The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1‐expressing anti‐NP antibodies from the secondary response, on the affinity of these antibodies was examined by site‐specific mutagenesis and recombinant antibody construction. A tryptophan––leucine exchange at position 33 in the VH segment of all but one of the high‐affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10‐fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high‐affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one‐codon deletion) in VH and the particular D‐JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain‐bearing anti‐NP antibodies which by itself leads to an increase of hapten‐binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.

No abstract

While somatic antibody mutants are rare in the preimmune repertoire and in primary immune responses, they dominate secondary and hyperimmune responses. We present evidence that somatic hypermutation is restricted to a particular pathway of B-cell differentiation in which distinct sets of B-cell clones are driven into the memory compartment. In accord with earlier results of McKean et al. (1984) and Rudikoff et al. (1984), somatic mutation occurs stepwise in the course of clonal expansion, before and after isotype switch, presumably at a rate close to 1 X 10(-3) per base pair per generation. At this rate, both selectable and unselectable mutations accumulate in the rearranged V region genes. The distribution of replacement mutations in the V regions shows that a fraction of the mutations in CDRs is positively selected whereas replacement mutations are counterselected in the FRs. By constructing an antibody mutant through site-specific mutagenesis we show that a point mutation in CDR1 of the heavy chain, found in most secondary anti-NP antibodies, is sufficient to increase NP binding affinity to the level typical for the secondary response. Somatic mutation may contribute to the immune repertoire in a more general sense than merely the diversification of a specific response. We have evidence that clones producing antibodies which no longer bind the immunizing antigen can be kept in the system and remain available for stimulation by a different antigen. Somatic mutations are 10 times less frequent in DJH loci than in either expressed or non-expressed rearranged VDJH or VJ loci. We therefore conclude that a V gene has to be brought into the proximity of the DJH segment in order to fully activate the hypermutational mechanism in these loci.

Murine gammaherpesvirus causes an acute respiratory infection followed by a latent infection in B lymphocytes. In the first 2-3 weeks after infection mice develop a marked splenomegaly, where the spleen cell number increases by 2-3-fold. Cytofluorimetric analysis during splenomegaly revealed an increase in numbers of B lymphocytes and of both CD4 + and CD8 + T lymphocytes. The largest increase relative to uninfected spleens was in the CD8 + population. The number of latently infected cells in the spleen peaked at day 10 post-intraperitoneal infection, then declined to 1/106-1/107 cells per spleen. Depletion of CD4 + T lymphocytes prevented the splenomegaly and greatly reduced the peak infective centre level, while having no effect on the long-term level of latently infected cells. Given the similarity between MHV-68-induced splenomegaly and Epstein-Barr virus-induced infectious mononucleosis, these data highlight the usefulness of MHV-68 as a mouse model for the study of gammaherpesvirus immunology and pathobiology.

Infection of mice by murine gammaherpesvirus 68 (MHV-68

The murine gammaherpesvirus 68 (MHV-68) is an ideal model system for the study of interactions between gammaherpesviruses and their hosts. Intranasal infection of mice with MHV-68 results in replication of the virus in the lung epithelium followed by latent infection of B cells. Resolution of productive MHV-68 infection depends on the adaptive immune system, but little is known about the role of innate immune mechanisms and the early interaction between the host and the virus. In this report, we have used mice that are deficient in components of the early defence system, the common type I interferon (IFN) receptor (IFN R), the transcriptional activator IRF-1, and the inducible nitric oxide synthase, to investigate the contribution of these mechanisms to control of MHV-68 infection. We show that while wild-type mice are highly resistant to infection with MHV-68, mice unresponsive to type I IFNs (IFN-alpha/beta R(-/-) ) are highly susceptible to the virus. At high multiplicities of infection (m.o.i. ; 4 x 10(6) PFU), 80-90% of IFN-alpha/beta R(-/-) mice succumb to infection, and at low m.o.i. (4 x 10(3) PFU), 50% mortality rates occur. Both high and low doses of virus lead to 100- to 1000-fold higher lung virus titres in IFN-alpha/beta R(-/-) mice than are found in wild-type mice and result in systemic dissemination of the virus. Latently infected cells are detectable in the spleens of IFN-alpha/beta R(-/-) mice earlier than in wild-type mice, and the numbers of latently infected cells are 10-fold higher in the IFN-alpha/beta R(-/-) mice during the acute phase of infection. We find IRF-1 has a critical role in protection from fatal disease, whereas inducible nitric oxide synthase does not appear to be important. The results indicate that innate immune mechanisms are critical for the early control of MHV-68 and may play a role in the establishment of latency.

