High rate of recombination throughout the human immunodeficiency virus type 1 genome.

The diploid nature of human immunodeficiency virus type 1 (HIV-1) indicates that recombination serves a central function in virus replication and evolution. Previously, while examining the nature of obligatory primer strand transfers during reverse transcription, a high rate of recombination was observed at the ends of the viral genome within the viral long terminal repeats, prompting the following question: does recombination occur at a high rate throughout the genome? To address this question, two vectors based upon different strains of HIV-1 were utilized. The vectors were comprised predominantly of autologous HIV-1 sequence and were approximately the same size as the parental genome. The proviral progeny of heterodimeric virions were analyzed after a single cycle of replication, and the sequence heterogeneity between the two strains allowed direct examination of recombination crossovers. The results obtained indicate that HIV-1 undergoes approximately two to three recombination events per genome per replication cycle. These results imply that both HIV-1 RNAs are typically utilized during reverse transcription and that recombination is an important aspect of HIV-1 replication.

CD8(+), radiosensitive T cells of parental origin, oppose cells capable of down-regulating cytotoxicity in murine acute lethal graft-versus-host disease.

Murine graft-versus-host (GVH) disease takes two forms depending upon the parental/F1 strain combination employed. Anemia, lymphopenia, hypogammaglobulinemia, profound anti-F1 cytotoxicity, and the loss of cytotoxic potential against third party alloantigen is seen in acute lethal GVH disease. In contrast to this, in chronic GVH disease there is polyclonal B cell activation, auto-antibody production, no anti-F1 cytotoxicity, and retained cytotoxicity against allotargets. We have previously reported that this marked disparity in disease expression results from a radiosensitive host veto cell which protects the F1 mouse from parental anti-F1 cytotoxicity in mice undergoing CGVH disease. This cell could be induced in vitro or in vivo in CGVH disease. Using an in vitro system, we now demonstrate that a CD4(+), radiation-sensitive, T cell does emerge in acute lethal GVH disease which is capable of down-regulating cytotoxicity. The cell does not appear to be a veto cell in that it attenuates cytotoxicity directed against nonself alloantigen. The function of this cell does not appear to be influenced by minor lymphocyte stimulatory gene products. We further report that, in ALGVH disease, regulation by this cell is not readily apparent due to the emergence of a CD8(+) T cell of parental (B6) origin, which opposes its action.

The nature of human immunodeficiency virus type 1 strand transfers.

The diploid nature of human immunodeficiency virus type 1 (HIV-1) suggests that recombination serves a central function in virus replication and evolution. A system was developed to examine HIV-1 strand transfers, including the obligatory DNA primer strand transfers as well as recombinational crossovers during reverse transcription. Sequence heterogeneity between different strains of HIV-1 was exploited for examining primer transfer events. Both intra- and intermolecular primer transfers were observed at similar frequencies during minus-strand DNA synthesis, whereas primer transfers during plus-strand DNA synthesis were primarily intramolecular. Sequence analysis of long terminal repeats from progeny proviruses also revealed a high rate of homologous recombination during minus-strand synthesis, corresponding to an overall rate of approximately three crossovers per HIV-1 genome per cycle of replication. These results imply that both viral genomic RNAs serve as templates during HIV-1 reverse transcription and that primer strand transfers and recombination may contribute substantially to the rapid genetic variation of HIV-1.

Use of single-cycle analysis to study rates and mechanisms of retroviral mutation.

Retroviruses evolve at rapid rates. This allows them to escape immune surveillance, thwarts vaccine development, and leads to rapid emergence of drug-resistant virus. Information regarding the retroviral mutation rates and the underlying mechanisms of mutagenesis will undoubtedly expedite the development of strategies to combat retroviral-mediated diseases. In this review, we discuss how the unique retroviral life cycle can be adapted such that retroviral variation can be studied in a single cycle of replication. By limiting replication to a single cycle, retroviral mutation rates can be directly measured, and the consequences of mutations can be observed. In addition, retroviral recombination rates as well as the nature of primer strand transfer during reverse transcription can be studied using this system. Molecular analysis of the spectrum of mutations arising during a single cycle of virus replication also sheds light on the mechanisms of mutagenesis and retroviral replication.

The effect of erythropoietin administration on murine bone marrow chimeras.

All patients develop anemia after autologous or allogeneic bone marrow transplantation and red blood cell transfusion is almost always required in the post-transplant period. Recently Epo therapy has been employed in the setting of bone marrow transplantation. As bone marrow transplant patients typically suffer from malignancies and are immunocompromised, further immunosuppression should be avoided. Recent reports have suggested that Epo may modulate immune response. We have studied the effects of Epo on immune response in murine bone marrow chimeras. Epo administration resulted in an increase in hematocrit. There was no significant alteration in lymphocyte numbers, although a shift in lymphocytes toward T cell predominance was observed. Epo administration resulted in enhanced cell proliferation in response to T and B cell mitogens, although no alteration in cytotoxicity or natural killer cell activity was observed. No example of Epo-induced impaired immunity was observed.

