Protection of CD3 delta knockout mice from lymphocytic choriomeningitis virus-induced immunopathology: implications for viral neuroinvasion.

For a virus to establish a neuronal infection, it must spread from its primary site of infection to the central nervous system (CNS) before immune-mediated clearance occurs. Lymphocytic choriomeningitis virus (LCMV) is a murine pathogen that can result in persistent neuronal infection in newborn mice and in adults that lack CD8(+) T cells. To determine the neuroinvasive capacity of LCMV in the presence of an existent, but compromised, cytotoxic T lymphocyte response, the course of LCMV infection was examined in mice that possess 10% of the normal complement of T lymphocytes, due to the lack of the CD3 delta (delta) subunit of the T cell receptor complex (CD3 delta KO mice). Unlike immunocompetent mice that produced a massive immune response that caused death by 6-7 days postinfection, CD3 delta KO mice mounted a weak response and survived. The presence of viral antigen gradually shifted from the class I MHC-positive meninges and ependyma to class I MHC-deficient CNS neurons 10-30 days postinoculation. The infected CD3 delta KO mice developed a delayed T cell response that suppressed virus replication in peripheral tissues but not in the CNS; subsequent adoptive transfer experiments supported the hypothesis that the lack of clearance from neurons was due to sequestration of LCMV in an immune-privileged cell type. Based on these results, we propose that a critical parameter in the pathogenesis of neurotropic viruses is the rate of immune activation; individuals with impaired T cell responses may be more vulnerable to persisting CNS infections.

Limiting TCR expression leads to quantitative but not qualitative changes in thymic selection.

Thymic selection is controlled in part by the avidity of the interaction between thymocytes and APCs. In agreement, the selective outcome can be modulated by altering the expression levels of selecting ligands on APCs. Here we test the converse proposition, i. e., whether changing TCR levels on thymocytes can alter the selective outcome. To this end, we have generated mice in which all thymocytes express two transgenic TCRs simultaneously (dual TCR-expressing (DTE) mice), the class I-restricted HY TCR and the class II-restricted AND TCR. Due to mutual dilution, surface expression levels of the two individual transgenic TCRs are diminished in DTE relative to single TCR-expressing mice. We find that thymic selection is highly sensitive to these reductions in TCR surface expression. Positive selection mediated by the AND and HY TCRs is severely impaired or abolished, respectively. Negative selection of the HY TCR in male DTE mice is also partly blocked, leading to the appearance of significant numbers of double positive thymocytes. Also, in the periphery of male, but not female, DTE mice, substantial numbers of single positive CD8bright cells accumulate, which are positively selected in the thymus but by a highly inefficient hemopoietic cell-dependent process. Overall our results favor the interpretation that the outcome of thymic selection is not determined solely by avidity and the resulting signal intensity, but is also constrained by other factors such as the nature of the ligand and/or its presentation by different subsets of APCs.

Altered functional responsiveness of thymocyte subsets from CD3delta-deficient mice to TCR-CD3 engagement.

CD3delta-deficient (delta degrees) mice are defective in alphabeta T cell development. Here we explore the capacity of TCR-CD3 signaling complexes expressed on delta degrees thymocytes to mediate the following functional outcomes in response to antibody cross-linking: (i) the transition from the CD4-CD8- to CD4+CD8+ stage, (ii) the transition from the CD4+CD8+ to CD4+CD8- or CD4-CD8+ stages and (iii) the induction of apoptosis. We provide evidence that CD3deltaepsilon complexes are dispensable for mediating the anti-CD3-mediated CD4-CD8- to CD4+CD8+ transition. On the other hand, CD3delta is critical at the CD4+CD8+ stage. We demonstrate that CD4+CD8+ thymocytes from delta degrees mice, unlike delta degrees CD4-CD8- thymocytes and wild-type CD4+CD8+ thymocytes, require prolonged or consecutive stimuli to elicit functional responses. Depending on the nature of the secondary stimulus, delta degrees thymocytes can be induced to undergo apoptosis or preferential maturation to the CD4-CD8+ stage. Taken together these results indicate that the signaling capacity of the TCR-CD3 complex is noticeably altered in the absence of CD3delta. The essential role of CD3delta at the CD4+CD8+ stage of development correlates with the onset of TCRalpha rearrangement, consistent with a critical structural and/or functional relationship between CD3delta and TCRalpha.