Murine gammaherpesvirus is a natural rodent pathogen which causes a primary infection in the lungs and establishes a persistent infection in B lymphocytes. During the primary infection, large amounts of gamma interferon (IFN-␥) are produced by spleen, mediastinal, and cervical lymph node cells. To investigate the role of IFN-␥ in control of the virus infection, mice lacking the cellular receptor for IFN-␥ (IFN-␥ R؊/؊ mice) were infected with murine gammaherpesvirus 68 (MHV68). IFN-␥ R؊/؊ mice showed no difference from wild-type mice in the titers of infectious virus in the lungs or in the rate of clearance of the lung infection.In the spleen, however, clear differences were observed. By 14 days postinfection, spleens from IFN-␥ R؊/؊ mice were pale, shrunken, and fibrous. Histological examination showed that there was an early (day 10) infiltration of granulocytes followed by widespread destruction of splenic architecture (days 14 to 17). A marked decrease in the number of splenic B cells and CD4 ؉ and CD8 ؉ T cells occurred. These changes were accompanied by a 10to 100-fold greater load of latently infected cells in IFN-␥ R؊/؊ mice than in wild-type mice at 14 to 17 days postinfection, but this was reduced to the levels found in wild-type mice by 21 days postinfection. Treatment of the mice with the antiviral drug 2-deoxyl-5-ethyl-beta-4-thiouridine from 6 days postinfection did not prevent the occurrence of these changes. The changes were, however, completely reversed by depletion of CD8 ؉ T cells prior to and during the primary infection. Depletion of CD4 ؉ T cells also reversed the major pathological and virological changes, although in this case there was evidence of some histological changes. Thus, the lack of IFN-␥ receptor had profound consequences in spleens of MHV68-infected mice. The possible mechanisms involved in these changes are discussed.

Unfractionated mononuclear (UM) cells and T cells freshly prepared from the blood of adult donors were co-cultivated in microtest plate wells with progressively lower numbers of cells from the autologous EB-virus-transformed B-cell line. The fresh cells present in co-cultures from EB virus antibody-negative (seronegative) donors regularly facilitated autologous cell line outgrowth, monitored after 4 weeks, whereas outgrowth was markedly inhibited in the corresponding co-cultures from seropositive donors. Larger-scale co-cultures, set up at a ratio of 80-100 fresh UM cells to one autologous virus-transformed B cell, were harvested after 8 to 12 days and the T-cell subpopulation was examined for cytotoxicity both by growth inhibition and by chromium release assays. Cytotoxic T cells were generated exclusively in seropositive donor co-cultures and were strongly active against the autologous virus-transformed cell line without affecting either autologous uninfected B cells or any of a range of EB virus genome-negative target cell lines chosen as sensitive indicators of non-specific cytotoxicity. Recognition of allogeneic EB-virus-transformed cells was restricted to those whose HLA-A and/or B and/or B and/or C antigen expression matched that of the effector cells themselves;; moreover target cell lysis was specifically inhibited in the presence of monoclonal antibodies binding to these HLA antigens. The results indicate that EB-virus-specific HLA-restricted memory T cells, present in the blood of previously-infected individuals, can be reactivated in vitro using the established autologous virus-transformed cell line as a stimulus. THe reactivated cytotoxic cells appear to recognize a virus-induced lymphocyte-detected membrane antigen, LYD-MA, analogous to that first invoked to explain the cytotoxic response to primary EB virus infection observed during infectious monoucleosis.