By
From the * Division of Hematologic Products, Center for Biologics Evaluation and Research,
Food and Drug Administration, Bethesda, Maryland 20892; Department of Obstetrics and
Gynecology, Johns Hopkins University, Baltimore, Maryland 21287; § Laboratory of Immunopathology,
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
Maryland 20892; and
Flow Cytometry Section, Laboratory of Molecular Structure, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
To examine whether a retroviral disease can be controlled in animals in which cells from a resistant strain coexist in a state of immunological tolerance with cells from a susceptible strain, allophenic mice were constructed and infected with LP-BM5 murine leukemia viruses which induce a fatal disorder, termed murine acquired immunodeficiency syndrome (MAIDS), characterized by lymphoproliferation and immunodeficiency in susceptible inbred strains of mice. We found that in two different strain combinations, resistance to MAIDS was contingent on the presence in individual animals of >50% of lymphocytes of resistant strain origin and correlated with reduction or elimination of retrovirus. In contrast, animals harboring substantial, but less than predominant, numbers of genetically resistant lymphocytes developed disease and died within the same time frame as susceptible control mice with uncontained proliferation of retrovirus.
Can a retroviral infection be controlled or eradicated by
the presence of immune cells that resist infection?
Current thought on therapies for human retroviral disease
considers the possibility that rendering at least part of a patient's immune system resistant to infection will prove beneficial (1, 2). To gain insight into the ability of resistant cells
to alter the outcome of retroviral disease, allophenic mice
were constructed from inbred strains of mice susceptible
and resistant to the syndrome of lymphoproliferation and
immunodeficiency induced by LP-BM5 murine leukemia
viruses (MuLV)1 termed murine acquired immunodeficiency syndrome (MAIDS) (3). Induction of MAIDS is
dependent on expression of a replication defective virus,
BM5def, that encodes a unique Gag polyprotein (4, 6).
Allophenic mice (designated strain A These studies establish that MAIDS can be controlled by
the presence of immune system cells that resist disease, and
suggest thresholds for the percentage of cells of resistant
genotype required to prevent progressive disease. Critically, assessment of retroviral burden over the course of disease reveals that although infection is initially established in
allophenics with significant numbers of cells of susceptible
genotype, those mice with a preponderance of cells of resistant genotype are able to contain the viral burden and remain healthy.
Generation of Allophenic Mice and Determination of Lymphoid Chimerism.
The allophenic mice were constructed either by embryo fusion at the four to eight cell stage of development in the
case of B6 strain B) bear
cells of two different genotypes, with individual cells deriving from and expressing the characteristics of one donor
strain or the other, but not of both, as in an F1 animal. We
used as the susceptible donor the prototypic MAIDS-sensitive strain, C57BL/6 (B6). The 129/SvJ (129) and A/J
strains were selected as resistant donors because they failed
to exhibit any signs of disease for
38 wk after infection.
A/J allophenics, or by injection of embryonic stem
cells into a blastocyst in the case of the B6
129 allophenics (7).
129 allophenics was determined
from tail blood samples obtained before infection. PBL were obtained from heparinized blood, and isolated on Lympholyte-M
(Cedarlane Labs. Ltd., Hornby, Ontario, Canada) cushions, stained
with fluorescent antibodies to the cell surface antigen Ly-9.1
(PharMingen, San Diego, CA), and analyzed on a cell sorter (FACS®
440; Becton Dickinson, San Jose, CA). B6 mice do not express Ly-9.1 (10, 11).
Studies of Infected Mice.
Mice were judged to have MAIDS on
the basis of progressive lymphadenopathy and splenomegaly and
changes in cellular populations and cell surface antigens characteristic of MAIDS: the development of a B220 dull population of
cells, excessive numbers of CD11b (Mac-1)+ cells, and the emergence of a CD4+, Thy 1.2 population of cells (13). Because
spleen biopsies were performed, spleen weights at death are not
presented, as they would not provide an accurate measure of lymphoproliferation.
Statistical Analysis.
The statistical significance of the association between 50% lymphocytes of resistant strain origin and development of disease, and >50% lymphocytes of resistant strain
origin and resistance to disease was assessed by Fisher's Exact Test
(StactXact3 software; CYTEL Software Corporation, Cambridge, MA).
We first asked whether induction of MAIDS could be controlled by the presence of cells from the 129 strain of inbred mice. 129 mice are of the Fv-1nr (22) genotype and therefore restrict replication of the B-tropic helper viruses in LP-BM5 MuLV. Furthermore, they are also resistant to MAIDS development following infection with BM5def pseudotyped with a N-tropic helper virus (23); the mechanism of this resistance is not known.
