T cell Development and Positive Selection in the Thymus

A synopsis of the lecture by Dr. Steve Cobbold for the FHS Physiology Immunology Option

1/ Positive selection as the critical and unique step in T cell development?

Positive selection is the process that focusses the T cell receptor to recognise peptides bound to MHC molecules - without
this T cells might be distracted by binding soluble antigens, the job for which B cells are specialised. Positive selection
takes place in the thymus and is required for T cells to develop, mature and exit into the periphery - otherwise they die
(apoptose) through "neglect".

2/ T-cell receptor rearrangements and their relationship to positive selection

The T cell repertoire is generated randomly and positive selection acts as the main check that an individual T cell has made
a "sensible" receptor. The first checkpoint in the generation of ab T cells is after the rearrangement of the Vb gene, to
check that this can be expressed at the pre-T cell surface in combination with a (non-variable) pre-Ta chain. Such a
succesful pTaVb receptor generates a signal to stop rearranging the Vb chains, leading to effective allelic exclusion (ie
only one correctly expressed Vb chain per T cell). The last component to rearrange is the TCR a chain, and T cells will
repeatedly rearrange multiple Va genes to try and find one that "works" with the earlier rearranged Vb. If a working
receptor is generated that also binds appropriately to MHC plus peptide (see below) then the T cell recieves a signal for
positive selection (which involves the MAPKinase pathway and p21ras) which switches off the recombinase (RAG) genes,
upregulates the amount of T cell receptor, and allows maturation and exit from the thymus.

3/ Positive selection and the maturation of T cells from CD4/CD8 DP to CD4 and CD8 SP T cells

Various surface markers are associated with T cell maturation from DN (double negative for CD4/8) through DP (double
positive CD4/8) with low CD3 (TCR) to CD3 high, SP (single positive for either CD4 or CD8) mature T cells. Instructive
versus stochastic models of positive selection: does recognition of self MHC-I or MHC-II cause down-regulation of CD4 or
CD8 respectively or does down-regulation occur randomly and only those that can form the correct complexes (eg. MHC-
II+CD4+TCR) get signals for further maturation? There is evidence for both models from CD8 transgenic "rescue
experiments", although functional commitment ("helper" vs. "cytotoxic") seems more directly associated with CD8 or CD4
downregulation. Recent evidence suggests an alternative model where the initial duration of TCR signalling in the thymus
determines subsequent development into CD8 (short duration) or CD4 (longer duration) mature T cells.

4/ Are specialised Antigen Presenting Cells or particular antigen peptides required for positive selection?

Evidence that thymic epithelial cells (TEC) tend to positively select while bone marrow derived dendritic cells (BMDC)
tend to negatively select. Bone marrow chimeras and thymus grafting experiments, direct thymic injection of cell types,
reconstituted foetal thymic organ culture (FTOC). Elution and sequencing of self peptides from MHC molecules and
showing that these are no different in the thymus from elsewhere and can positively select in TAP-1-/- FTOC (where the
normal peptide transporter is missing so the MHC-I molecules are "empty" and so do not select).

5/ Avidity model of positive vs. negative selection

HIGH avidity recognition by the T cell receptor of MHC plus self antigen is postulated to cause deletion (negative selction
and tolerance) while LOW avidity recognition of even the same MHC/antigen is postulated to be required for positive
selection. Model (and recent crystal structure) or MHC+peptide+TCR complex and consideration of what "avidity" means,
particularly with respect to so called "agonist" and "antagonist" peptides. Experimental evidence for model: TCR
transgenics with high or low avidity interactions (for MLS or Hb variants), recent direct measurements of TCR affinities for
MHC plus various peptides using BIAcore.

6/ Role of self peptides in positive selection

How does the MHC+self peptide generate a low avidity binding for positive selection? Recent experiments show data
suggesting a number of possibilities: 1) a "gemisch" or mixture of self peptides with no specific motifs for TCR recognition
that act either to stabilize the MHC molecule for surface expression or to dilute the avidity of specific recognition of any
single peptide; 2) specific self peptides that behave as altered peptide ligands (APLs) and pre-determine the reactivity of
the selected T cell; 3) bona-fide self peptides that are recognized with a low avidity simply becuase they are represented at
very low density/MHC occupancy.
T cell development and Positive Selection in the Thymus - Selected References

Mini review:
Bevan, M.J., In thymic selection, peptide diversity gives and takes away.
Immunity 7, 175-178 (1997)
This volume also contains a number of articles concerning the role of peptides in positive selection.
See in particular papers by:
Tourne et al: Selection of a Broad Repertoire of CD4+ T cells in H-2Ma0/0 mice
Surh et al: Thymic selection by a single MHC/peptide ligand produces a semidiverse repertoire of CD4+ T
cells
Cook et al: Quantitation of the cell surface level of Ld resulting in positive versus negative selection of the
2C transgenic T cell receptor in vivo

Other papers:
Chan, S., Benoist,C., & Mathis, D. A challenge to the instructive model of positive selection. Immunol.
Rev. 135, 119 (1993). This volume of Immunological reviews contains a number of excellent articles that
cover most aspects of positive selection and T cell development. See also papers by Ashton Rickardt and by
Elliot in the same volume.

Yasutomo, K., Doyle, C., Miele, L., & Germain, RN. The duration of antigen receptor signalling
determines CD4+ versus CD8+ T-cell lineage fate. Nature 404, 506-510 (2000)

Smith, L. CD4 murine T-cells develop from CD8 precursors in vivo. Nature 326, 798 (1987).

