoid-induced expression of CD38 mRNA transcript and the production of CD38 protein in HL-60 cells. Various tissues from transgenic mice that expressed an antisense construct of RAR-α lacked or produced very low levels of CD38. As expected, the tissues from transgenic mice contained 50% to 80% reduced levels of RAR-α. These results suggest that regulation of CD38 expression, both in vitro and in vivo, is under the direct control of RAR-α retinoid receptors.

Involvement of Retinoic Acid Receptor-α–Mediated Signaling Pathway in Induction of CD38 Cell-Surface Antigen

AbstractHuman leukocyte antigen CD38, a 45-kD single-chain, transmembrane glycoprotein, is a bifunctional ectoenzyme that participates in signal transduction pathways involved in the regulation of cell growth and differentiation. In this study, we demonstrate the nature of retinoid receptors involved in retinoic acid–induced expression of CD38 protein in the human myeloblastic leukemia cell line HL-60. We used a variant HL-60 cell line, HL-60R, in which retinoid receptor function has been abrogated by a trans-dominant negative mutation. We introduced the normal retinoic acid receptors (RAR)-α, -β, and -γ or retinoid X receptor (RXR)-α into HL-60R cells by retroviral vector-mediated gene transfer. Based on experiments using these cell lines and receptor-specific synthetic retinoids that preferentially bind to one of the RARs or RXRs, we conclude that RAR-α is involved in retinoid-induced CD38 expression in HL-60 cells. Further evidence included our demonstration that blocking of RAR-α with the antagonist Ro 41-5253 completely suppressed the retinoid-induced expression of CD38 mRNA transcript and the production of CD38 protein in HL-60 cells. Various tissues from transgenic mice that expressed an antisense construct of RAR-α lacked or produced very low levels of CD38. As expected, the tissues from transgenic mice contained 50% to 80% reduced levels of RAR-α. These results suggest that regulation of CD38 expression, both in vitro and in vivo, is under the direct control of RAR-α retinoid receptors.

Antigen presentation in retroviral vector-mediated gene transfer in vivo.

We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte/macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vector-immunized mice were able to prime naive T cells in vitro, and adoptive transfer of in vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.

Possible Involvement of Bcl-2 Pathway in Retinoid X Receptor Alpha-Induced Apoptosis of HL-60 Cells

Retinoids induce granulocytic differentiation and subsequent apoptosis in human myeloid (HL-60) leukemia cells. Differentiation is induced due to activation of retinoic acid receptors (RARs) whereas, activation of retinoid X receptors (RXRs) seems to be essential for driving these cells into apoptosis. In order to understand the mechanism of RXR induced apoptosis, we used a variant HL-60 cell line (HL-60R) with a trans-dominant negative mutation. The retroviral vector-mediated gene transfer was used to introduce the functional RARs or RXRα into HL-60R cells. We studied the effect of receptor-selective retinoid treatment on the expression of Bcl-2 and Bax oncogenes by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis in RARs and RXRα transfected HL-60 cells. Our results show that activation of RXRα results in apoptosis via down-modulation of Bcl-2 mRNA as well as its gene product expression with no change in Bax mRNA expression.

Correction of Sanfilippo A skin fibroblasts by retroviral vector-mediated gene transfer.

The recent cloning of the sulfamidase gene has made possible the consideration of gene-based therapies for Sanfilippo A syndrome (mucopolysaccharidosis type IIIA), one of the most common of the mucopolysaccharidoses. In this paper, we present the construction of a retroviral vector in which a sulfamidase cDNA is under the transcriptional control of the Moloney murine leukemia virus long terminal repeat. This construct was used to make a high-titer (4 x 10(5) colony-forming units/ml) producer cell line, PA317/LNSSN#19, in the amphotropic packaging cell line PA317. This producer cell line was shown to be helper virus free using an assay for horizontal spread of virus. Virus supernatant from PA317/LNSSN#19 was used to transduce Sanfilippo A fibroblasts, resulting in complete correction of both the enzymatic defect and the storage phenotype as assessed by intracellular accumulation of 35SO4(-)-labeled material. Phenotypic correction was seen even when the levels of viral transduction were low. These results show that gene therapy of the Sanfilippo A syndrome is practicable, although the nature of the disorder suggests that careful consideration needs to be given to the choice of the cellular target for gene transfer.