The initial study determined time to lymphadenopathy
and time to death following infection of unmanipulated
B6 129 allophenics. To meaningfully assess these two
clinical endpoints of infection, mice were not subjected to
procedures such as splenic biopsy which have an impact on
the course of viral disease (Sechler, J.M.G., and A.S. Rosenberg, unpublished observation). Instead, the mice were
bled only after development of lymphadenopathy for assessment of cellular populations indicative of MAIDS.
Four mice with <50% PBL of 129 origin developed
lymphadenopathy and died in a time course similar to that
of fully susceptible B6 mice. Only the allophenic harboring
68% PBL of 129 origin failed to develop disease (Fig. 2, A
and B). 10 additional B6 129 mice with primarily B6
PBL also developed lymphadenopathy secondary to MAIDS
in the same time frame as B6 mice, whereas 6 mice with 57-82% 129 PBL never developed lymphadenopathy and
remained healthy (Fig. 2 C). Association between development of disease and having
50% of lymphocytes of resistant strain origin, and resisting disease and having >50% of
lymphocytes of resistant strain origin, was highly significant
(P = 0.0002 by Fisher's Exact Test).
These data indicate that protection against MAIDS in this strain combination requires that a majority of lymphoid cells be of the resistant strain and that mice with lower frequencies of 129 cells have no survival advantage over inbred susceptible animals. The frequency of cells from the resistant strain required to prevent induction of MAIDS is ~60%, as suggested by the susceptible mouse with 62% 129 cells and three resistant mice with 57-59% of PBL from strain 129.
Serial Studies of B6To develop an understanding of the relationships
among percent chimerism, virus expression, and development of MAIDS, we studied a series of B6 129 mice by
examining splenic biopsies taken soon after infection for
the presence of the three viruses that make up the LP-BM5
virus mixture and then observing the mice for development of disease; some mice were biopsied serially (Table 1). Mice were identified at autopsy as having MAIDS or not by
gross observations and FACS® analysis of lymphoid tissues.
While all allophenics tested 4 wk after inoculation expressed infectious ecotropic MuLV indicating successful infection of all animals, recovery of infectious MCF MuLV
was less common and generally correlated with high expression of BM5def.
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Interestingly, detection of BM5def at this early timepoint was not predictive of subsequent development of disease (20). First, the two allophenics with 59% PBL of 129 origin in which BM5def was detectable did not develop disease. Second, the allophenic with 46% PBL of 129 origin, in which BM5def was not detected at the 4 wk timepoint, did go on to develop MAIDS as revealed by flow cytometry of PBL 23 wk after inoculation. Since previous experience has shown that mice with fully developed MAIDS uniformly express BM5def at high levels (24; Morawetz, R., and H.C. Morse, unpublished observations), we assume that infection of this mouse early in disease was either at levels below our limit of detection or localized to tissues not assayed.
The B6 129 mouse with 59% 129 cells studied at three
timepoints after infection is of special interest. Tests showed
that a moderate proportion of spleen cells obtained at 4 wk
after infection produced infectious ecotropic virus, whereas
cells recovered at 19 wk from spleen and 35 wk from
lymph node produced no detectable virus. In addition,
transcripts for BM5def were detected in spleen at 4 and 19 wk but were present in lymph node at only trace levels at
35 wk. Thus, despite initial infection, the burden of both ecotropic and defective viruses was reduced, and the mouse
remained healthy until killed.
Long-term disease-free survival was also associated with undetectable ecotropic virus in the mouse with 68% 129 cells which was sacrificed at 24 wk after inoculation without lymphadenopathy or splenomegaly. This contrasts with the high level expression of helper virus in the mouse with 49% 129 cells which had MAIDS and was moribund when tested at the same time after infection. Prolonged resistance to MAIDS was thus associated with control of ecotropic virus spread and limited expression of BM5def.
Studies of Time to Disease in B6To examine whether similar levels of
genotypically resistant cells are required for disease protection if resistance is predicated on an immunologic mechanism, B6 A/J chimeras were assessed. The disease resistance of A/J mice requires the presence of CD8+ T cells
and is presumably immunologically mediated (25). In addition, A/J mice restrict replication of both the ecotropic and
BM5def components of the LP-BM5 virus mixture (26, 27).
PBL of B6 A/J mice ranged in chimerism from 3 to
97% A/J with 14 of 17 mice bearing predominantly B6
PBL (Fig. 3 B). Of eight unmanipulated chimeras (Fig. 3 A),
seven with cells of predominantly B6 origin developed
lymphadenopathy within the same time frame as infected
B6 and B6AF1 control animals. In contrast, the allophenic with 54% PBL of A/J origin exhibited resistance to MAIDS
until sacrifice at 63 wk after infection.