Marrack, P., et al. Comparision of peptides bound to spleen and thymus class II. J. Exp. Med. 178, 2173
(1993).

Carlow, D.A., Teh, S.J., and Teh, H.S. Altered thymocyte development resulting from expressing a deleting
ligand onselecting thymic epithelium. J. Immunol. 148, 2988 (1992).

Blackman, M.A., Marrack, P., and Kappler, J. Influence of the major histocompatibility complex on
positive selection ofVb17a+ T-cells. Science 244, 214 (1989).

Itano, A., Kioussis, D., Robey, E. Stochastic component to development of class I major histocompatibility
complex specific T cells. Proc. Natl. Acad. Sci. 91(1), 220 (1994).

Corbella, P., Moskophidis, D., Spanopoulou, E. et al. Functional commitment to helper T cell lineage
precedes positive selection and is independent of T cell receptor specificity. Immunity, 1(4), 269-76
(1994).

Kawai, K., Ohashi, P. Immunological function of a defined T-cell population tolerized to low affinity self
antigens. Nature 374, 68 (1995)

Hsu, B.L., Evavold, B.D., and Allen, P.M. Modulation of T cell development by an endogenous altered
peptide ligand. J. Exp. Med. 181(2), 805-810 (1995)

Alam, S.M., Travers, P.J., et al. T-cell-receptor affinity and thymocyte positive selection Nature 381,
616-620 (1996)

Alberola-Ila, J., et al. Positive and negative selection invoke distinct signalling pathways. J. Exp. Med.
184, 9-18 (1996)

Liui, X., Bosselut, R. Duration of TCR signaling controls CD4-CD8 lineage differentiation in vivo.
Nature Immunology 5(3): 280-288 (2004)
T cell receptor rearrangements with respect to thymic ontogeny and positive selection

gd

gd
Positive selection on CD1?

g ab
a rearrangements
Checkpoint 2

gd rearrangements
d
+ +

g g
CD4 8
Checkpoint 1

d bd
CD4 CD4
MHC-II

a
CD4/8 double negative
Pre-T a
b b
MHC-I

Vb CD8 CD8
Positive selection

b rearrangements Neglect
 Which cells in the thymus positive vs. negative select?

Proof that mature CD4+ and CD8+ T cells come from

a common thymic CD4+8+ precursor (Smith 1987)

The avidity model of positive selection

Evidence for Evidence for
directed model: stochastic model:

CD8.1 transgene CD8.1 transgenic

introduced into mice crossed to TCR
"normal" mice transgenic (anti-fluNP
increased presented by MHC-I
positive slelction and anti-IE+MTV)
into CD8+ showed "rescue" of
(MHC-I CD4+8-(TgCD8+) but
restricted) MHC-I directed T
compartment cells in non-deleting
(IE-) background.
However, the function
of these CD4+8-
rescued cells directed
to MHC-I was still
"helper" - ie.
determined at the time
of CD8 down-
regulation.

Duration of TCR signalling determines CD4 v CD8

(Yasutomo et al Nature 404, 506 [2000])
First Thymic Second Thymic
Reaggregate Reaggregate
Organ Culture Organ Culture
Various normal or mutant Various normal or mutant
Thymic Stromal Cells Thymic Stromal Cells
(TSCs) (TSCs)

Sorted
Immature h oulrsTSCs CD4++ CD4-+
4 +
1 orma ng CD8 CD8
Thymocytes CD69 hi
wi n R on stroDCs)
t h
Y-TnCd (MaleD4 or CD
8 TSCs or DCs CD4 SP T cells
H
CD4++ r
o liga plus C-linkin g with appropriate
MHC for continued
CD8 3 X
CD69 lo or CD mAb TCR interaction
wi 1.5 (regardless of CD4/8)
'AND' TCR Tg or A th norm hour
s
CD4 ND TC a l T
or C signal (IRA witho SCs
(PCC+MHC-II)
or CD4++ CD4+-
HY-TCR Tg D3 m b mu ut CD8 CD8
Ab X tant
-linkin ) CD69 hi Notch-1
(HY+MHC-I) g CD8 SP T cells
 T he role of (s elf) peptides in pos itive s elec tion
T hymoc yte
(eg. anti S V -M)
" G emis c h" model
(A s hton-R ic kardt, 1993) B X D S ynthetic
F Y
X S mixture of
R ec ons tituting thymic organ c ultures A D MHC -binding
for pos itive s elec tion works bes t with peptides
a c omplex mixture: either s elf peptides E pithelium
eluted from MHC or s ynthetic random mixture
T hymoc yte
S pec ific (A P L ) model " polyc lonal"
(Hs u et al, 1995)
(T ourne et al, 1997)
(S urh et al, 1997) C lip C lip C lip
C lip C lip C L IP or
C lip C lip E -alpha
P os itive s elec tion by even a s ingle low affinity
peptide c an generate a wide, B UT L IMIT E D, C lip C lip peptide
repertoire determined by that peptide. only
F or example, 0/3 rearranged trans genic T C R s E pithelium
were s elec table on C L IP , and 0/2 on E -alpha.
T hymoc yte
(anti-Ld + p2C a)
L ow dens ity (avidity) model
(C ook et al, 1997) p2C a
L ow dens ity
p2C a
P os itive s elec tion is s pec ific bec aus e the p2C a of s pec ific
s elec ting peptide is either the s ame or very MHC + peptide
s imilar to that rec ognized by the mature T c ell. E pithelium (B eta2 K O mic e)
L ow avidity is ac hieved due to low dens ity.