CD28-B7 costimulatory blockade by CTLA4Ig delays the development of retrovirus-induced murine AIDS

Abstract

Mouse AIDS (MAIDS) induced in C57BL/6 mice by infection with a replication-defective retrovirus (Du5H) combines extensive lymphoproliferation and profound immunodeficiency. Although B cells are the main target of viral infection, recent research has focused on CD4(+) T cells, the activation of which is a key event in MAIDS induction and progression. A preliminary observation of increased expression of B7 molecules on B cells in MAIDS prompted us to address the possible involvement of the CD28/B7 costimulatory pathway in MAIDS. Mice infected with the MAIDS-inducing viral preparation were treated with murine fusion protein CTLA4Ig (3 x 50 microg/week given intraperitoneally), a competitive inhibitor of physiological CD28-B7 interactions. In CTLA4Ig-treated animals, the onset of the disease was delayed, lymphoproliferation progressed at a much slower rate than in untreated mice, and the loss of in vitro responsiveness to mitogens was reduced. Relative expression of Du5H did not differ between treated and untreated animals. These results suggest that the CD28/B7 costimulatory pathway contributes to MAIDS development.

FIG. 1

MAIDS is associated with overexpression of B7 costimulatory molecules on B cells. The staining profile of SP cells from uninfected C57BL/6 mice (A, C, and E) and mice with MAIDS at 10 weeks postinfection (B, D, and F) are compared. These are representative results obtained from four or five mice analyzed separately in each group. MAIDS is associated with an expansion of B cells expressing B220 at low densities (B220^dim cells), accounting for 25% of the lymphocyte population (B) versus 4% in controls (A). B220⁺ cells, contained within the bold boxes, were evaluated for B7.1 (C and D) and B7.2 (E and F) expression. B7.1 was expressed on 46% ± 1.22% of the B220⁺ cells in mice with MAIDS (D) versus 15% ± 0.75% of those in controls (C). In mice with MAIDS, overexpression of B7.1 was due mainly to expansion of the B7.1⁺ B220^dim population (D, arrow). In controls (E), 24% ± 2.8% of the B220⁺ cells were B7.2⁺; in infected mice (F), this fraction rose to 46% ± 2.7%, equally distributed between B220^dim and B220^hi cells. FITC, fluorescein isothiocyanate.

FIG. 2

Systemic administration of CTLA4Ig inhibits MAIDS-associated lymphoproliferation. Each column represents the mean SP weight ± the standard error of the mean for each experimental group. The number of infected mice studied at each time point is indicated at the top of the corresponding column. Administration of CTLA4Ig to uninfected controls did not induce any variation in SP weight. In infected animals, CTLA4Ig administration delayed the onset of the lymphoproliferative process and significantly reduced its amplitude. Statistical significance of the differences between untreated and treated, infected groups was tested by a two-tailed Student t test or the Mann-Whitney test. ∗, P < 0.05; ∗∗, P < 0.025.

FIG. 3

Histologic comparisons of SPs from untreated (A and C) or CTLA4Ig-treated (B and D) mice infected for 15 weeks. Tissue samples were fixed in 4% paraformaldehyde, embedded in glycolmethacrylate (JB Polyscience), and semithin sectioned before staining with hematoxylin and eosin. (A) SP from an untreated infected mouse showing obliteration of the white pulp-red pulp demarcation (magnification, ×100) due to diffuse infiltration by large, blastic cells displaying immunoblastic or plasmocytoid differentiation. (C) Several mitoses were observed (magnification, ×400). (B) SP from a CTLA4Ig-treated, infected animal showing white pulp expansion consisting of follicular enlargement (F) and widening of the adjacent periarteriolar lymphoid sheaths (arrow) (magnification, ×100). (D) Follicular and periarteriolar cells that are blastic, similar to those in untreated mice, and display high mitotic activity. Extension of the lymphoid infiltration around central arterioles is much less prominent than in panel C (magnification, ×400).

FIG. 4

Chronic administration of CTLA4Ig partially preserves immune function in Du5H/G6T2-infected mice. Proliferative responses of LN lymphocytes from treated and untreated infected mice to ConA and LPS in vitro are compared. Data are expressed as comparative proliferation indexes. Columns represent means of the data obtained from two to five mice in each group + the standard error of the mean. In infected animals, the onset of immune anergy is early, affecting both B- and T-cell responses. The short-term effect of CTLA4Ig administration is preservation of immune responses in vitro early after infection (week 4). At later time points, CTLA4Ig does not prevent the immune anergy but the level of proliferative responses is higher in treated than in untreated, infected animals.

FIG. 5

Expansion of the CD4⁺ Thy-1⁻ population is delayed by CTLA4Ig treatment. LN single-cell suspensions were stained with a phycoerythrin-conjugated rat anti-mouse CD4 monoclonal antibody (GK 1.5) and a fluorescein isothiocyanate-conjugated rat anti-mouse Thy-1.2 monoclonal antibody (30-H12) (Pharmingen) as previously described (32). The fraction of CD4⁺ cells that were Thy-1⁻ was determined by FACS analysis. The columns represent the mean calculated fractions plus the standard error of the mean.

FIG. 6

CTLA4Ig treatment does not reduce defective Gag mRNA expression. cDNA obtained from reaction of RNA samples of individual mice with RT were amplified by either defective-Gag- or HPRT-specific primers for 30 cycles. The bands for the two products appeared on a 2% agarose gel at the expected migration points, corresponding to 237 and 163 bp, respectively. Lanes: 2 and 3, samples from untreated, infected mice (SP weights, 450 and 477 mg); 4 and 5, samples from CTLA4Ig-treated, infected mice (SP weights, 165 and 199 mg); 1, negative control for PCR (water); 6, specificity control for PCR using a J1 plasmid containing the defective Du5H sequence. No amplification was obtained after PCR of RNA samples (data not shown). These results are representative of two to five mice per group.