Gene transfer into hematopoietic stem cells of nonhuman primates.

Nonhuman primates provide an appropriate preclinical large-animal model to test the efficacy of bone marrow gene therapy procedures. Successful retroviral vector-mediated gene transfer into monkey pluripotent hematopoietic stem cells (PHSC) has closed the gap between gene transfer experiments in mouse models and clinical application of bone marrow gene therapy. After initial bone marrow transplant failures, ex vivo bone marrow culture conditions were found that sufficiently supported maintenance of the long-term repopulating ability of genetically modified autologous monkey grafts. The efficiency of gene transfer into primate PHSC has, however, remained at least one order of magnitude lower than has been achieved in mice. Similar gene transfer efficiencies have been obtained with total bone marrow grafts, CD34+ bone marrow grafts, and mobilized peripheral blood progenitor cell grafts; however, various attempts to increase the transduction efficiency have been without significant success. Primate PHSC seem to require quite different culture conditions for their maintenance and transduction than mouse PHSC, in particular regarding hematopoietic growth factor addition. In contrast to observations in other species, some form of conditioning appeared essential for engraftment of transduced PHSC in monkeys. Although it has been shown that mouse retroviruses can replicate in monkeys and are capable of inducing neoplasms, experiments in monkeys have sufficiently confirmed the safety of current gene transfer procedures to allow their clinical application.

Retroviral Vector-Mediated Transfer of the Tumor Necrosis Factor α Gene Into Human Cancer Cells Restores an Apoptotic Cell Death Program and Induces a Bystander-Killing Effect

Tumor necrosis factor alpha (TNFalpha) may induce tumor cell death by apoptosis, the physiologic program of cell death usually lost during neoplastic progression. However, many tumor cells are resistant to its effect unless high doses are administered. By retroviral vector-mediated gene transfer, we have transduced the TNFalpha gene into the DNA of human tumor cells to investigate whether the indefinite neoplastic cell proliferation could be blocked and the lost physiologic program of cell death restored. Evidence is provided that high-TNFalpha-producing clones generated from a human lymphoma T-cell line (ST4) can undergo apoptosis following transduction of the TNFalpha gene. Internucleosomal DNA cleavage was documented by May-Grünwald-Giemsa and by propidium iodide staining, as well as by gel electrophoresis. The induced apoptotic phenomenon is TNFalpha-mediated, since it can be reverted following incubation with anti-TNFalpha monoclonal antibodies (MoAbs), and it occurs with cytokine levels released in the supernatant by the engineered cells much lower(>100 times) than those required to promote the same effect on parental ST4 cells following administration of exogenous recombinant TNFalpha. The process is associated with a downregulation of the apoptosis-preventing gene, bcl-2, while the expression of bax and p53, genes usually involved in promoting apoptosis, persists. Mixed-culture experiments performed coincubating TNFalpha-transduced and untransduced ST4 cells allowed documentation of a bystander-killing effect on the parental cells. This phenomenon still occurred at transduced to parental cell ratios as low as 1:20 and was blocked in the presence of an anti-TNFalpha MoAb. These findings indicated that TNFalpha may play a regulatory role in the proliferation of human tumor cells, and suggest potential new antitumor therapeutic strategies based on the direct delivery of the TNFalpha gene into cancer cells.

A Peripheral Blood-Derived Monolayer Supports Long-Term Cultures of Human CD4+ and CD8+ T Lymphocytes

