T Cell Development I: The Generation of TCR+ Thymocytes

Questions for the next 2 lectures: How do you generate a diverse T cell population with functional TCR rearrangements? How do you generate a T cell population that is self-MHC restricted? How do you ensure that those diverse T cell receptors are not-self reactive? How do you coordinate lineage specification with MHC specificity and coreceptor expression? •  vs.  T cell • CD4 vs. CD8

The pathway of T cell development 1. Bone marrow All the cells of the immune system are derived from stem cells in the bone marrow. The bone marrow is the site of origin of red blood cells, white cells (including B and T lymphocytes and macrophages) and platelets. T-cell originates from bone marrow but all the important events in their development occurs in the thymus. Thymus dependent lymphocytes = T lymphocytes. Thymus Thumus localised above the heart. In the thymus gland lymphoid cells undergo a process of maturation and education prior to release into the circulation. This process allows T cells to develop the important attribute known as self tolerance. The thymus varies in size and undergoes structural alterations with age. It undergoes rapid growth until the end of the second year, after which time the rate of growth slows until approximately the fourteenth year. After this, the thymus begins to involute or decrease in size, and, gradually, the lymphatic tissue is largely replaced by fat and connective tissue. In old age, very little thymic tissue may be present. Removal of the thymus after puberty is not associated with any loss of T-cell function. Thus it seems that once the T-cell repertoire is establish, immunity can be substained without the production of significant number of new T-cell; The pool of peripheral T-cell is maintained by the division of mature T-cell. 3.DiGeorge syndrome DiGeorge syndrome, which is a birth defect that affects in particularthe immune system.The syndrome is marked by absence or underdevelopment of the thymus and parathyroid glands. It is named for the pediatrician who first described it in 1965. Children born with complete DiGeorge Syndrome often face a host of medical challenges that can include heart problems, developmental disorders and deafness, but without treatment, infection resulting from immune deficiency is by far the factor that most often causes these children to die. DiGeorge syndrome is caused by inheritance of a defective chromosome 22 -- from the mother in most cases. The third and fourth pharyngeal pouches fail to develop normally during the 12th week of pregnancy, which results in completely or partially absent thymus gland and parathroid glands. The child is born with a defective immune system and an abnormally low level of calcium in the blood. These defects usually become apparent within 48 hours of birth. The infant's heart defects may lead to heart failure, or there may be seizures and other evidence of a low level of calcium in the blood (hypocalcemia).The prognosis is variable; many infants with DiGeorge syndrome die from overwhelming infection, seizures, or heart failure within the first year. Advances in heart surgery indicate that the prognosis is most closely linked to the severity of the heart defects and the partial presence of the thymus gland. In most children who survive, the number of T cells, a type of white blood cell, in the blood rises spontaneously as they mature. Duke University Medical Center physicians have reported in the Aug. 1, 2003 issue of the journal Blood, successfully treating the immune disorder complete DiGeorge Syndrome in seven of 12 children who underwent an experimental thymus transplantation procedure. As many as one in 4,000 children in the United States are born each year with varying degrees of DiGeorge Syndrome, a condition in which the body does not produce adequate quantities of T cells, the cells that help the body fight infections. Between five and 10 children are born in the United States each year with complete DiGeorge Syndrome, a condition in which babies' immune systems do not develop at all because they are born without a thymus. Without intervention, few children with complete DiGeorge Syndrome live to age 1, and none survive past 3 years of age. The seven surviving Duke patients are all well and living at home two to 10 years after receiving their transplants. Five patients in this study died, all from underlying congenital problems. Transplantation is made possible because a small amount of thymus tissue is ordinarily discarded during neonatal heart surgeries. It must be excised in order for surgeons to expose the heart. Markert asks parents of babies undergoing heart surgery for permission to use any discarded thymus tissue to help a child with DiGeorge Syndrome. The thymus tissue is then sliced thinly and cultured, and tested for any abnormalities or diseases. After preparing the tissue, surgeons implant the slices of thymus tissue into the quadriceps muscles of both legs of the complete DiGeorge Syndrome baby. Without an immune system, the body cannot reject new organs. Therefore, matching the donor thymus tissue to the complete DiGeorge Syndrome baby is not necessary. T cells...precursors are born in the bone marrow and “educated” in the thymus. The thymus “involutes” with age DiGeorge Syndrome, Nude mice

