Figures and NotesThymic Selection II: The Thymus Selects the Useful,

Destroys the Harmful and Ignores the UselessApril 24, 2003; Carrie Miceli,cmiceli@ucla.edu

Reading assignment on lat lectures handouts (for this and last lecture):

How do we experimentally determine the progressive stages of cell differentiation?

• Method 1: Inject the sorted or fractionated double negative thymocytes and follow their development in vivo. The more mature a thymocyte is, the more rapidly it will give rise to single positive T cells.

• Method 2: Determine the extent of TCR and gene rearrangement in sorted populations by southern blots.

• Method 3: Study gene knockouts ie CD3, , or RAG

Phases during which thymocytes undergo cell division = curved arrows. The extent of T cell development in thymus of mice (red ) or humans (blue) deficient for a few selected genes is shown.

Stringent developmental blocks=continuous bars; leaky mutations =dashed bars.

For mouse T cell development time line: 1, Ikaros; 2, c-kit + c and GATA-3; 3, winged-helix nude; 4, IL-7, IL-7R, c, JAK-3, and c-kit; 5, c +. pT; 6, RAG-1, RAG-2, SCID (DNA-PK catalytic subunit), Ku80, TCR enhancer, CD3-5 + CD3-/, TCRa + TCRb, Lck + Fyn, and ZAP-70 + Syk; 7, pT; 8, CD3-/, Vav, Lck, CD45, and TCF-1 + LEF-1; 9 MHC class II, CD4, and H-2M; 10b (only CD8+ T cells), MHC class I, 2m, TAP-1, and CD8; and 11, LKLF.

For human T cell 9, TCF-1 + LEF-1; 10 (affecting CD4+ and CD8+ Ts), TCR enhancer, CD3, and ZAP-70; 10a (only CD4+ T cells), development time line: 1, reticular dysgenesis; 3, Di George syndrome; 4, SCID-X1 (c) and SCID JAK3; 6, RAG-1, RAG-2, and other SCID; 10a, MHC class II; and 10b, ZAP-70 and TAP-2.

Book keeping-- more thymocytes are producedthan are present. Therefore most are dying. 95-99%

How do we know this?

Measure proliferating cells by ( BrdUlabeling), measure efflux by FITC andcounting the labeled cells. Only 1-3% of thecells produced actually leave. All of thesesystems have experimental problems, but theconclusion is likely to be correct. More than95% of thymocytes dye in the thymus.

If there is so much death, why isn't the thymusa necrotic dying mess?

Thymocytes undergo apoptosis, not necrosis.apoptotic cells are recognized and rapidlyengulfed by macrophages.

Fig. 7.10 Developing T cells that undergo apoptosis are ingested by macrophages in the thymic cortex. A) a section through the thymic cortex and part of the medulla in which cells have been stained for apoptosis using TUNEL. Thymic cortex is to the right. Apoptotic cells are scattered throughout the cortex, rare in the medulla. b)higher magnif. stained red for apoptotic cells and blue for macrophages. Apoptotic cells are seen within macrophages. Sprent & Suhr. Nature 1994, 372:100. • In normal B6 mouse can count only 0.5%-1% of total thymocytes undergoing apoptosis• if assume Tunel positive cells only visible for an hour can account for 12-24% of cells dying.• Conclusion: apoptosis is fast and so are those macrophages. •Dynamic process

Why all the death? Quality control, receptor checking

• First test of quality is at preT stage to see if is functional (and can bind ligand? No ligand to date). Those that don’t probably die, definitely don’t expand.

• TCR chain is required for thymus maturation. deficient mice have 50X smaller thymus and lack DP thymocytes

• TCR gets to the surface with a surrogate chain (pT) which transduces a (lck/fyn dependent) signal for the cell to rapidly proliferate and continue development by rearranging alpha. This gives functionally rearranged an opportunity to pair with several .

• Mice deficient for preT or lck and fyn similar to

Thymocytes from preT-/- are blocked at the DN to DP transition.same as:

chain -/-LckFyn

The pre-TCR requires pT+ for surface expression and signaling a Lck and Fyn dependent proliferative burst.

After TCR emerges on the surface it needs to be checked

out for two reasons • During “positive selection” cells expressing TCRs with a “minimal” ability to interact with peptide/MHC are selected on thymic epithelial cells to undergo a number of activation and differentiation events including rescue from apoptosis and maturation into CD4 or CD8 single positive cells. – Can a TCR bind self MHC at all– Is it worthy of a spot in the periphery (i.e. likely to be useful?)

