How alpha beta T cells deal with induced TCR alpha ablation.

On deletion of the gene encoding the constant region of the T cell antigen receptor (TCR)alpha chain in mature T cells by induced Cre-mediated recombination, the cells lose most of their TCR from the cell surface within 7--10 days, but minute amounts of surface-bound TCR beta chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naive T cells decay over time, the decay rates being faster for CD8(+) cells (t(1/2) approximately 16 days) than for CD4(+) cells (t(1/2) approximately 46 days). TCR(+) naïve cells are either maintained (CD8(+)) or decay more slowly (CD4(+); t(1/2) approximately 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD8(+) cells; t(1/2) approximately 52 days) or not at all (CD4(+) cells), but at the population level, these cells fail to expand as their TCR(+) counterparts do. Together with earlier data on T cell maintenance in environments lacking appropriate major histocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the alpha beta TCR contributes significantly to T-cell homeostasis.

Immune responses and cytokine induction in the development of severe hepatitis during acute infections with murine cytomegalovirus.

Salivary gland-derived murine cytomegalovirus (SGV) infections of mice have been widely used as models of human cytomegalovirus infections and in the study of CMV biology. Still, many aspects of SGV pathogenesis are not clearly defined. Fatal and non-fatal SGV infections were investigated to characterize pathogenetic correlates of mortality and to assess the role of the immune response in disease progression. Suppression of immune responses was observed in both lethal and sublethal infections. Depletion of immune cell populations in spleen, however, correlated with severe CMV-induced hepatitis and mortality. In addition, T cell depletion studies indicated a requirement for this immune cell subset in control of liver damage and survival of infected mice. Examination of cytokine responses revealed a previously undescribed shock-like syndrome in lethally-infected mice characterized by high levels of tumor necrosis factor alpha and interferon gamma. Furthermore, the sites of tumor necrosis factor alpha gene induction did not strictly correlate with either viral load or the sites of tissue damage during infection. Taken together, these findings define the pathogenetic progression of disease as it relates to disease outcome and suggests that organ-specific differences in cytokine induction play a significant role in the late stages of acute lethal MCMV infections.

The immunoevasive function encoded by the mouse cytomegalovirus gene m152 protects the virus against T cell control in vivo.

Cytomegaloviruses encode numerous functions that inhibit antigen presentation in the major histocompatibility complex (MHC) class I pathway in vitro. One example is the mouse cytomegalovirus (MCMV) glycoprotein gp40, encoded by the m152 gene, which selectively retains murine but not human MHC class I complexes in the endoplasmic reticulum-Golgi intermediate compartment/cis-Golgi compartment (Ziegler, H., R. Thäle, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U.H. Koszinowski. 1997. Immunity. 6:57-66). To investigate the in vivo significance of this gene function during MCMV infection of the natural host, we constructed recombinants of MCMV in which the m152 gene was deleted, as were the corresponding virus revertants. We report on the following findings: Deletion of the m152 gene has no effect on virus replication in cell culture, whereas after infection of mice, the m152-deficient virus replicates to significantly lower virus titers. This attenuating effect is lifted by reinsertion of the gene into the mutant. Mutants and revertants grow to the same titer in animals deprived of the function targeted by the viral gene function, namely in mice deficient in beta2-microglobulin, mice deficient in the CD8 molecule, and mice depleted of T cells. Upon adoptive transfer of naive lymphocytes into infected mice, the absence of the m152 gene function sensitizes the virus to primary lymphocyte control. These results prove that MHC-reactive functions protect CMVs against attack by CD8(+) T lymphocytes in vivo.

Hierarchical and redundant lymphocyte subset control precludes cytomegalovirus replication during latent infection.