HD mice: a novel mouse mutant with a specific defect in the generation of CD4(+) T cells.

We have identified a spontaneous mutation in mice, which we term HD for "helper T cell deficient." This mouse is distinguished by the virtual absence of peripheral T cells of the CD4(+)8(-) major histocompatibility complex (MHC) class II-restricted T helper subset due to a specific block in thymic development. The developmental defect is selective for CD4(+)8(-) cells; the maturation of CD4(-)8(+) and gamma delta T cells is normal. The autosomal recessive mutation underlying the HD phenotype is unrelated to MHC class II, since it segregates independently of the MHC class II locus. Moreover, the HD phenotype is not caused by a defect of the CD4 gene. Bone marrow transfer experiments demonstrate that the defect is intrinsic to cells of the hematopoietic lineage, i.e., most likely to developing thymocytes themselves. The frequency of CD4(+)8(low) intermediate cells is markedly increased in HD mice, suggesting that class II-restricted thymocytes are arrested at this stage. This is the first genetic defect of its kind to be described in the mouse and may prove highly informative in understanding the molecular pathways underlying lineage commitment.

CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage.

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR-mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3 delta chain is analyzed by generating a mouse line specifically lacking this component (delta-/- mice). Strikingly, CD3 delta is shown to be differentially required during development. In particular, CD3 delta is not needed for steps in development mediated by pre-TCR or gamma delta TCR, but is required for further development of thymocytes expressing alpha beta TCR. Absence of CD3 delta specifically blocks the thymic selection processes that mediate the transition from the double-positive to single-positive stages of development.

Inter- and intra-patient sequence diversity among parainfluenza virus-type 1 nucleoprotein genes.

Parainfluenza viruses (PIV) have been categorized into four discrete types (types 1-4), based on antigenic similarities. Here is described an evaluation of nucleoprotein (NP) sequence variability among nine patients infected with the type 1 virus. The examination of short segments of the NP sequence was sufficient to define significant variability both within and between patient samples. These data, in conjunction with previous studies of hemagglutinin-neuraminidase and fusion protein sequences from PIV-infected patient populations suggest a lack of absolute stability among isolates within each virus type. Potentially, antigenic variability exists to the extent that an immune response elicited toward one isolate may not be fully protective against another of the same type. Thus, sequence variability could contribute to natural re-infections with PIV, as well as to previous vaccine failures. Results highlight the importance of analyzing viruses that break through vaccine-induced immunity, in order to measure the influence of virus diversity on PIV vaccine outcome.

Gene rearrangement patterning and DNase-I hypersensitive sites within the T-cell receptor J alpha locus.

For several years, the relationship between alpha beta and gamma delta T-cell progenitors has been a topic of debate. Some argue that a subset of T-cell progenitors is "pre-committed" to the alpha beta lineage and is thus programmed to rearrange alpha, but not delta genes. It is further argued that the deletion of the delta locus by a unique rearrangement, delta rec-psi J alpha, may be the critical forerunner to V-J alpha joins in alpha beta committed cells and that a hypersensitive site (HS) termed 5'TEA might regulate such rearrangement. Here we present an alternative hypothesis. We first emphasize that directed J alpha gene rearrangements do not exclusively target the psi J alpha gene, but that clustered gene rearrangements occur throughout the J alpha locus during T-cell development. We describe the existence of not one, but at least two HS sites distributed along the J alpha locus which might serve as regulators for the gene rearrangement event. Finally, we suggest that progenitor T-cells are not committed to a particular delta or alpha gene rearrangement, but that a flexible progenitor responds to complex interactions between environmental signals and multiple regulatory elements interspersed among delta/alpha genes.