• If no, die of neglect (via apoptosis) , if yes receive development signals– Ensure “correct” coreceptor choice

• Class II restricted TCR develop to CD4+ (and helper function)• Class I restricted TCR develop to CD8+ (and cytotoxic function)

• During “negative selection”, thymocytes with TCRs highly reactive with self MHC/peptide ligands presented on dendritic (and in some instances thymic epithelial cells) are purged from the repertoire.– Is a TCR highly auto-reactive and likely to attack self in the periphery?– If yes die as a result of active signal for apoptosis– Thymocytes make transition from most undifferentiated T cells to mature T immunocompetent cells.

Tolerence must be induced here• Only 1% get out alive

Strategies for studying selection/tolerance in the thymus

• The problem is (was) diversity/ T cells each have a different specificity• To determine criterion for selection, one must be able to correlate specificity with

outcome during development• TCR transgenics to the rescue!!

– Make a mouse with fully rearranged TCR and transgenes that produce TCR with a given specificity

– rearranged chain expression inhibits endogenous chain rearrangement via “allelic” exclusion mechanisms, so all T cells are TCR tg +

chain rearrangement not turned off simply by / surface expression. Rather, turned off with “proper” ligand binding (positive selection turns off RAG genes). Thus, “allelic exclusion” of is leaky.

– Can get rid of cells expressing endogenous rearrangement • By staining with a “clonotypic” antibody against Tg TCR or w/ anti-

chain• By crossing to a Rag1/2-/- mice, rearrangement of endogenous genes

impossible• Super-antigen expression in the thymus leads to deletion cells expressing particular

Vb genes.

Positive and Negative Selection in the H-Y (Rag-/-) Transgenic model

Small thymus-negative selection

CD8low Tg T cells

Normal thymus-positive selection

CD8hi Tg T cells

T cell APC

CD8

CD3 Tg TCRHY

peptide MHC

C57BL/6 control male

HY TCR transgenic Female

HY TCR transgenicmale

Expression of TCR Tg in females with H-2Db

results in development of CD8+T (increased CD8/CD4)

Expression of TCR tg and autoantigen in males results in deletion of DP thymocytes and blocks development of Tg+ CD8 SP

Harold von Boehmer HY TCR transgenic mice

In Rag-/-HY TCR transgenics all thymocytes express the TCR tg. More dramatic skewing to

CD8 in females and deletion in males

Female HY TCR tg

CD4

0.6%;

5.8% 2.8%;

91%;

CD4

CD8

Male HY TCR tg7X108 2X107

CD8

9%,CD4

MalesFemales

Peripheral T cells from spleen of HY-TCR transgenic mice

• wt mice have 2:1 CD4:CD8

•Normal levels of CD8 on CD8+ T cells•HY/Db responsive (stimulated bymale APCs)

• CD8 down-regulated on those cells that do escape• HY/Db nonresponsive

Negative Selection as a Central Tolerance mechanism• Thymic Dendritic cells function to present self peptides present in

“professional APC”• Thymic Medulary Epithelial cells can also negatively select

– Antigens seen in epithelial tissues?– Or more? Every peptide in the body? (Aire transcriptional regulator)

• How do we purge the repertoire of self antigens found only in peripheral tissues

– How professional are they at antigen presentation? – Small amounts or peptide loaded APC travel to the thymus and enter both class

I and class II pathways?– Do these cells have the capacity to express bits of every gene expressed in the

periphery?– How do we get a high enough concentration of any given peptide? – Do peripheral mechanisms exist to protect against inefficiency

» Anergy/Tregs?

Positive selection

• Definition: Positive selection is manifested by the tendency of mature T cells to recognize antigens in the context of ones own MHC molecules as opposed to allogeneic (foreign) MHC molecules.

• MHC molecules are polymorphic and unlinked to the TCR genes, so this must occur as a result of a developmental process.

• Take home lesson: DP undergo programmed cell death unless the TCRs are engaged in the thymus– Receptor checking process – Positive selection involves “choice” of appropriate co-receptor

– Mature CD4 SP have class II restricted TCRs– Mature CD8 SP have class I restricted TCRs

How is TCR specificity coupled to proper co-receptor expression and developmental program?