Reactivation from latent cytomegalovirus (CMV) infection is often associated with conditions of immunosuppression and can result in fatal disease. Whether the maintenance of systemic CMV latency is mainly governed by factors of the infected cell or by immune control functions is unknown. Likewise, the putative immune control mechanisms which could prevent the induction and spread of recurrent CMV infection are not clearly identified. We took advantage of latently infected B cell-deficient mice and a sensitive method for virus detection to study CMV reactivation after ablation of lymphocyte subsets. A crucial role of both T lymphocytes and natural killer (NK) cells was demonstrated. Within 5 d after depletion of lymphocytes, productive infection occurred in 50% of mice, and 14 d later 100% of mice exhibited recurrent infection. A hierarchy of immune control functions of CD8(+), NK, and CD4(+) cells was established. Reactivation was rare if only one of the lymphocyte subsets was depleted, but was evident after removal of a further subset, indicating a functional redundancy of control mechanisms. The salivary glands were identified as the site of most rapid virus shedding, followed by the detection of recurrent virus in the lungs, and eventually in the spleen. Our findings document a previously unknown propensity of latent CMV genomes to enter productive infection immediately and with a high frequency after immune cell depletion. The data indicate that only the sustained cellular immune control prevents CMV replication and restricts the viral genome to a systemic state of latency.

Lack of MHC class I complex expression has no effect on spread and control of cytomegalovirus infection in vivo.

It has been claimed that MHC class I proteins serve as receptors for murine cytomegalovirus (MCMV) and that this interaction is the most important mechanism for virus entry in most cells. This claim is based on the observation that the MHC haplotype contributes to the susceptibility to cytomegalovirus (CMV) infection in vivo. Results from in vitro studies support the concept that stable expression of correctly folded MHC class I molecules contributes to infection, since the individual properties of MHC class I alleles, the availability of beta 2-microglobulin (beta 2m) and also the degree of peptide charging of the MHC class I heavy chain beta 2m heterodimers determined the infection phenotype of cell lines. To assess the biological relevance of proper MHC class I expression we investigated CMV infection in beta 2m-deficient mice which fail to express ternary MHC class I complexes and lack peripheral CD8+ T lymphocytes. We found that organ virus titres and virus clearance kinetics were not altered in beta 2m mutant mice. In addition, there was no indication of diminished virus propagation in beta 2m-/- embryonic fibroblasts. beta 2m-/- mice suffered from the lack of CD8+ T lymphocytes that was partially compensated for by the function of CD4+ T lymphocytes. An organ-specific anti-virus function of natural killer (NK) cells was observed, independent from the beta 2m deletion. The immune control unique for salivary gland infection was maintained. From the data presented here, we confirm the role of MHC class I molecules in the immune surveillance of CMV infection but question the biological impact of correct MHC class I complexes for productive infection.

Immunosuppressive and antiproliferative effects of somatostatin analog SMS 201-995.

The effects of long acting somatostatin analog SMS 201-995 were examined in vivo on: 1) lymphoid morphostasis and functional reactivity of cells obtained from SMS treated donors, 2) on humoral, and 3) cellular type of immunity; and in vitro on: 1) blastic transformation of lymphocytes stimulated by activators of different transmembrane pathways (CD2 by PHA and CD3/TCR by anti-CD3 monoclonal antibody and by allogeneic cells) and 2) on growth and secretory activity of several hybridoma cell lines. The data have shown that SMS in vivo decreases the proportion of CD4+, CD5+ and Ig+ cells in spleen. The reactivity of these cells to Con A was suppressed, but their spontaneous blastic transformation was increased. SMS suppressed also the plaque forming cells generation and proliferation of cells in popliteal lymph nodes during the local host versus graft reaction. The former immunosuppression was abrogated with the use of growth hormone, while in the latter, the time dependent changes in spleen composition were also noticed. The data obtained in vitro revealed that SMS may inhibit only the CD2-induced blastogenesis (in early and late interval after the use of PHA). SMS inhibited also the spontaneous growth and/or secretion of antibodies in some hybridoma cell lines.

Antibodies are not essential for the resolution of primary cytomegalovirus infection but limit dissemination of recurrent virus.

