Immunological characterization of human vaginal xenografts in immunocompromised mice: development of a small animal model for the study of human immunodeficiency virus-1 infection

Abstract

A small animal model for the in vivo study of human immunodeficiency virus-1 and other fastidious infectious agents in human host target tissues is critical for the advancement of therapeutic and preventative strategies. Our laboratory has developed a human vaginal xenograft model that histologically recapitulates features of the human vaginal epithelial barrier. Vaginal xenografts were surgically implanted into C.B.-Igh-1(b)/IcrTac-Prkdc(scid) (SCID) and NOD/LtSz-scid/scid (NOD/SCID) mice, with and without human peripheral blood mononuclear cell reconstitution. Immunohistochemical staining of vaginal xenografts demonstrated that in the SCID strain healed vaginal xenografts did not retain intrinsic human immune cells at baseline levels, whereas the NOD/SCID strain supported retention of intrinsic human immune cell populations within the xenografts for at least 2 months after engraftment. In peripheral blood mononuclear cell-reconstituted NOD/SCID mice with vaginal xenografts, flow cytometric analyses detected human immune cell populations in the peripheral blood and immunohistochemical methods detected infiltration of human CD45+ cells in the mouse spleens and vaginal xenografts for at least 2 months after reconstitution. This optimized NOD/SCID human vaginal xenograft model may provide a unique small animal in vivo system for the study of human immunodeficiency virus-1 transmission and infection.

Figure 1.

The establishment of human vaginal xenografts in immunocompromised mice. Representative H&E-stained sections of vaginal xenografts harvested at 0 (A), 3 (B), 7 (C), and 14 (D) days after engraftment demonstrate the graft-healing process. The initial deterioration of the vaginal epithelium begins ∼3 days after engraftment and continues down to the basal layer of cells ∼7 days after engraftment. However, by 14 days after engraftment, the differentiated vaginal epithelium is restored within the xenografts. Histological and cytochemical analyses demonstrate that human vaginal xenografts are representative of healthy human vaginal tissues. Original magnifications, ×200.

Figure 2.

Immunohistochemical human CD45 staining of human vaginal xenografts in SCID animals. Vaginal xenografts were surgically implanted into SCID mice as described in Materials and Methods. Grafts were harvested immediately after implantation into SCID animals at day 0 (A), day 3 (B), day 7 (C), and day 14 (D) after engraftment. Immunohistochemical staining of these tissues demonstrates that human CD45-positive cell populations were present at time 0 (A) and were maintained within the tissue to day 7 (B and C) after engraftment. However, by 14 days after engraftment, the intrinsic human immune cell populations were significantly reduced (D). Arrows indicate CD45+ human immune cells. Original magnifications, ×200.

Figure 3.

Representative PBMC-reconstituted SCID mouse with advanced lymphoma development. All data panels collected from one representative animal injected with 9 × 10 PBMCs. The presence of human immune cells in peripheral blood was measured by FACS analysis 2 months after inoculation (A). The first graph demonstrates very high levels of human CD45+ lymphocytes in the peripheral blood. In addition, the second graph demonstrates higher levels of reconstituted CD8+ T-cell populations over CD4+ T cells. On autopsy, the thymus (B), liver (C), and spleen (D) demonstrated diffuse lymphomas infiltrated with dysmorphic, enlarged cell populations (arrows). Original magnifications, ×400.

Figure 4.

The establishment of human vaginal xenografts in NOD/SCID mice. NOD/SCID-immunodeficient mice were able to tolerate human vaginal xenograft tissue transplantation without rejection. H&E staining of vaginal tissue samples harvested 2 months after engraftment (E and F) demonstrates that the histology of the grafts was well preserved in this animal strain. The graft-healing time-course samples demonstrate that, overall, the process is similar to that observed in the SCID strain: time 0 (A), day 3 (B), day 7 (C), and day 14 (D). The epithelial layer is generally restored throughout the graft by 3 weeks after engraftment in the NOD/SCID strain. Original magnifications: ×200 (A–D and F), ×100 (E).

Figure 5.

Immunohistochemical analyses of human vaginal xenografts in NOD/SCID mice. Immunohistochemical staining for human CD45 on harvested xenografts demonstrated that the intrinsic levels of human immune cells within the vaginal xenografts were conserved throughout the course of the graft-healing process in NOD/SCID animals. Tissues were harvested and stained with H&E at days 0 (A), 3 (B), 7 (C), and 21 (D) after engraftment. Arrows indicate CD45+ human immune cells. Original magnifications, ×200.

Figure 6.

Immunohistochemical analyses of specific human immune cell populations within the vaginal xenografts. Column A represents freshly excised human vaginal tissue. Column B represents vaginal xenografts from the same donor as in column A, 21 days after engraftment in NOD/SCID animals. Human CD4+ cells were identified on 0 (A) and 21 days (B) and retained to 2 months after engraftment. CD8+ cells (C and D) and CD68+ monocytes/macrophages (E and F) were also detected within the vaginal xenografts throughout the graft-healing process. Finally, CD1a Langerhans’ cells (G and H) and CD21 mature B lymphocytes (I and J), were also detected within these human xenografts throughout the graft-healing process. The immune cell populations were retained at baseline levels (equivalent to freshly excised vaginal tissue) throughout the 2-month monitoring process. Original magnifications, ×200.

Figure 7.

Representative FACS analyses of peripheral blood from NOD/SCID mice: unreconstituted or reconstituted with 5 × 10 human PBMCs per mouse (same PBMC donor). Column 1: Donor PBMC profile before injection into NOD/SCID mice. Column 2: Representative unreconstituted NOD/SCID mouse at 2 months after inoculation of PBS alone. Column 3: Representative PBMC-reconstituted NOD/SCID mouse at 2 months after inoculation. Human CD14/45 and CD4/8 expression was measured in rows A and B, respectively.

Figure 8.

Immunohistochemical analyses of unreconstituted and PBMC-reconstituted NOD/SCID animals with healed vaginal xenografts. CD45 immunohistochemical staining of harvested spleens from NOD/SCID animals 2 months after injection with PBS or human PBMCs. Animals injected with PBS alone (unreconstituted) did not stain positively for human immune cell populations in the spleen (A). Animals injected with 5 × 10 human PBMCs stained positively for human CD45 expression in the spleen 2 months after injection (C and E). The vaginal xenografts from both unreconstituted and of reconstituted animals (B, unreconstituted; D and F, reconstituted) stain positively for human CD45 expression 2 months after engraftment. Graft tissues from reconstituted animals possess increased numbers of human immune cells compared to the unreconstituted sample. Original magnifications: ×200 (A–D), ×100 (E and F).