• Instructive Model: during positive selection TCR/CD4 engagement by MHC II sends an instructive signal for CD8 off/CD4 on; engagement by MHC I sends an instructive signal for CD4 off/CD8 on . What is the nature of this signal and how does it cue a developmental program which includes functional maturation (helper or CTL)? (probably strength of Lck signal CD4>CD8; strong Lck makes you go CD4 helper program)

• Stochastic/selective model:CD4 or CD8 is randomly turned off prior to positive selection. Those with “matching” TCR and coreceptor expression are selected during positive selection. Those that randomly made the wrong choice die of neglect.

During positive selection CD4/CD8 coreceptor expression is coupled to T cell specificity

• Thymocytes expressing class II restricted TCRs develop into CD4 SP• Thymocytes expressing class I restricted TCRs develop into CD8 SP

• CD4 development requires class II expression, only CD8s present in class II knockouts• CD8 development requires class I expression, only CD4s present in class I knockouts

Got peptides??.• Peptides are important for positive selection, need some level of specificity. Recent data indicate that likely more than one peptide can positivelyselect any given TCR (low binding threshold required, so many

may fit the bill)

• Complexity of petide(s) positively selecting a given T cell receptor bearing clone. • What is the nature of the peptide(s) in the groove during positive selection. ?

• Specificity of peptide important. (Not any peptide will work for TCR transgenics)

• Can mice bearing "a single petide in I-A MHC groove" postively select a diverse repertoire? • select a surprisingly high number of CD4 T cells (but not normal), though diversity of peptides is reflected in diversity of

response). 1.) Single petide in a MHC made by "sewing/covalently linking peptide" into I-A and expressing as a transgene on invariant

chain knockout.

2.) H-2 M (DM) knockouts (primarily but not exclusively presenting clip peptide),

• Problem: neither 1 or 2 really only express 1 peptide.

Requirement for Diverse, Low-Abundance Peptides in PositiveSelection of T Cells. Gregory M. Barton, Alexander Y. Rudensky *

Science Jan 1, 1999• Residues 86 to 102 of Ii have been

replaced with peptide E. When expressed in Ii- in Ii-knockout mice (Tg·IiKO), this transgene restored surface expression of the class II allele, I-Ab, to wild-type levels on splenocytes. The E peptide was efficiently loaded onto I-Ab .knockout mice (Tg·IiKO), this transgene restored surface expression of the class II allele, I-Ab, to wild-type levels on splen-ocytes. Tg·dblKO= Tg·IiKO·H2MKO

(B not shown) The percentage of class II bound with non-E peptides was calculated and ranged from 2 to 9% in three independent experiments and were comparable for both Tg·IiKO and Tg·dblKO splenocyte lysates.

Fig. 2. CD4 T cell development is impaired in Tg·dblKO mice. CD4 versus CD8 plots of (A) thymocytes and (B) splenocytes from the

indicated mice are shown. Conclusion: In Tg·IiKO mice, there was a small population of I-Ab molecules loaded with diverse, high-affinity non-E peptides that are dependent on H-2M and therefore not detectable in Tg·dblKO mice.

These peptides appear to be critical for positive selection of the normal number of CD4 T cells seen in Tg·IiKO mice.

TgIiko TgIiko TgIidblkoTgIidblko

dblkodblkoIiko Iiko

Models for thymic selection(not mutually exclusive)

Restatement of the problem: you can not negatively select everything you positively select.

A. MHC is recognized, peptide is irrelevant. We know, however, that peptides are required to get MHC molecules to the cell

surface. Therefore, according to this model the best peptides are those which keep out of the way. Disproven.

B. The avidity required for positive selection is less than the avidity required for negative selection. This means that for a single TCR, there will be more ligands capable of positive selection than are

capable of negative selection. (avidity model, previous incarnations referred to as affinity models)

C. The "quality" of the signal is critical, rather than the quantity. At the molecular level, what does this mean? We do not know, but in a series of cogent papers it was shown that antagonist peptides are

best at positive selection. Perhaps a conformational change (dimerization, multimerization) with agonist, not with antagonist.

D. Positive and negative selection happen at distinct stages of thymocytes development and in different anatomical locations.

Difference in positive and negative selection could reflect:

1) differential responses to differing populations of APC that either contain different peptides or that

express different costimulatory molecules.

2) T cell at different stages of development respond to TCR engagement differentially.