An in vitro culture system has been developed for the long-term maintenance of primary, human peripheral blood and umbilical cord blood T lymphocytes, which does not rely on the use of stimulatory cytokines, antigen, or mitogens. In these cultures, a monolayer of adherent cells, some spindle-shaped and some resembling macrophages, developed within a week. All adherent cells were positive for the extracellular matrix proteins laminin and fibronectin, the intermediate filament vimentin, and for the surface markers major histocompatibility complex class II, platelet-endothelial cell adhesion molecule l (CD31), and E-Selectin (ELAM-1; CD62E). They were negative for the leukocyte common antigen (CD45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Factor VIII-related antigen. These monolayers supported the maintenance of nonadherent, resting, mature T cells for up to 3 months, and these cells retained their ability to respond to mitogens and allogeneic cells. Both CD4+ and CD8+ cells were supported. The proportion of CD4+ and CD8+ cells remained unchanged after 3 months in culture. We have also used T cells from 2-month-old cultures as target cells for retroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retroviral vector. The infection efficiency was similar to that obtained for fresh peripheral blood T cells, indicating that the long-term-cultured cells might be suitable for certain gene therapy applications.

Retroviral transfer of HSV1-TK gene into human lung cancer cell line.

We used a recombinant retrovirus as one of the potential vectors for human gene therapy to transfer a drug sensitivity gene into human lung cancer cells. The gene encoding the thymidine kinase (TK) of herpes simplex virus type 1 (HSV1) was used as the drug sensitivity gene. The antiherpes drugs acyclovir (ACV) and ganciclovir (GCV) were chosen to test the HSV1-TK activity transferred into the human lung cancer cell lines. The rationale for this approach was that ACV and GCV are nucleoside analogs specifically converted by HSV1-TK to a toxic form capable of inhibiting DNA synthesis or disrupting cellular DNA replication. The results obtained from our experiments demonstrate that the retroviral vector-mediated HSV1-TK gene transfer leads to ACV- and GCV-dependent cytotoxicity in human lung cancer cell lines, including both small-cell carcinoma and non-small-cell carcinoma. Although the gene transfer of HSV1-TK gene into tumor cells would be one model for gene therapy to control lung cancer, further investigations are necessary for the proper choice of the therapeutic gene and vector targeting such as tumor cell specific delivery of the gene or tumor cell specific expression of the transduced gene.

Gene therapy using retroviral vectors.

Gene therapy is a novel approach for treating various congenital and acquired genetic disorders, including cancer, heart disease, and acquired immune deficiency syndrome. Amongst possible gene delivery systems, retroviral vector mediated gene transfer has been the most extensively studied and has been approved for use in over 40 current Phase I/II clinical trials for the treatment of various disorders, primarily cancers. Recent technological improvements include the optimization of vector production by concentration and lyophilization, resulting in high titers of vectors, as well as the large-scale production of vector-produced cells for the treatment of brain cancer. Present clinical protocols require specialized care centers with expertise in molecular biology and cell transplantation. Considerable effort is under way to develop retroviral vectors that can be both injected directly into the body and targeted to specific cell types within the body. Such vectors could be administered to patients by physicians in their offices. Successful development of this new technology would greatly expand the clinical potential of gene therapy.

Cross-Correction of β-Glucuronidase Deficiency by Retroviral Vector-Mediated Gene Transfer

An in vitro model for cross-correction of lysosomal storage disorders from genetically modified cells was developed to approximate the physiological conditions needed for gene therapy in vivo. β-Glucuronidase (GUSB)-deficient mucopolysaccharidosis (MPS) type VII (Sly disease) cells were studied to determine the amount and stability of enzyme transfer. Gene transfer by retroviral vectors encoding rat or human GUSB corrected the enzymatic deficiency in cultured MPS VII fibroblasts from humans, dogs, and mice. The vector-transduced cells released GUSB into the culture supernatant in amounts proportional to the amounts of cellular GUSB activity. The activity of the exported GUSB was stable in tissue culture medium for at least two weeks. Uninfected MPS VII target cells incorporated GUSB from the medium when the vector-infected donor cells were separated from the deficient target cells by a fluid-permeable membrane. The high level of GUSB in the cross-corrected target cells decreased rapidly after the enzyme donor cells were removed.

A murine model for B-lymphocyte somatic cell gene therapy.

