Positive Selection Is Not Required for Thymic Maturation
ofTransgenic ~8 T Cells
By Edina Schweighoffer and B.J. Fowlkes
From the Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0420
Previously published reports describing thymic differentiation in two TCtL~/8 transgenic
mouse models have suggested that ~/8 T cells require MHC-mediated positive selection to
reach full maturity. Recent studies indicate that recognition of antigen by mature ~/8 T cells is
not MHC restricted, raising the issue of why developing ~/8 T cells would even require MHC-
driven positive selection. Therefore, we have reinvestigated the requirements for development
and selection in G8 "y8 T cell receptor (TCtL) transgenic mice. Analyses of absolute cell num-
bers, phenotypic subsets, and functional competence of thymic and peripheral G8 ~/~ T cells
indicate that these cells can fully mature in class I MHC-deficient mice. Moreover, mixed bone
marrow chimeras demonstrate that y5 T cells of mutant B2-microglobulin ([32m ~ origin are
partially deleted in the presence of H-2d-bearing thymocytes (previously beheved to be the
haplotype mediating positive selection). We conclude that there is no requirement for class I-like
molecules for the maturation/development of these transgenic y8 T cells and that the differ-
ences in thymocyte phenotype and number observed are, instead, attributable to effects of
defined, while the maturation process for ~/8 T cells is less
well understood. Since the proportion of y8 T cells in the
thymus is <1% (1), studies on the development of these
cells has been greatly aided by ~/8 TCIL transgenic mice.
For our investigation, we have used the G8 mouse (2) that
carries a transgene containing productively rearranged
and 8 TCtL genes derived from a T cell clone (3, 4) pro-
duced by immunization of BALB/c nu/nu mice with
B10.BP,. spleen cells. The reactivity of the original T cell
clone and of the "y5 T cells from the transgenic mice has
been mapped to T22/T10 in the nonclassical (class Ib) re-
gion of the MHC (TL-region). The strongest reactivity
was observed with H-2 b, intermediate with H-2 k, and no
reactivity with H-2 a antigen-presenting cells (APCs) (4).
Like classical MHC class I (class Ia) molecules, the putative
class Ib ligand of the G8 TCIL associates with [32-micro-
globulin (132m) 1 (5); therefore, the absence of[32m may in-
fluence the developmental fate of the transgenic ",/8 T cells.
Consistent with this idea were the reported phenotypic dif-
ferences between the G8 [32m + and G8 [32m ~ thymuses that
demonstrated a higher proportion of HSA-CD44hiCD451LB hi
~/8 T cells in the class I + mice (6). Moreover, the G8 ~32m ~
he major events in the intrathymic differentiation of
ot[3 T cells have been intensely investigated and well
~ Abbreviation used in this paper: 132m, [~2-microglobulin.
thymocytes did not proliferate to allogeneic stimulation,
and these mice had very few, if any, peripheral lymphoid
~/~ T cells (7). These data lead to the conclusion that ~/B T
cells, like ot[3 T cells, require MHC-dependent positive se-
lection to complete their maturation. Similar conclusions
were reached using another TCtL~/B transgenic strain,
KN6, which has a similar pattern of recognition to G8 and
was shown to react to T22, as well (8, 9). In contrast to
these results, an investigation of ~/~ development in non-
transgenic ~2m ~ mice indicated that many or most yB cells
do not require class I or class I-like MHC for their devel-
opment (10). These conflicting results could be explained if
only a subset of ~/8 T cells use MHC for positive sel .~ction.
In our efforts to investigate the cellular and molecular in-
teractions involved in thymic ~/~ T cell development, sur-
prisingly, we found no requirement for [32m-associated
molecules for full ~/8 T cell maturation in G8 mice. Since
the only other example of MHC requirement for ~/8 devel-
opment arises from a transgenic strain with very similar
ligand reactivity to G8 (11), our findings raise the issue of
whether any ~/8 T cells require MHC-driven positive se-
Materials and Methods
Mice. G8 transgenic mice (2) were backcrossed five times to
B10.D2 mice and crossed two times to 132m ~ (H-2 b) mice (previ-
ously backcrossed five times to C57BL/10 (B10) mice) to gener-
J. Exp. Med. y The Rockefeller University Press i 0022-1007/96/05/2033/09 $2.00
Volume 183 May 1996 2033-2041
ate TClq, y8 transgenic, [32m ~ o~pring (H-2 b or H-2a). C57BL/6
mice were obtained from the National Cancer Institute (Freder-
ick, MD) and B10.D2 mice from The Jackson Laboratory (Bar
Harbor, ME). Mice were bred and housed in an NIAID P,e-
search Animal Facility according to AAALAC specifications.
R_AG-2 ~ (H-2 a) (12) and 132m ~ (13) mice (both backcrossed five
times to C57BL/10) were bred and maintained on NIAID con-
tract at Bioqual Inc. (Rockville, MD).
Monoclonal Antibodies And Flow Cytometry.
ric analyses, cells were stained according to standard protocols
For flow cytomet-
(14) using the following labeled antibodies: anti-TCRyS-biotm
(GL3), anti-TC1LVy2-biotin or -FITC
TCR.otlB-FITC or-PE (H57-597), anti-CD5(Ly-1)-biotin or-lYE
(53-7.3), anti-CD44(Pgp-I)-biotin or -PE (IM7), anti-CD45R.B-
biotin or-PE (16A), anti-CD24/HSA-biotin or-PE (M1/69),
anti-Ka-FITC (SF1-1.1), all obtained from PharMingen (San Di-
ego, CA); anti-TCRyS-FITC (GL3), streptavidin-TC or-PE,
from Caltag Labs. (San Francisco, CA); and avidin-APC (Molec-
ular Probes, Eugene, OR). For cell sorting, cells were stained
with anti-TClLyS-biotin/APC- avidin, anti-HSA-PE, and anti-
TCRoL~-FITC and sorted into yS+ot[3-HSA + and yS+o~[3-HSA
cell populations, respectively. To avoid FcR-mediated binding of
antibodies, cells were pretreated with anti-Fc-receptor mono-
Figure 1. Two-color flow cytometric analyses reveal phenotypic dif-
ferences between thymocytes from G8 j32m + (H-2 d) and G8 j32m ~ mice.
Freshly prepared thymocytes were stained with anti-TCR~8 mAb and
the indicated antibodies. FACS ~ density plots are shown; data were col-
lected using live gating for the TCR~/8 + cells. (Data are plotted in two-
color format to facilitate comparison with data from chimeras shown in
Figure 2. Thymocyte numbers in G8 J32m + (H-2 a) and G8 J]2m ~
mice. (Top) Total number of3~8 thymocytes isolated from G8 (32m + (H-2 a)
and G8 j32m ~ mice, respectively. (Bottom) Number of HSA- y~ thy-
mocytes per mouse derived from the product of the percentage of HSA +
and HSA 3'8 thymocytes and the total number ofthymocytes. Each cir-
cle represents one mouse. Average values for each group are shown as col-
umns: (top) 2.9 X 106 vs. 15.3 • 106; (bottom) 31.5 • 104 vs. 25.3 X 104.
2034 Thymic Selection ofy~ T Cells
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2041 Schweighoffer and Fowlkes