Models for thymic selection (continued)

Differential Avidity Model (goldilocks)

To hot-neg selectTo cold -die of neglectJust right- positive selection

• To simplify the system , several groups examined the selection of a single TCR ( TCR transgene) selected by a single predominant peptide that can be varied at will.

• This required the use of fetal thymic organ culture (FTOC) and TCR trangenic mice

• FTOC-take thymus (fetal) at 16 postcoitum, culture in vitro for 9 days. Immature thymocytes at start, some mature at the end.

• The surface expression of class I molecules can be stabilized by the addition of (your favorite) peptide in (TAP deficient) mice or peptide plus B2M (B2m-/-) to cells in vitro, including cells in a FTOC

Evidence for a Differential Avidity Model of T cell Selection in the thymus. Ashton-Rickardt…..Zinkernagel, Tonegawa Cell 76,651 (1994)

Cross H2-Db/LCMV pep33-41 specific TCR transgenic to TAP-/-

CD4

CD8

Positive selection impaired in TAP-/- FTOC and is restored by nominal antigen peptide. Remember that levels of stabilized class I MHC/peptide still lower than normal

Gated on TCR tg hi gets rid of leak looks specifically at postively selected cells

Positive selection Negative selection

LCMV p-33-241

Irrelevant Db binding peptides; so specific peptide matters

(Get negative selection with LCMV 33-241 on TAP+/+)

CD8+ TCR transgenic P14 Cells generated in Tap-/-FTOC

Increase concentration, increase avidity; drive positive selection to negative selection

T Cell Receptor Antagonist Peptides Induce Positive Selection

Cell 76: 17-27 1994Hogquist, Jameson, Heath, Howard, Bevan, Carbone

FTOC using OT-1 TCR transgenic X 2m-/-

TCR specific for H2-Kb/ova 257-264

Need peptide and 2m to stabilize surface class I MHC

2m2m

Negative selection Because less than 10% as many total cells

Agonist peptide

Agonist and antagonist properties of peptides used in FTOC. The capacity of various peptides to induce or antagonize lysis by a CTL line derived from a transgenic mouse

All peptides can bind MHC and stabilize MHC equally

Only antagonist positively select; quality of peptide

Some peptides K1 follows avidity model in so far as positive selection on -/-, but negative on higher avidity +/+, but not in every case i.e.R4

No direct correlation between how good of a “positive selector” and how good of an antagonist (ie P7 vs R4). Wouldn’t this be a prediction of an avidity model

The sequel “ Strong Agonist ligands for TCR do not mediate positive selection of functional CD8 T cells

Hogquist, Jameson, BevanImmunity 3: 79-86 (1995)

• No matter how low in concentration you drop the agonist peptide concentration in this OT-1 TCR/2M-/- FTOC model, agonist never gives + selection; Either negative selection or no effect.

• One “weak angonist” could postitively select, but resulting peripheral Ts were non-functional.

• Is this consistent with an avidity model??• One model in support of avidity, another

against…what’s up with that??

MODEL D. Positive and negative selection happen at distinct stages of thymocytes development and in different

anatomical locations

The thymus (cortical) epithelium is responsible for imprinting self on T cells

Thymus transplants show that nonlymphoid cells, or the radiation resistant portion of the organ is responsible for positive selection. Used irradiated mice reconstituted with bone marrow stem cells and a thymus graft 1970s doherty

Perhaps positive and negative selection induced by different cells types, perhaps sequential according to order the thymocytes are presented with these “APCs” in the thymus

Anatomical correlates

UNOPPOSED POSITIVE SELECTION AND AUTOREACTIVITY IN MICE EXPRESSING CLASS II

MHC ONLY ON THYMUS CORTEX NATURE 1996 p81-5Terri Laufer…Laurie Glimcher

• Approach: mouse with class II under keratin 14 promotor as a trangene crossed to a class II-/- mouse to re-express class II only in cortical thymic epithelial cells = K14 mouse

K14 5-18%

Mouse % SP CD4 cells in the periphery (spleen, ln)

Class II-/- 1- 4%

AutoreactivityProlif to self

36-5

-+-

C. Normal mouse has class II expressed in Cortex +Medulla

d. 36-5 tg expresses on C+M, but nowhere else in the body

e. A-/-

a. K14 MHC in cortical epith only(weaker)

Conclusions• Cortical epithelial cells can positively select, but can’t

negatively select. • Medulary epithelial cells can negatively select (MTEC, specialized

thymic APC in which Aire directs the expression of tissue specific antigens (relevant to autoimmune uvitis, EAU inter-photo receptor binding protein proteins (IRBP) and other tissue specific autoimmunity..can barely visualize antigen in the thymus. J Exp Med 03, Avichezer…Caspi

• Other’s experiments indicate the cells of hematopoetic origin are good negative selectors (thymic dendritic cells) (evidence that some antigens travel to thymus via BM derived APC (relevant to EAE, MBP 121-150) and negative selection purges reactive clones?

