Richard K. Barth

Structure, Organization and Polymorphism of Murine and Human T‐Cell Receptor a and β Chain Gene Families

T lymphocytes and B lymphocytes, the two major classes of effector cells in the vertebrate immune system, are capable of recognizing and responding to a virtually infinite range of antigenic determinants. Both populations of cells possess a membrane-bound antigen receptor which mediates the specific response of a given T cell or B cell to a particular antigen, resulting in subsequent antibody production and activation of cell-mediated defenses. While it has long been known that the antigen receptor of B lymphocytes is a membrane-bound immunoglobulin of the IgM class, the T-cell antigen receptor (TCR) has proven elusive to molecular characterization until recently. In 1983, using anticlonotypic monoclonal antibodies which affected antigen-specific activation of individual T-cell clones, Meuer et al. (1983) were able to demonstrate that the T-cell antigen receptor is a disulfide-linked heterodimeric glycoprotein composed of a and P chains, each with a molecular weight of approximately 45 kilodaltons. Tryptic peptide analysis of receptors from different T-cell populations revealed the presence of both highly conserved constant and considerably heterogenous variable structure regions consistent with the properties expected for the T-cell antigen receptor (Mclntyre & Allison 1983). Preparation of T cell-specific cDNA libraries by subtractive hybridization with total B cell poly(A)+ RNA and differential screening led to the determination of the nucleotide sequences of a, fi and y chains of the T-cell antigen receptor (Yanagi et al. 1984, Hedrick et al. 1984, Saito

The Roman god Janus: a paradigm for the function of CD43

Abstract Although the structural characterization of the major leukocyte cell surface mucin CD43 has proceeded rapidly, understanding its physiological function has been hindered by contradictory results. Here, Julie Ostberg and colleagues propose that the Roman god of two faces, Janus, provides a model that reconciles the roles attributed to CD43.

Fibroblast heterogeneity in the healing wound

Although fibroblasts are traditionally described as static cells providing framework and support for tissues, there is an accumulating body of evidence showing that fibroblasts are a dynamic cell type which exist in functionally and morphologically heterogeneous subpopulations. Fibroblast subsets have been shown to play a critical role in the production and regulation of extracellular matrix components, in wound repair and regeneration, and have been implicated in the pathogenesis of fibrotic conditions. We have reviewed the evidence supporting heterogeneity of fibroblasts from pulmonary, periodontal, and dermal tissues. In addition, we will explore the role fibroblast subpopulations may play in the complex process of wound repair and regeneration.

Restriction fragment length polymorphisms of the mouse T-cell receptor gene families

We have studied the restriction fragment length polymorphisms (RFLPs) found in the germline T-cell receptor genes of 25 inbred Mus musculus strains and 8 wild Mus species. Included in the inbred mice tested were several strains which spontaneously develop systemic autoimmune disease. Extensive polymorphism was evident for the variable (V) gene segments of the α gene family for both the inbred strains and wild mouse species. Changes in the total number of bands hybridizing with probes for Vα gene segments suggest that members of a Vα gene segment subfamily are not closely linked, but are interspersed with members of other subfamilies; that expansion and contraction of the multimembered subfamilies may be an important diversifying factor. Our data obtained with β gene probes revealed genomic diversity that is much more limited than that seen for the α locus. Analysis of inbred mice with probes for the γ gene locus revealed some RFLPs, but little evidence of expansion or contraction in the numbers of gene segments. Among the autoimmune mice, NZW, NZB, and BXSB/MpJ all display distinctive differences with α gene probes. NZW mice have a large deletion of the β gene family, which has been reported previously. We found no differences to distinguish the MRL/MpJ lpr/lpr mice from non-autoimmune strains.

Differential epitope expression of Ly-48 (mouse leukosialin)

Ly-48 is a major sialoglycoprotei expressed on the surface of variety of mouse hematopoietic cells that exhibits many characteristics isoforms and may function in signal transduction and cell adhension. Ly-48 is recognized by the 3E8-specific monoclonal antibody (mAb) and it has been suggested that it is the same antigen recognized by another mAb known as S7. In this report, we demonstrate definitively by transfection of a Ly-48 cDNA that S7 and two previously uncharacterized mAbs, S11 and S15, recognize the same antigen as teh 3E8-specific mAb. However, 2-D gel immunoblot analyses demonstrate the complex nature of Ly-48. Although all four mAbs react similarly with lysates from the M-45 B-cell myeloma line, 2-D immunobot analyses of the EL-4 T-cell line reveal three distinct patterns of reactivity. Further, while transfection of Ly-48 into the K562 erythroleukemic cell line conferred reactivity to all four mAbs, transfection of the Ly-48 cDNA into the nonhematopoietic cell line, Line 1, conferred reactivity only to the S11 and S15 mAbs. Thus, the Line 1 transfectants suggest the importance of posttranslational modifications in the expression of the 3E8 and S7 epitopes. Interestingly , developing fetal liver cells show the same pattern of differential Ly-48-specific mAb reactivity. The developing early fetal liver cells are reactive with S11 and S15 but are negative, to very weakly, reactive with the 3E8-and S7-specific mAbs. These results show that Ly-48 epitopes can be expressed independently on cell lines in vitro and are differentially expressed on healthy cells in vivo.

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes

Abstract Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans.

Molecular analysis of skewed Tcra-V gene use in T-cell receptor beta-chain transgenic mice.

