Medizin - Open Access LMU - Teil 05/22 Archiv

The distribution of the functional subsets of porcine T cells, the cytolytic/suppressor (Tc/s) and the helper/inducer (Th/i) cells was studied in cryostat sections of thymus, lymph nodes, tonsils, Peyer's patches, spleen and liver using the indirect immunoperoxidase technique. Three murine monoclonal antibodies (mAb) were used. The mAb 8/1 reacts with an antigen present on all T cells and on cells of the myeloid lineage; the antigen has not yet been characterized biochemically. The mAb 295/33 (anti-T8) binds to the porcine T8 antigen and defines the Tc/s subset, while mAb PT-4 (anti-T4) detects the porcine T4 antigen and defines the Th/i subset. Practically all thymocytes were stained by mAb 8/1. The majority of cortical thymocytes apparently co-expressed T8 and T4, whereas distinct fractions of medullary cells were labelled by either anti-T8 or anti-T4. In peripheral lymphoid organs all three mAb reacted with cells in the thymus-dependent areas and with cells scattered in the lymphoid follicles. In lymph nodes, tonsils and Peyer's patches, anti-T8 and anti-T4 each labelled approximately half of the cells stained by mAb 8/1. In the periarteriolar lymphoid sheath of the spleen, anti-T4 labelled more cells than did anti-T8. The reactivity of mAb 8/1 with the Kupffer cells of the liver demonstrated the expression of the 8/1 antigen on cells of the monocyte lineage. The T8 and T4 antigens could not be detected in acetone-fixed and paraffin-embedded sections, while the antigen recognized by mAb 8/1 remained preserved. Altogether, despite an inverted microanatomical structure of porcine lymph nodes, the frequency and distribution of T8+ and T4+ cells in thymus-dependent areas proved to be similar to those found in other species.

The expression of an antigen on porcine T lymphocytes detected by murine monoclonal antibody (mAb) 8/1 was investigated by functional studies and dual-parameter immunofluorescence. mAb 8/1 reacts with greater than 95% of thymocytes and in peripheral blood with all T lymphocytes and with cells of the monocyte/macrophage lineage, but not with B cells, erythrocytes, and platelets. Pretreatment of peripheral blood lymphocytes with mAb 8/1 plus complement abrogated the proliferative response in vitro to mitogen, soluble antigen, and MHC determinants. Dual-parameter immunofluorescence revealed that resting porcine T8+ as well as T4+ lymphocytes express the 8/1 antigen, whereas after in vitro activation, cell surface expression of the antigen was low or absent in both T cell subsets. Thus, the 8/1 antigen represents a marker that discriminates between resting and activated T lymphocytes. Distribution and functional criteria indicate that 8/1 represents a novel marker not described before for any other mammalian species.

The existence of four subpopulations in resting porcine T lymphocytes is documented. In addition to the two known subpopulations which are typified by a mutually exclusive expression of either the CD8 or the CD4 differentiation antigen, CD4-CD8+ and CD4+CD8- T lymphocytes, respectively, two unusual subpopulations were prominent not only in peripheral blood, but also in lymphoid tissues: CD4-CD8- T lymphocytes expressing neither of these antigens and CD4+CD8+ T lymphocytes coexpressing both antigens. While CD4+CD8+ lymphoblasts have been found also in other species, resting T lymphocytes with that phenotype are without precedent among all species analyzed to date. This unique composition of the porcine T lymphocyte population has to be taken into consideration when the swine is used as a large animal model in experimental medicine.

Cloned genomic fragments from the region (0.769 to 0.818 map units) coding for immediate-early (IE) transcripts of murine cytomegalovirus (MCMV) were used to analyze the physical organization of this region, the direction of transcription, and the proteins synthesized in vitro. Three IE transcription units could be identified. From IE coding region 1 (ie1; 0.781 to 0.796 map units) a dominant 2.75-kilobase (kb) RNA was transcribed from right to left on the prototype arrangement of the MCMV genome which directed the synthesis of an 89,000-molecular-weight polypeptide (89K polypeptide), the major IE protein. This phosphoprotein (pp89) has been shown to be active in the regulation of transcription. Upstream of ie1 and separated by the MCMV enhancer sequence was a second IE coding region, ie2, which was mapped at 0.803 to 0.817 map units. From ie2 a 1.75-kb RNA of moderate abundance was transcribed in the direction opposite to that of the ie1 RNA. After hybrid selection of the ie2 transcript, a 43,000-molecular-weight translation product was detected. A third coding region, ie3, was located directly downstream of ie1 (0.773 to 0.781 map units). The series of RNAs with low abundance, terminating in ie3, probably used the ie1 transcription start site and ranged from 1.0 to 5.1 kb in size. The 5.1-kb RNA apparently represents the nonspliced transcript from both coding regions ie1 and ie3. A 15K polypeptide was translated in vitro from RNA that was hybrid selected by ie3 sequences. Immunoprecipitation with monoclonal antibody revealed that 31K to 67K polypeptides were related to pp89. Some of these proteins were translated from RNAs that were smaller than 2.75 kb. Polypeptides related to pp89 were also synthesized in vivo. Because polypeptides unrelated to pp89 that were translated from RNA that was selected by ie2 and ie3 sequences were not immunoprecipitated by murine antisera, we assumed that the amount of these proteins synthesized in vivo during infection was probably very low