• Authors conclude “positive selection of thymocytes occurs before, independently of, and in distinct anatomical sites from negative selection.

Cathepsin L: Critical Role in Ii Degradation and CD4 T Cell Selection in

the Thymus. Nakagawa… Rudensky, Science 1998 April 17; 280: 450-453.

Fig. 2. Reduced number of CD4+ T cells in CTSL-deficient mice.

Fig. 3. Radioresistant thymic epithelial cells are responsible for the defect in development of CD4+ T cells in ctsl/ mice.

Fig. 4. Within the thymus, CTSL activity was found in cTECs and thymocytes, but not in BM-derived thymic DCs; whereas CTSB was ubiquitously expressed

Fig. 5. Altered MHC class II processing in thymic cortical epithelial cells of ctsl/ mice.

Fig. 1. Normal expression of surface MHC class II molecules and antigen presenting function of ctsl/ SCs. (not shown)

Mizuochi et al J Exp Med 1992 175:1601

Cresswell summarizes implications of Rudensky studies

They appear to use cathepsin S for the late stages of invariant chain degradation

D. Positive and negative selection happen at distinct stages of thymocytes development and in different anatomical locations

(Laufer).

Therefore, difference in positive and negative selection could reflect:

1) differential responses to differing populations of thymic APC that either contain different peptides (rudensky) or

APC that express different costimulatory molecules (ie CD30 needed for negative, but not positive selection) Amakawa, Cell

1996.

2) T cell at different stages of development respond to TCR engagement differentially, possible if really sequential.

Perturbing TCR signal transducers and assessing affects on negative and positive selection; familiar models re-

invented/refined

• Strength of Signal Model of Thymocyte Development (the TCR is an on/off switch with high, medium, and low settings– Direct extension of the Avidity Model– Receptor occupancy translates into strength of signal; distinguish

high avidity from low avidity by counting receptors engaged and counting intensity of signal

– Negative selection induced by a strong signal (high avidity; agonist)

– Positive selection induced by a weaker signal (lower avidity; partial agonists/antagonists

– Insufficient signal leads to death by neglect– Quantitative differences

What are the predictions for each model regarding how modulators of TCR signal transduction would affect positive vs negative selection?

See assigned reading for a review including recent signaling data.

• Positive and Negative Selection invoke distinct signaling pathways (the TCR is not a simple on/off switch

• recruitment of different transducers under different circumstances.– Alternate signals result in distinct functional outcomes.– Qualitative differences– Kinetic proofreading; an individual TCR can distinguish

positively and negatively selecting ligands by “reading” the occupancy time

– Receptor could generate an ordered set of biochemical signals such that the earliest signal lead to positive selection and the later signals would induce negative selection.

Positive and Negative selection rely on TCR

transducers/costimulators to a different extent. • Alberola-Ila et al., J Exp. Med. 184: 9-18, 1996. Positive, but not,

negative, selection is blocked in mice expressing dominant-negative Ras and Mek-1 transgenes simultaneously.

•Backstrom et al Science, 1998 281:835-8. Transgenic mice expressing an TCR lacking the -CPM (or CD3 also Science,281 1998), thymocytes are deficient in positive, but not negative, selection (and sustained but not transient ERK.

•RasGrp -/- affects positive, but not negative selection (sustained ERK?) Nature Immunology1, 317, 2000, Immunity 17, 617, 2002.

• Grb-2 haplo-insufficiency (+/-) affects negative but not positive selectionNature Immunology 2, 29, 2001 (transient ERK regulator?)

•Amakawa R; et al. Cell, 1996 Feb 23, 84(4):551-62. CD30-deficient mice are defective at negative but not positive selection.

HighAvidity

Low Avidity

AvidityAvidity