As with the B6 129 allophenics, an understanding of
the relationships among percent chimerism, virus expression, and development of MAIDS was sought. All 14 B6
A/J mice with <51% A/J PBL developed MAIDS,
whereas three mice with >50% A/J cells did not (Fig. 3 B).
The mouse with 51% A/J cells had splenomegaly at the
time of biopsy 4 wk after infection, but did not progress to
peripheral lymphadenopathy by the time of a second biopsy 19 wk later, although the spleen was still slightly enlarged and had nodules. Together, these findings suggest
that about 50% PB cells of A/J origin are essential to mediate disease resistance. Again, there was a highly significant
association between development of disease and having
50% of lymphocytes of resistant strain origin, and resisting disease and having >50% of lymphocytes of resistant
strain origin (P = 0.0015 by Fisher's Exact Test).
The relationship between virus expression and percent
chimerism in B6 A/J mice was generally consistent with
results in the B6
129 allophenics. In particular, the two
MAIDS-resistant mice showed a reduction in the frequency of ecotropic-virus-producing cells between the
spleen samples obtained earlier and later in infection. In
contrast, the mice with 40 and 44% PBL of A/J origin showed little or no ability to control expression of either
helper virus or BM5def (Table 2). Thus, as with resistant
B6
129 chimeras, resistant B6
A/J chimeras were clearly
infected, had an initial burst of virus replication, but were
able to contain retroviral burden.
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One approach to treatment of human retroviral disease involves reconstructing the immune system with lymphocytes that cannot be infected to mediate critical immune functions. Unfortunately, animal models to test such an approach for HIV or SIV are lacking. Thus, despite limitations as a model of disease induced by HIV, we tested a murine retroviral disease which shares some features of human AIDS. Allophenic mice comprised of cells from MAIDS-resistant and -susceptible strains were studied rather than radiation bone marrow chimeras to avoid problems associated with delayed repopulation of lymphocyte subsets critical for induction and progression of disease. Thus, resistance versus susceptibility was studied in a "best case" scenario in which an intact but chimeric immune system with mutual tolerance was present at the time of infection.
Our results suggest that a critical balance in the proportion of lymphoid cells from the MAIDS-resistant and -sensitive strains determines whether mice infected with LPBM5 MuLV develop MAIDS or exhibit long-term resistance to disease. Indeed, MAIDS was prevented or controlled only in the chimeras that had a predominance of cells of the resistant genotype at the time of infection; no significant benefit with regard to disease progression or survival was conferred on animals in which lymphoid cells of the resistant genotype were in the minority.
It thus seems possible that a race between the rate of virus spread among cells of susceptible genotype and the rate at which virus-specific effector cells and mechanisms are activated determines the outcome of infection with LPBM5 MuLV. When the proliferation of virus-infected cells outpaces the activation and expansion of effector mechanisms, the animal develops lymphoproliferation and immunodeficiency. This model predicts that disease develops in chimeras with a large pool of genetically susceptible B cells, shown to be the principal early targets of infection (28, 29), coupled with a smaller pool of genotypically resistant effector cells. In these mice the frequency of virus-specific precursors would be limited, lessening the chance that sufficient virus-specific effectors could be generated to prevent disease. In allophenics with a predominance of cells of resistant genotype, there would be a sufficient level of virusspecific effectors and/or effector mechanisms to contain virus spread and prevent development of disease.
An alternative view of the requirement for a majority of resistant genotype cells to mediate resistance is based on the suggestion that B6 mice may provide a factor such as a cytokine necessary for disease induction (24, 30). Thus, resistant allophenics may not have this factor in an amount sufficient to facilitate disease progression, or the resistant strain may provide a factor such as IL-12 (31) that inhibits disease induction. Indeed, a genetically controlled negative regulatory factor has been implicated in resistance to induction of erythroleukemia by Friend virus complex (32).
We can conclude that the presence of genetically resistant immune system cells in sufficient numbers can confer long-term resistance to retroviral disease, even though viral integration and replication has occurred. This may have important implications for novel approaches to therapy of human retroviral disease.
Address correspondence to Dr. Amy S. Rosenberg, 29 Lincoln Drive, Bldg 29A, Rm 2B12, Bethesda, MD 20892.
Received for publication 1 May 1996
We gratefully acknowledge Joan Austin and Charles Shifler for expert technical assistance; Jason Yip and Kerstin Cehrs for help in preparing the figures and tables; David Stephany for guidance in flow cytometry analysis; and Gwendolyn Jackson for expert animal care. We thank Jawahar Tiwari for performing the statistical analyses. We thank Dennis Klinman, Elizabeth W. Shores, and Giovanna Tosato for critical reviews of the manuscript.This work was supported in part by a collaborative research and development agreement between the Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, and IMMUNO-USA, and by contract NO1-AI-72622 to Microbiologic Associates, Inc.
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