Mature primary B lymphocytes represent a potentially important cellular target for somatic cell gene therapy, which could prove advantageous for the treatment of certain metabolic and immunologic disorders. Their capacity to serve as antigen-presenting cells could be utilized for triggering and/or potentiating immune responses to tumors and viruses. Alternatively, B cells expressing an autoantigen could be manipulated to induce antigen-specific unresponsiveness for treatment of autoimmune diseases. Efficient expression of an exogenous gene product in long-lived B lymphocytes could be particularly useful for providing a corrected gene product in the bloodstream. Despite these advantages, efficient gene transfer into mature primary B cells has not been reported. One reason for this is that current protocols for retroviral vector-mediated gene transfer into lymphocytes rely on in vitro expansion and/or drug selection. This precludes the use of mature primary B cells as targets, since they cannot be readily cultured for long periods of time. In this report, we describe an efficient and rapid protocol for the introduction of exogenous genes into primary B cells without the need for drug selection. We have used retroviral vectors containing the human adenosine deaminase gene as a marker gene, since the biological activity of this enzyme is easy to measure and is readily distinguishable from that of the endogenous mouse adenosine deaminase. Upon adoptive transfer into SCID mice, infected B cells continuously expressing one to three copies of the human adenosine deaminase gene could be found in the spleens of recipient animals for at least 3 months.

Pantropic Retroviral Vector-Mediated Gene Transfer, Integration, and Expression in Cultured Newt Limb Cells

Limb regeneration is a unique developmental phenomenon restricted to certain urodeles in which limb cells dedifferentiate and produce the blastema and then redifferentiate into the tissues that compose the missing part. Genetic modification of the blastema cells would greatly facilitate understanding the programmed gene expression that results in the reconstitution of the limb. To test whether pantropic retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein could mediate gene transfer into blastema cells, we infected a stable newt limb cell line and demonstrated integration and expression of the provirus. Thus, pantropic retroviral vectors offer a new tool for the study of limb regeneration and other developmental phenomena in amphibia.

Efficient Retrovirus-Mediated Gene Transduction Into Murine Hematopoietic Stem Cells and Long-Lasting Expression Using a Transwell Coculture System

The neomycin phosphotransferase (neo) gene was transduced into murine hematopoietic stem cells by culturing a recombinant retrovirus-producing cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene transduction into stem cells retaining long-term reconstitution ability was successfully performed by using protocols of total 15 to 18 days of culture including establishment of the Dexter culture, transduction, and G418 selection. In the irradiated recipients of these cells, a large majority of the BM, thymus, and spleen cells as well as peripheral blood (PB) leukocytes were of donor origin and the neo gene was present in these organs up to 21 weeks after cell transfer. One third to two thirds of the in vitro colony-forming cells in the BM of the recipient mice were resistant to cultivation with G418. It was further found that the hematopoietic system of secondary recipients given BM cells from a primary recipient mouse was predominated by original donor-type cells. The transduced neo gene was detected in the PB, BM, thymus, and spleen cells of these secondary recipients. These results indicate that our procedure of retroviral vector-mediated gene transfer is highly effective in safely introducing a gene into pluripotent hematopoietic stem cells.

Efficient retrovirus-mediated gene transduction into murine hematopoietic stem cells and long-lasting expression using a Transwell coculture system

The neomycin phosphotransferase (neo) gene was transduced into murine hematopoietic stem cells by culturing a recombinant retrovirus- producing cell line in a Transwell (Costar, Cambridge, MA) (bottomed with a porous membrane) hung into a Dexter-type long-term bone marrow (BM) culture. Gene transduction into stem cells retaining long-term reconstitution ability was successfully performed by using protocols of total 15 to 18 days of culture including establishment of the Dexter culture, transduction, and G418 selection. In the irradiated recipients of these cells, a large majority of the BM, thymus, and spleen cells as well as peripheral blood (PB) leukocytes were of donor origin and the neo gene was present in these organs up to 21 weeks after cell transfer. One third to two thirds of the in vitro colony-forming cells in the BM of the recipient mice were resistant to cultivation with G418. It was further found that the hematopoietic system of secondary recipients given BM cells from a primary recipient mouse was predominated by original donor-type cells. The transduced neo gene was detected in the PB, BM, thymus, and spleen cells of these secondary recipients. These results indicate that our procedure of retroviral vector-mediated gene transfer is highly effective in safely introducing a gene into pluripotent hematopoietic stem cells.