Virus shedding from the epithelial cells of the serous acini of salivary glands is a major source for the horizontal transmission of cytomegalovirus. These cells are, different to other tissues, exempt from CD8 T lymphocyte control. CD4 T lymphocytes are essential to terminate the productive infection. Here, we prove that T-B cooperation and the production of antibodies are not required for this process. For the infection with murine cytomegalovirus, mutant mice were used which do not produce antibodies because of a disrupted membrane exon of the immunoglobulin mu chain gene. Also, in these mice the virus clearance from salivary glands is a function of CD4 T lymphocytes. However, these mice clear the virus and establish viral latency with a kinetics that is distinguishable from normal mice. Reactivation from virus latency is the only stage at which the absence of antibodies alters the phenotype of infection. In immunoglobulin-deficient mice, virus recurrence results in higher virus titers. The adoptive serum transfer proved that antibody is the limited factor that prevents virus dissemination in the immunodeficient host.

Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-gamma and tumour necrosis factor.

We have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon-gamma (IFN-gamma). We now demonstrate that IFN-gamma and tumour necrosis factor alpha (TNF-alpha) display synergism in their antiviral activity. As little as 2 ng/ml of IFN-gamma and TNF-alpha reduced the virus yield by about three orders of magnitude. There was no effect on immediate early (IE) and early (E) gene expression as far as the candidate genes IE1, E1 and those encoding the major DNA-binding protein and the DNA polymerase were concerned. Late gene transcription, assayed by the candidate genes encoding glycoprotein B and the MCMV homologue of ICP 18.5, was blocked and MCMV DNA replication was found to be reduced but not halted. The most prominent finding of the cytokine effect, seen by electron microscopy, was an alteration of nucleocapsid formation. Altogether, the synergism is multifaceted and acts at more than one stage during viral morphogenesis. Because the cytokines clearly do not act at an early stage of infection we conclude that the mode of cytokine activity differs between alpha- and betaherpesviruses.

Participation of endogenous tumour necrosis factor alpha in host resistance to cytomegalovirus infection.

Interferon gamma (IFN gamma) represents an essential cytokine involved in murine cytomegalovirus (MCMV) clearance from the salivary gland and the control of horizontal transmission. Because IFN gamma cannot be responsible for all cytokine effects during recovery from MCMV infection we have now tested the potential participation of tumour necrosis factor alpha (TNF alpha) in the antiviral defence. Neutralization of endogenous TNF alpha abolished the antiviral activity of CD4 T cells in immunocompetent as well as in CD8 subset-deficient mice. These data suggest that the antiviral effect of the CD4 subset requires the presence of at least two cytokines, namely IFN gamma and TNF alpha. Depletion of endogenous TNF alpha in adoptive cell transfer recipients diminished the antiviral function of CD8 T lymphocytes suggesting that TNF alpha also participates in CD8 T cell effector functions. Furthermore, endogenous cytokines were found to be required for survival after infection with lethal doses of MCMV, whereas immunotherapy with recombinant TNF alpha and IFN gamma could not limit virus replication in vivo. The results suggest that, similar to IFN gamma, TNF alpha is an integral part of the protective mechanisms involved in cytomegalovirus clearance.

Gamma interferon-dependent clearance of cytomegalovirus infection in salivary glands.

Cytomegalovirus (CMV), similar to other members of the Herpesviridae family, can establish both persistent and latent infections. Each of the CMVs that are found in many animal species replicates in the salivary gland, and oral secretion represents a source of horizontal transmission. Locally restricted replication characterizes the immunocompetent individual, whereas in the immunocompromised host, protean disease manifestations occur due to virus dissemination. The virus is cleared by immune surveillance, and CD8+ T lymphocytes play a major role. Remarkably, certain cell types of salivary gland tissues are exempt from CD8+ T-lymphocyte control of murine CMV infection and require the activity of CD4+ T lymphocytes. The results presented here suggest that this activity is a function of Th1 cells. Neutralization of endogenous gamma interferon abrogated the antiviral activity of Th1 cells but not that of CD8+ T lymphocytes in other tissues. Neutralization of endogenous gamma interferon did not interfere with the induction of the cellular and humoral immune response but acted during the effector phase. Recombinant gamma interferon could not replace the function of Th1 cells in vivo and had limited direct antiviral activity in vitro. The results therefore suggest that gamma interferon represents one, but not the only, essential factor involved in salivary gland clearance, establishment of CMV latency, and, eventually, the control of horizontal transmission.