Nude Mice The nu mutation was first reported in 1966 in a closed stock of mice in a laboratory in Glasgow, Scotland. It was not until 1968, however, that it was discovered that the homozygous nude mouse also lacked a functional thymus, i.e., it was athymic. The mutation produces a hairless state, generating the name "nude." The other, unique defect of nude mice is the failure of the thymus to develop normally to maturity. The thymus remains rudimentary and produces reduced numbers of mature T cells. This means nude homozygotes (animals with identical mutant genes at corresponding chromosome loci) do not reject allografts and often do not reject xenografts (tissue from another species). The discovery that human neoplasms (tumors) could be grown in nude mice was immediately recognized as an important research tool. Thus, the spontaneous mutation of nu among laboratory mice was a serendipitous development that led to the nude mouse becoming the first animal model of a severe immunodeficiency. In the decades since, the nude mouse has been widely utilized by researchers studying factors regulating transplantable human tumor growth and cancer metastasis. Although it lacks T cells, the nude mouse has a normal complement of bone-marow-dependent B cells. It thus presented a unique tool for the study of the role of the thymus on lymphocyte differentiation, investigations of B cell functions (including interactions with other immune cells) and studies of the actions of other immune cells, including the natural killer (NK) cells. Nude mice have elevated levels of both macrophages and NK cells; their macrophages are more potent than those from mice with a normal thymus. They have a normal complement activity A. Human (mammal) skin after 60 days. B. Cat (mammal) skin at 51 days. C. Chicken (bird) at 32 days; D. Chameleon (reptile) at 41 days. E. Fence lizard (reptile)at 28 days. F. Tree frog (amphibian) at 40 days.

The Thymus is required for T cell development scid mutation: DNA PK no gene rearrangement nude mutation: transcription factor required for terminal epithelial differentiation (whn) NO B or T CELLS NO THYMUS

T cell development occurs in the thymus The Thymus has a Capsule with Trabeculae. The trabeculae divide the thymus into Lobules. Per lobule you have an outer cortex and an inner medulla. The cortex is formed of dense lymphoid tissue which lacks nodules. Since the stroma of the medulla is less heavily infiltrated with lymphocytes than the cortex, the medulla stains more lightly than the cortex. Immature lymphoid cells enter the cortex proliferate, mature and pass on to the medulla. From the medulla mature T lymphocytes enter the circulation. Arteries supplying the thymus follow the connective tissue septa and give off branches that enter the lobular cortex and break up into capillaries, which supply the cortex. Epithelial reticular cells sequester developing lymphocytes and form a sheath covering capillaries and lymphatic vessels. The sheathing forms what is called the blood-thymus barrier, preventing antigen contamination of developing and programmed T lymphocytes. The blood-thymus barrier is not found in the medulla, which appears to have a richer blood supply than the cortex. The capillaries terminate in thin-walled veins located in the connective tissue septa along with arteries. Lymphatic vessels arise within the thymic lobule and join to form larger vessels, which accompany the arteries and veins in the septa. In contrast to lymph nodes, the thymus contains no lymph sinuses or afferent lymphatic vessels. The following cell types are present: lymphoid cells, epithelial cells, macrophages, other supporting cells Thymic epithelial cells have different appearances in different locations within the gland. They form a continuous sub-capsular layer and a network in the cortex and medulla. Deep in the medulla they are also aggregated into Hassall's corpuscles. Morphologically, the thymus differs from the lymph node in its lack of nodules and afferent lymphatic vessels, and by the presence of Hassall's corpuscles. Efferent lymphatic vessels take origin close to the trabeculae and leave through them. Blood vessels also enter and leave through the trabeculae. Capillaries but no venules can be seen in the cortex. In the medulla, venules are present. Blood vessels in the thymic parenchyma have a sheath of reticulo-epithelial cells external to the basal lamina of their endothelium; this establishes the blood-thymic barrier. Most developping T-cell dies within the thymus. T cell precursor arriving in the thymus from the BM spend up to a week differentiating there before they enter a phase of intense proliferation. Approximately 98% of the lymphocyte will fail their maturation and die whithin the thymus. They die by apoptosis and their residual bodies are seen inside the macrophages throughout the thymic cortex. This apparent waste is a crucial part of T-cell development as its reflects the intensive screeming that each new thymocyte undergoes fopr the ability to recognize self-MHC and for self tolerance.

Figure 5-3 part 1 of 2

The epithelial cells & developing thymocytes

T cell development can be characterized using flow cytometry When progenitors enter the thymus from the BM, they lack most of the surface molecules characteristic of mature T-cellsvand their receptor genes are unrearanged. If injected into the blood \stream these precursor can give rize to B-cell and NK cell. Interaction with the thymic stroma trigger an initial phase of differentiation along the T-cell lineage followed by cell proliferation and the first expression of molecules specific for Tcell. At the end of this phase, which last about a week, the thymocytes bear distinct markers of the T-cell lineage, but they do not express CD4, CD8 or CD3--> They are called Double negative. In the fully developped Thymus DN are 5% of the cells. They also comprise fully developed gammadeltaTCell and NK-T cell. NK T-cell recognize CD1 molecules

CD4 CD8 Real data 8 88 3 1 Double-positives CD4 single-positive Double-negatives CD8 single-positive

DN’s can be further subdivided into DN1 through DN4 CD25 (a chain of IL2R) “Gate” on CD4-CD8-, analyze for CD44 and CD25

The real data (gated on DN cells) CD25

Double negative cells appear in the fetal thymus before double positive cells One of the ways we can establish the relationship between DN and DP is by looking early in development. K. Hogquist

T cell populations during development and in the lymphoid tissue THYMUS SPLEEN CD4 CD8

Thymocytes at different developmental stages are found in distinct parts of the thymus Maturation

Today Mention gd lineage decision to be discussed at end Thursday

Most cells fail to complete thymocyte maturation That’s because they fail to complete the steps necessary for moving forward.