Abstract The influence of β-chain diversity on the expressed T-cell receptor (TCR) α-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR β-chain gene. Analysis of these mice using α-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded β chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards α chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in β-transgenic mice is exhibited almost exclusively by CD4+ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8+ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed β chains can profoundly influence the selection of companion α chains expressed in the periphery, and that α-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3α domain participates in direct contact with the MHC.

Scattering of primary care: doctor switching and utilization of health care by children on fee-for-service Medicaid.

ObjectiveTo determine whether children on fee-for-service Medicaid who switch primary care doctors use less health care and are less up to date with preventive care visits than children who do not switch primary care doctors.DesignRetrospective cohort study using Medicaid claims data.Setting51,027 children enrolled on Medicaid in Monroe County, New York.Patients14,187 children enrolled continuously on fee-for-service Medicaid between January 1992 and December 1994.Main Outcome MeasuresUtilization of primary care, emergency department (ED) services, and specialty care and proportion up to date with preventive care visits according to American Academy of Pediatrics guidelines.ResultsDuring the 2-year study period, 22% of children switched primary care doctors. Compared with children who did not switch primary care doctors, those who switched had more primary care visits (4.7 vs. 3.2 visits/year,P<.01), age-adjusted preventive care visits (1.2 vs. 1.0 visits/year), ED visits (0.72 vs. 0.47 visits/year,P<.01), and specialist visits (0.99 vs. 0.31,P<.01). On multivariate analysis, doctor switching was associated with increased odds of being up to date with preventive care visits (odds ratio [OR]=1.7; 95% confidence interval [CI] 1.3 to 2.1). However, on multivariate analysis stratified by age, the association was significant only for older children (ages 11 to 14). Altogether, 68% of all children and 44% of infants less than 1 year old made the recommended number of preventive care visits during the study period.ConclusionsAll groups of children received less preventive care than recommended by the American Academy of Pediatrics. Children who switched primary care doctors had higher utilization of health care, including primary care, ED, and specialty care. Contrary to expectations, they were more likely to be up to date with preventive care visits. The heavy utilization of health services by doctor switchers indicates that this subgroup of children on Medicaid may not be at risk for poor access to health care, but additional research is needed to determine whether the quality of care is related to doctor switching.

Increasing the frequency of T‐cell precursors specific for a cryptic epitope of hen‐egg lysozyme converts it to an immunodominant epitope

Efforts to understand the mechanisms that govern how immunodominant T‐cell epitopes are selected from protein antigens have focused mostly on differences in the efficiency of processing and presentation of peptide/major histocompatibility complex (MHC) complexes by antigen‐presenting cells, while little attention has been directed at the role of the T‐cell repertoire. In this report, the influence of the T‐cell repertoire on immunodominance was investigated using transgenic mice that express the β chain from a T‐cell receptor specific for a cryptic Ek restricted epitope of hen‐egg lysozyme, HEL85‐96. In these mice, the frequency of HEL85‐96‐specific T‐cell precursors is increased 10–20‐fold over non‐transgenic mice. Transgenic mice respond as well as non‐transgenic controls to intact HEL, even though they respond poorly or not at all to a variety of other antigens, including the dominant H‐2k restricted epitopes of HEL. Following immunization with native HEL, the only HEL peptide that could recall a response in vitro in the transgenic mice was HEL85‐96. Therefore, this normally cryptic epitope is the sole immunodominant epitope in the transgenic mice, and this alteration in immune response is due solely to an increase in the frequency of specific T‐cell precursors. An analysis of four additional H‐2k restricted cryptic epitopes of HEL suggests that three are similarly limited by T‐cell frequency, and that only one is consistent with a defect in efficient antigen presentation. This indicates that there are at least two different types of cryptic epitopes, one in which crypticity is caused by inefficient processing or presentation, and another in which the frequency of specific T‐cell progenitors is limiting.

The Impact of Conjugate Pneumococcal Vaccination on Routine Childhood Vaccination and Primary Care Use in 2 Counties

BACKGROUND. Pneumococcal conjugate vaccine immunization recommendations were rapidly implemented by primary care providers. Before the recommendations, concern was expressed that adding pneumococcal conjugate vaccine might result in delays in other vaccinations or preventive services. OBJECTIVES. The study objectives were to measure whether incorporation of pneumococcal conjugate vaccine by primary care providers delayed other vaccinations or added primary health care visits. DESIGN AND METHODS. In 2 counties surrounding Rochester and Nashville, we reviewed a representative sample of primary care charts for children born before and after licensure of pneumococcal conjugate vaccine. Receipt of vaccinations and health care visits were compared for the 2 age-matched cohorts. RESULTS. We reviewed 1459 records from Rochester and 1857 records from Nashville. The pre–pneumococcal conjugate vaccine and post–pneumococcal conjugate vaccine cohorts had similar demographic characteristics. The median age for receipt of any vaccination was not older for the postvaccine cohort than for the prevaccine cohort in either community. The percentage of children up-to-date for vaccinations by 18 months for postvaccine versus prevaccine cohorts was similar in Rochester (72% in each cohort) and in Nashville (58% postvaccine and 65% prevaccine). The number of well-child care visits or other health care visits during the first 18 months of life was not statistically different between the 2 cohorts. CONCLUSIONS. Implementation of pneumococcal conjugate vaccine was not associated with delays in other childhood vaccinations or more primary care visits.