Viral cross-reactivity and antigenic determinants recognized by human parainfluenza virus type 1-specific cytotoxic T-cells.

To obtain information relevant to vaccination against human parainfluenza virus type 1 (hPIV-1), cytotoxic T-lymphocyte (CTL) responses to individual viral components were tested. The CD8-positive T-cell fraction was first enriched from human, adult PBL and grown for several passages in the presence of hPIV-1-infected stimulator cells. T-cell lines were then tested for CTL activity toward hPIV-1 and toward the related viruses hPIV-3 and Sendai virus (the murine parainfluenza type 1 virus). All tested cultures which responded to hPIV-1 also responded to hPIV-3 and Sendai virus, demonstrating sequence conservation between all three viruses among major antigenic determinants for CTL. Specificity for particular viral components was defined using recombinant vaccinia viruses expressing individual proteins from either mouse or human parainfluenza type 1 viruses. Strong CTL responses toward hemagglutinin-neuraminidase, phosphoprotein, and nucleoprotein (NP) were demonstrated. The testing of vaccinia constructs expressing truncated proteins then showed that there were multiple CTL determinants within NP. Several T-cell lines from one donor recognized an NP peptide (amino acids 321-336) conserved between the hPIV-1 and Sendai virus. In total, the results demonstrated that the human CTL response is directed to multiple determinants within several distinct hPIV-1 proteins.

Restricted usage of T-cell receptor V alpha sequence and variable-joining pairs after normal T-cell development and bone marrow transplantation.

TCR V alpha 3 and V alpha 5 transcripts in PBLs from healthy individuals of multiple age groups and from BMT recipients were analyzed. PCR, cloning, and sequencing studies revealed significant V-J junctional diversity among TCR transcripts from all tested blood samples, as provided both by N/P-region addition and exonuclease activity. However, results illustrated restrictions in TCR alpha diversity at several additional levels. First, V alpha 5 and V alpha 3 gene families, which were expected to be composed of multiple members, were dominated in each case by a single sequence at the transcript level. Second, restrictions existed in V-J pairing in that J alpha genes, which were encoded toward the 5' region of the locus, were rearranged frequently with V alpha 3 and rarely with V alpha 5. Conversely, J alpha genes encoded toward the 3' region of the locus preferentially rearranged with V alpha 5. Healthy individuals showed few differences with regard to V-J pairing patterns, while one of three BMT recipients demonstrated a skewed usage of 3' J alpha genes. In total, results demonstrated qualitative restrictions that may limit the working TCR repertoire in human peripheral tissues, both among BMT recipients and their healthy donors.

Thymic nuclear matrix associated activity is not V(D)J recombinase.

It was previously reported that nuclear matrix isolated from young rat thymus contained an activity that supported V(D)J recombination at a high efficiency (Dave et al., BIOCHEMISTRY 30: 4763-4767, 1991). A similar type of activity is also detected in the matrix prepared from fetal calf thymus. However, restriction enzyme mapping analyses of the recombined product clearly suggest that the double antibiotic resistance exhibited by the matrix treated plasmid substrate is not a consequence of V(D)J signal sequence recombination.

Nuclear matrix bound V(D)J recombination activity in rat thymus nuclei: an in vitro system.

We report here that a high level of V(D)J recombination activity is tightly associated with high-salt-resistant nuclear matrix isolated from thymus glands from 2- to 3-week-old rats. The soluble nuclear fractions either were devoid of or contained a very low level of recombinase activity. This is the first time that the process mimicking V(D)J recombination has been achieved in an in vitro system. The matrix-bound V(D)J recombinase activity was further found to be lymphoid specific, detectable only during early stages of development. These observations suggest that in vitro recombination of V(D)J segments of genes encoding antigen-binding proteins could be a matrix-bound process and that the nuclear matrix may be an important intranuclear domain for the functional organization of the V(D)J recombinase system.