Macrophages...garbage collectors, contain the dying cells Most developing thymocytes die in the thymus apoptotic cells (DNA fragmentation) macrophages Cortex of the thymus. Macrophages...garbage collectors, contain the dying cells

T cell development involves the sequential generation, assembly and testing of the newly rearranged TCR

TCR rearrangement is very similar to Ig rearrangement We can’t talk about T cell development without talking about rearrangement. We can think of the beta chain as analogous to the heavy chain. Alpha chain analogous to the light chain.

Figure 5-6 part 1 of 3 First step: TCRbeta rearrangement DN thymocytes begin rearranging TCRbeta locus.

Figure 5-6 part 2 of 3

How do you test for successful TCRbeta chain rearrangements if you have not rearranged TCRalpha? Pre-Talpha Analagous to the surrogate light chain for immunoglobulin Pre-T cell receptor

TCR  chain is required for DN to DP transition: Experimental Evidence WT TCRa-/- TCRb-/- TCRa/b-/- TCRb/d-/- 108 108 <107 <107 106 Total thymocyte numbers Expressing TCRgd CD4 CD8

(work of Nigel Killeen, UCSF) Does the pre-TCR have a ligand? Remove extracellular domains… …and examine T cell development (work of Nigel Killeen, UCSF)

Pre-TCR extracellular domains are not required! pTaT;bT receptor restores CD4+8+ development and thymocyte expansion Important to note differences in cellularity. Remember that there is a huge expansion during Beta selection (Irving (1998) Science 280:905)

b-selection leads to proliferation DN2 Gated on CD4/CD8 DNs CD25 DN3 Proliferating cells are bigger Forward scatter (cell size)

TCR b locus has two D-J clusters Allows a 2nd rearrangement if 1st is nonproductive

We are here

Progression through development correlates with rearrangement TCRbeta rearrangement begins during DN stages.

At the DP stage, TCRalpha rearrangement begins

TCR a locus has 50 Ja segments

TCRa locus-- replacement rearrangement

DP thymocytes express surface TCR CD3 lo cell-- pre-TCR CD3 hi cells-- TCR

TCR  chain is required for DP to SP transition: Experimental Evidence WT TCRa-/- TCRb-/- TCRa/b-/- TCRb/d-/- 108 108 <107 <107 106 Total thymocyte numbers Expressing TCRgd CD4 CD8

Differential expression of Src family kinases Signals at each step lead to gene induction. Differential expression of Src family kinases

Remember what Lck does

Role of Lck in T cell development RAG KO TCRTransgene RAG KO x Lck KO TCRTransgene RAG KO CD4 CD8 Total thymocyte number 0.4 x 106 288 x 106 10 x 106 30-fold reduction in DPs!

 vs  Thursday

A model for  versus  lineage commitment Both gamma and delta genes have to rearrange before preTCR signals commitment to ab lineage Distinct signals from gammadelta vs preTCRab determine fate.

TCR rearrangement removes the delta locus Further ensures commitment to alphabeta

TCR lineage comprises the majority of T cells 90% 10%

during early fetal development gd T cells are favored during early fetal development

Waves of  T cells with specific TCR usage develop early gestation

Waves of  T cells home to different tissues

Early waves of  T cells gd cells bearing specific receptors end up in skin (Vg5), gut (Vg2), uterus (Vg6), etc. Limited junctional diversity: no N nucleotides (no TdT) The mechanisms controlling this limited rearrangement are poorly understood.

Antigen recognition by  T cells is different than  T cells  T cells are not MHC restricted! Antigen is recognized directly, more like an antibody In some cases ligands for the  TCR are self proteins upregulated under stress conditions In humans, circulating  cells recognize a phospholipid antigen from Mycobacterium tuberculosis Conceptually quite different than what we have been talking about. Probably OK because at least the initial waves of gd T cells have fixed specificity.

Dendritic epidermal T cells (DETC) Vg5 gd T cells home to the skin and wedge among keratinocytes Involved in wound healing as well as tumor protection Let’s talk about one subset.

DETCs promote wound healing Using this to illustrate recognition of upregulated self ligands.

Roles of gd T cells in cancer • gd-knockout mice have a higher incidence of skin cancers induced by carcinogens, including DMBA/TPA (tumor initiator/promoter) (shown in figure) or methylcolanthrene (MCA). • The protection is mediated by DETC resident in the mouse skin Appearance of tumor cells in knockout versus wildtype mice treated with carcinogen Girardi et al, Science 294:605 (2001)