In murine cytomegalovirus, abundant immediate-early transcription originates from 0.769 to 0.815 map units of the genome. This region contains the immediate-early gene (gene ieI) which encodes pp89, a phosphoprotein active in transcriptional regulation. In this paper we report on the precise location, structural organization, and sequence of gene ieI. The predominant ieI transcript, a 2.75-kilobase mRNA, is generated by splicing and composed of four exons. The precise termini of the 2.75-kilobase mRNA and the positions of the exons were determined by nuclease digestion experiments with either 5' or 3' end-labeled DNA fragments or in vitro transcribed cRNA probes. Exons of 300, 111, 191, and 1,703 nucleotides are separated by introns of 825, 95, and 122 nucleotides. The first AUG is located in the second exon of 111 nucleotides, and a single open reading frame of 1,785 nucleotides predicts a protein of 595 amino acids with a calculated molecular weight of 66,713. The N-terminal region of the protein contains sequences similar to a consensus sequence of histone 2B proteins. The regulatory function of pp89 and the role of this protein as an immunodominant antigen are discussed in relation to the amino acid sequence.

To confirm that immediate-early (IE) genes of murine cytomegalovirus (MCMV) give rise to antigens recognized by specific cytolytic T lymphocytes (CTL), a 10.8-kilobase fragment of MCMV DNA which is abundantly transcribed at IE times was transfected into L cells expressing the Ld class I major histocompatibility glycoprotein. The viral genome fragment contains sequences of the three IE transcription units of MCMV: ie1, ie2, and ie3. In the transfected cell lines, only the predominant 2.75-kilobase transcript of ie1 and its translation product pp89 could be detected. The transfectants were analyzed for membrane expression of an IE antigen by employing clone IE1, an IE-specific CTL clone, as the probe. Only cells that expressed both the MCMV IE gene(s) and the Ld gene were recognized by the CTL clone.

We have shown in a murine model system for acute, lethal cytomegalovirus (CMV) disease in the immunocompromised natural host that control of virus multiplication in tissues, protection from virus-caused tissue destruction, and survival are mediated by virus-specific CD8+ CD4-T lymphocytes. Protection from a lethal course of disease did not result in a rapid establishment of virus latency, but led to a long-lasting, persistent state of infection. The CD8- CD4+ subset of T lymphocytes was not effective by itself in controlling murine CMV (MCMV) multiplication in tissue or essential for the protective function of the CD8+ CD4- effector cells. The antiviral efficacy of the purified CD8+ CD4- subset was not impaired by preincubation with fibroblasts that presented viral structural antigens, but was significantly reduced after depletion of effector cells specific for the nonstructural immediate-early antigens of MCMV, which are specified by the first among a multitude of viral genes expressed during MCMV replication in permissive cells. Thus, MCMV disease provides the first example of a role for nonstructural herpesvirus immediate-early antigens in protective immunity.

We have shown in a murine model system for cytomegalovirus (CMV) disease in the immunocompromised host that in vivo application of recombinant human IL-2 (rhIL-2) can enhance the antiviral effect of a limited number of CD8+T lymphocytes, not only in prophylaxis, but also in therapy, when virus has already colonized host tissues. The observed net effect of IL-2 was consistent with the assumption of daily effector population doublings. The prospects for IL-2-supported immunotherapy of established CMV infection depend upon the tissues involved in disease. It appears that the prospects for controlling established CMV adrenalitis are less promising than for a therapy of interstitial CMV pneumonia.

We have constructed target cells by cotransfection of the MHC gene Ld and fragments of murine cytomegalovirus (MCMV) DNA coding for nonstructural immediate-early (IE) proteins. Transfectants were tested by using CTL clone IE1 with specificity for an IE epitope presented in association with Ld. Data show that clone IE1 recognizes a product of the ie1 transcription unit of MCMV, and that its specificity is shared by approximately 25% of polyclonal IE-specific CTL. The results provide the first definite evidence that expression of a herpes virus IE gene encoding a regulatory protein gives rise to antigen expression detectable by specific CTL

The murine immediate-early (IE) protein pp89 is a nonstructural virus- encoded phosphoprotein residing in the nucleus of infected cells, where it acts as transcriptional activator. Frequency analysis has shown that in BALB/c mice the majority of virus-specific CTL recognize IE antigens. The present study was performed to assess whether pp89 causes membrane antigen expression detected by IE-specific CTL. Site-directed mutagenesis has been used to delete the introns from gene ieI, encoding pp89, for subsequent integration of the continuous coding sequence into the vaccinia virus genome. After infection with the vaccinia recombinant, the authentic pp89 was expressed in cells that became susceptible to lysis by an IE-specific CTL clone. Priming of mice with the vaccinia recombinant sensitized polyclonal CTL that recognized MCMV- infected cells and transfected cells expressing pp89. Thus, a herpesviral IE polypeptide with essential function in viral transcriptional regulation can also serve as a dominant antigen for the specific CTL response of the host.