DNA synthesis in nuclei and nuclear matrices of regenerating rat liver: effect of whole-body gamma irradiation.

Partial hepatectomy (PH) of rats (Wistar strain) resulted in acceleration of DNA synthesis in liver which reached a maximum at 36 h after PH. Whole-body radiation exposure (10 Gy) of the rats at 12 h after PH completely arrested this stimulation in DNA synthesis. The elevation of DNA synthetic rate in response to PH and complete obliteration of this stimulation by whole-body radiation exposure were found to be the reflection of levels of DNA polymerase-alpha in nuclei and nuclear matrices isolated from the rat livers. Studies based on assays of DNA polymerase in nuclei and nuclear matrices, with and without exogenous DNA template (activated calf thymus DNA), revealed that whole-body irradiation blocked induction of DNA polymerase-alpha and, in turn, assembling of DNA polymerizing apparatus. Irradiation of nuclei (suspended in buffer) in vitro at doses as high as 500 Gy did not have any inhibitory effect on DNA polymerase-alpha activity.

Terminal deoxynucleotidyltransferase containing megadalton complex from young rat thymus nuclei: identification and characterization.

Nuclear matrix prepared from 2-3 week old rat thymuses contains tightly bound TdT activity which has been quantitatively solubilized with nonionic detergent and sonication. TdT is contained in a discrete complex with a sedimentation value of 23 S. The complex is retained on an anti-TdT antibody column and contains DNA ligase and 3'-5' exonuclease activities as well as DNA and several other proteins but is devoid of replicative DNA polymerases. Such a type of multienzyme complex is absent from the nuclear extracts of thymus prepared from older rats and also from liver and spleen extracts of young and old rats.

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. I. A possible site for TdT mediated function.

Approximately 80% of the terminal deoxynucleotidyl transferase (TdT) in thymus glands from 3-4 week old rats was found to be localized in the nucleus and the remaining 20% in the cytosol. Following endogenous nuclease digestion of the thymus nuclei, 70-85% of the nuclear TdT could be removed by low salt and high salt extractions, whereas 15-30% of the enzyme remained tightly bound to the residual nuclear matrix. Low salt and high salt extracts of the nuclei contained a mixture of 58, 56, 45 and 44 kDa species of TdT whereas only 58 kDa species of the enzyme was found to be associated with the matrix. In addition to TdT, 20-25% of the nuclear DNA polymerase alpha was also tightly bound to the isolated nuclear matrix. These observations lead us to propose that besides being the site of DNA replication via-matrix bound replicational complexes [Van der Velden H.M.W. & Wanka F., Molecular Biology Reports 12 (1987): 69], nuclear matrix may also be the site of TdT mediated function and that matrix bound TdT and free TdT could be the functional and nonfunctional forms of the enzyme, respectively, in the thymus gland.

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. II. Effect of ATP on free and matrix bound TdT.

Endogenous nuclease digestion of thymus nuclei from 3-4 week old rats followed by a step wise extraction with low salt, 0.5 M salt and 1 M salt removed approximately 70-85% of total nuclear terminal deoxynucleotidyl transferase (TdT) whereas approximately 15-30% of the enzyme remained tightly bound to the residual nuclear matrix. The cytoplasmic TdT as well as the bulk of nuclear TdT extracted in low salt and 0.5 M salt was found to be strongly inhibited at low concentration of ATP whereas matrix bound TdT and a significant portion of the enzyme in 1 M salt extract was completely insensitive to this nucleotide. The ATP resistant enzyme in the 1 M salt extract was unstable and slowly converted to ATP sensitive form upon prolonged preincubation on ice whereas under similar conditions it remained unaffected in the matrix bound form. These observations lead us to suggest that ATP resistant matrix bound TdT being capable of discriminating unnatural rNTPs against the natural dNTP substrates, may be the functionally organized form of the enzyme and that free TdT having lost the capability to distinguish between dNTP and rNTP may be the nonfunctional form of the enzyme in the thymus gland.