Characterization of recombinant plasminogen activator production by primate endothelial cells transduced with retroviral vectors

Retroviral vector-mediated expression of plasminogen activators (PAs) from endothelial cells (ECs) has been proposed as a potential therapeutic approach for intravascular thrombosis. To define the potential for gene transfer to increase fibrinolytic activity in a primate system, baboon ECs were transduced with retroviral vectors expressing wild-type and glycosylphosphatidylinositol-anchored urokinase, as well as wild-type and serpin-resistant tissue PA (t-PA). Expression of either t-PA or urokinase was increased by one log over baseline levels. There was no specific effect of either t-PA or urokinase overexpression on endogenous t-PA, urokinase, or PA inhibitor 1 (PAI-1) expression. Recombinant urokinase could be anchored to the cell surface at a level eight-fold above that of receptor-bound urokinase. The majority of secreted urokinase accumulated in conditioned medium as a free proenzyme, whereas both wild-type and serpin-resistant t-PA accumulated almost exclusively in complexes with PAI-1. In most but not all of the assays, the urokinase vectors conferred PA activity above that of the t-PA vectors. These data show that PA synthesis and activity are specifically increased subsequent to retroviral vector-mediated gene transfer in primate ECs. However, definition of an optimal PA vector will require in vivo experimentation.

Characterization of Recombinant Plasminogen Activator Production by Primate Endothelial Cells Transduced With Retroviral Vectors

Retroviral vector-mediated expression of plasminogen activators (PAs) from endothelial cells (ECs) has been proposed as a potential therapeutic approach for intravascular thrombosis. To define the potential for gene transfer to increase fibrinolytic activity in a primate system, baboon ECs were transduced with retroviral vectors expressing wild-type and glycosylphosphatidylinositol-anchored urokinase, as well as wild-type and serpin-resistant tissue PA (t-PA). Expression of either t-PA or urokinase was increased by one log over baseline levels. There was no specific effect of either t-PA or urokinase overexpression on endogenous t-PA, urokinase, or PA inhibitor 1 (PAI-1) expression. Recombinant urokinase could be anchored to the cell surface at a level eight-fold above that of receptor-bound urokinase. The majority of secreted urokinase accumulated in conditioned medium as a free proenzyme, whereas both wild-type and serpin-resistant t-PA accumulated almost exclusively in complexes with PAI-1. In most but not all of the assays, the urokinase vectors conferred PA activity above that of the t-PA vectors. These data show that PA synthesis and activity are specifically increased subsequent to retroviral vector-mediated gene transfer in primate ECs. However, definition of an optimal PA vector will require in vivo experimentation.

Conserved amino acid residues in the complementarity-determining region 1 of the TCR beta-chain are involved in the recognition of conventional Ag and Mls-1 superantigen.

Superantigens activate T cells by interacting primarily with the V beta region of the TCR. This report describes a series of studies performed to elucidate the role of the conserved amino acid motif (Asp-His-Asn) in the complementarity-determining region 1 (CDR1) of the TCR V beta chains that recognize murine endogenous superantigen Mls-1. By using site-directed mutagenesis of the Mls-1-reactive TCR V beta 6 gene, it is shown that the alterations of the conserved CDR1 motif disrupt Mls-1 superantigen and conventional Ag recognition in vitro. The loss of V beta 6 (mutant)+ TCR reactivity to Mls-1 superantigen is apparently dependent on the partner alpha-chain in the V beta 6/V alpha 3 TCR pairing shows some reactivity to Mls-1, whereas other TCR pairings do not. The examination of the developmental fate of the mutated form of the V beta 6 chain in Mls-1+ mice by using retroviral vector-mediated gene transfer confirms the critical role played by the CDR1 residues in Mls-1 recognition in vivo. Collectively, the results indicate that the CDR1 of the TCR V beta 6 chain is involved in interacting with peptide/MHC as well as in Mls-1 recognition. The observation that the conserved residues in selective TCR V beta chains are imparting a significant contribution to Ag recognition suggests a molecular basis for the intrinsic bias of some V beta chains for MHC molecules.