The host response in graft versus host disease. III. The in vitro induction of regulatory cells in chronic murine graft versus host disease.

Murine graft versus host (GVH) disease takes two forms depending upon the parental/F1 strain combination employed. Acute lethal GVH disease is characterized by anemia, lymphopenia, hypogammaglobulinemia, profound anti-F1 cytotoxicity, and the loss of cytotoxic potential against third-party alloantigen. In contrast to this, chronic GVH disease is characterized by polyclonal B cell activation, autoantibody production, no anti-F1 cytotoxicity, and retained cytotoxicity against allotargets. We have previously reported that this marked disparity in disease expression results from a radiosensitive host cell which protects the F1 mouse from parental anti-F1 CTX in mice undergoing CGVH disease. Using an in vitro system to induce the host protective cell, we now demonstrate that two distinct Thy-1+ cells emerge which regulate CTX against the host. One cell is of host origin, radiation sensitive, and functionally resembles a veto cell. The second regulatory cell, of parental origin, is radiation resistant and restricted in its ability to suppress anti-F1 CTX. We further demonstrate that the emergence of these cells is modulated by competitive immunoregulatory influences mediated by T contrasuppressor and I-J+ cells.

In vitro generation of soluble suppressor factor in murine chronic graft versus host (GVH) disease.

Veto cells are cells which suppress self directed cytotoxicity (CTX) regardless of the strain of origin of the cytotoxic effector cell. The mechanism by which veto cells inhibit self directed CTX is largely unknown. We have previously described the emergence of a veto cell in murine chronic graft versus host (CGVH) disease both in vivo and in vitro. Using an in vitro system in which veto cells are induced, we now report that a soluble factor is also induced which is capable of suppressing CTX. This cytotoxicity suppressing factor (CsF) is heat labile and has a molecular weight above 50 kD. The factor prevents the emergence of a cytotoxic effector cell. but is largely ineffective once the effector cell has functionally emerged. CsF is irreversible and is not restricted in its ability to inhibit anti-self CTX.

The host response in graft versus host disease. I. Radiosensitive T cells of host origin inhibit parental anti-F1 cytotoxicity in murine chronic graft versus host disease.

Murine graft versus host (GVH) disease takes two forms depending on the parental/F1 strain combination employed. Acute lethal GVH disease is characterized by anemia, lymphopenia, hypogammaglobulinemia, profound anti-F1 cytotoxicity, and the loss of cytotoxic potential against third-party alloantigen. In contrast to this, chronic GVH disease is characterized by polyclonal B cell activation, auto-antibody production, no anti-F1 cytotoxicity, and retained cytotoxicity against allotargets. We now report that this marked disparity in disease expression results from a radio-sensitive host mechanism which protects the F1 mouse from parental anti-F1 cytotoxicity in mice undergoing chronic GVH disease. Cellular analysis revealed that protection in chronic GVH disease is mediated by a phenotypically complex system of genetically unrestricted radiosensitive T cells of F1 origin. These cells fail to functionally emerge in mice undergoing acute lethal GVH disease.

The host response in graft versus host disease. II. The emergence of host protective cells is in part determined by background genomic compatibility.

Murine graft versus host (GVH) disease takes two forms depending on the parental/F1 strain combination employed. In an accompanying paper (Singh et al., Clin. Immunol. 151, 1993) many of the clinical features of these two forms of GVH disease are described. In addition to these clinical characteristics, acute lethal GVH (ALGVH) disease is characterized by diminished natural killer cell activity, whereas chronic GVH disease is characterized by normal or increased natural killer cell activity. Previously we have reported that this marked disparity in disease expression can be attributed to radiosensitive host cells which protect the F1 mouse from parental anti-F1 cytotoxicity (CTX) in mice undergoing chronic GVH (CGVH) disease. These cells fail to functionally emerge in mice undergoing ALGVH disease. We now report that the background genome, presumably the minor lymphocyte stimulatory loci, of the donor cells determines whether these host cells emerge and thereby dictates the form of GVH disease which is induced. C57BL/6 (B6) cells (H-2b, minor lymphocyte stimulatory locus (Mls)b) and B10.D2 cells (H-2d, Mlsb) were found to induce ALGVH disease when adoptively transferred to [C57BL/6xDBA/2]F1 (B6D2) (H-2b/d, Mls-1a/b, Mls-2a/b) recipient mice. DBA/2 cells (H-2d, Mls-1a, Mls-2a) and Balb/c cells (H-2d, Mls-1a, Mls-2b) induced CGVH disease in B6D2 mice. Using Mls congenic strains we have demonstrated that donor cell reactivity against Mls-2a was necessary and sufficient to induce ALGVH disease as determined by anemia, lymphopenia, anti-F1 cytotoxicity, and loss of cytotoxicity against allogeneic targets. Such Mls-2a reactivity correlated with the impaired induction of a host protective cell capable of vetoing self-directed CTX. Failure of this host protective cell to emerge in turn correlated with donor anti-host CTX and the emergence of ALGVH disease.