Biology Reference
In-Depth Information
tic progenitors contained in fetal liver or bone marrow tissue grafts are co-
implanted with fetal thymus tissue to create a chimeric animal (McCune et al.,
1988), and its use in AIDS research have recently been reviewed elsewhere
(McCune et al., 1997).
In 1988, Mosier and colleagues ®rst reported that human peripheral lym-
phocytes can stably be engrafted in SCID mice (Mosier et al., 1988). Human
peripheral lymphocytes were injected intraperitoneally in SCID mice and per-
sisted in the peritoneal cavity and organs for several months, converting them
in the so-called xenochimeric hu-PBL-SCID mice. Since its description, the
hu-PBL-SCID mouse model has been widely used to study selected aspects of
the human immune response, autoimmunity, and infectious diseases ( Duchosal
et al., 1990; Mima et al., 1995; Mosier, 1996; Saeki et al., 1992). In particular,
xenochimeric mice grafted with human peripheral blood lymphocytes ( PBLs)
can be successfully infected with HIV as originally reported by Mosier and
collegues (Mosier et al., 1991) and several immunological and viral parameters
can be easily monitored in these virus-infected chimeric animals.
HU-PBL-SCID MICE: MAJOR TECHNICAL ASPECTS OF THE MODEL
To obtain a hu-PBL-SCID mouse xenochimera, we injected 20±40 10
6
puri-
®ed human peripheral blood mononuclear cells into the peritoneum of 3- to 5-
wk-old SCID mice (higher numbers may result in a lethal graft versus host
disease). The injected cells rapidly (within 24 h after the intraperitoneal injec-
tion) spread from the peritoneal cavity, settling in the liver, lymph nodes, the
white pulp of the spleen and bone marrow. However, about 1 or 2 months later,
the peritoneal cavity remained the major source of human cells that did not
migrate to mouse organs, or possibly, that recirculated back to the peritoneum.
Cells can be recovered by peritoneal washing with small volumes (e.g., 1 ml) of
cold phosphate bu¨ered saline (PBS). After centrifugation, supernatants can be
stored at 80
C for cytokine or soluble factors analysis, whereas cells can be
pooled with a successive washing with a larger volume of cold PBS. Typically,
1±3 10
6
total cells are obtainable, consisting of both human and mouse cells.
By ¯uorescence-activated cell sorter ( FACS) analysis, according to light scatter
properties, human lymphocytes are detected as the higher ``forward light scat-
tered'' cells in a cloud of heterogeneously sized lymphoid cells (Fig. 19.1)
and are easily stained by monoclonal antibodies toward CD45, CD3, or CD4
and CD8 human surface markers. Analysis of the percentage of CD4 and CD8
cells is better performed by triple staining, electronically gating CD3 positive
cells.
SCID mouse spleens are smaller compared with those of immunocompetent
mice, and these organs occasionally reach abnormal size upon reconstitution.
However, few human cells can be recovered from spleen disruption, very often
accounting for <1% of the total splenic cell population. On the other hand,
lymph nodes are not easily detectable, because they are very small in size and
