1. In unimmunized mice: 1 in 26,300 spleen B cells could make anti-SRC IgM no detectable (<1 in a million) B cells that could make anti- SRC IgG (Martínez-Maza, et al. Scandinavian J. Immunol 17:251, 1983) In immunized mice: 1 in 219 B cells could make anti-SRC IgM (5d post-immunization) 1 in 112 B cells could make anti-SRC IgG (12d) 1 in 3,030 B cells could make anti-SRC IgG (180d) (Martínez-Maza, et al. Scandinavian J. Immunol 17:345, 1983)

2. Calame, K. 2001. Plasma cells: finding new light at the end of B cell development. Nature Immunology 2:1103.

4. T CELL DEVELOPMENT AND ACTIVATION There are a lot of similarities between T and B cells, in their development: –arise from hematopoietic precursors that are generated in the bone marrow –undergo similar DNA rearrangements to generate the genes for their antigen receptor molecules –have the capacity to respond to nearly any antigen –the initial stages of development are antigen- independent, with final differentiation occurring after exposure to antigen –cells that express antigen-receptors that react with self are eliminated

5. However, there are some significant differences: –since the T cell receptor can interact with antigen only when it is presented in association with self- MHC molecules, T cells need to be able to bind to a complex of self MHC + Ag peptide –in addition to this (perhaps because of this) T cells do not develop in the bone marrow, they undergo development in a specialized organ, the thymus.

6. T lymphocytes or T cells got their name from original observations that indicated that they were thymus- derived lymphocytes. T cell precursors travel from the bone marrow to the thymus:

7. Following development into mature, antigen-responsive T cells, these T cells emerge from the thymus and migrate to secondary lymphoid tissues, where they interact with antigen, antigen-presenting cells, and other lymphocytes:

8. The importance of the thymus in T cell development is demonstrated by inherited immune deficiencies: people that do not have a thymus (DiGeorge’s syndrome, aka Thymic Aplasia) do not develop functional T cells. DiGeorge’s syndrome results from a developmental defect – the failure of the third and fourth pharyngeal pouches to develop, which results not just in thymic defects, but also in absent parathyroids and in aortic arch defects. Thymectomy early in life reduces the ability to produce T cells. Thymectomy later in life does not markedly impair T cell number. In fact, the thymus decreases in size with age. However, the thymus can still produce new T cells up to middle-age, especially in situations where there is loss of T cells (HIV/AIDS).

9. While in the thymus, immature T cells, or thymocytes, undergo several changes that allow them to develop into mature T cells, ready for contact with antigen. Thymocytes interact with thymic epithelial cells and various other cells while in the thymus.

10. The thymus is composed of several lobes, each of which has cortical and medullary regions:

11. The cortex contains immature thymocytes in close contact with thymic epithelial cells. Medullary areas contain more mature thymocytes, epithelial cells, and dendritic cells and macrophages

12. During thymic differentiation, the great majority of thymocytes die by apoptosis, and are ingested by macrophages. Only a small minority of these T cell progenitors make it out as mature T cells

13. Thymic development occurs in two phases: 1)production of T cell receptors for antigen, by rearrangement of the TCR genes 2)selection of T cells that can interact effectively with self-MHC

14. Changes in the expression of cell-surface molecules accompany the thymic differentiation of T cells: –entering thymocytes are TCR, CD3, CD4, and CD8- negative –as thymocytes mature, and undergo rearrangement of their TCR genes to generate a functional TCR, they begin to express CD3, CD4, and CD8 –mature T cells ready to go to the periphery are TCR/CD3+, and either CD4 or CD8 positive

15. First phase of thymic development: rearrangement of TCR genes to produce a functional TCR Progenitor T cells enter the thymus (sub-capsular region of the outer cortex). These cells do not have rearranged TCR genes and lack expression of characteristic T cell surface molecules. Interaction with thymic stromal cells induces these progenitor T cells to proliferate. These immature thymocytes do not yet express CD4 or CD8, molecules that are expressed by mature T cells: double-negative thymocytes.

16. There are two types of T cell receptors:  and   TCR T cells are the most abundant, by far: (or  &  chain)

17. Unlike B cells, in which the genes that encode the BCR rearrange in a set order, the TCR , , and  genes start to rearrange at about the same time. If a productive  or  rearrangement occurs first, the T cell is committed to that lineage, and stops further rearrangement of the  TCR gene.

18. However, if  is rearranged first, then the T cell continues to proliferate, and undergoes further rearrangements. This results either in rearranged  TCR gene, yielding an  TCR lineage cell, or rearranging  and  genes, resulting in a  TCR cell.

19. Rearrangements that lead to an  T cell begin the rearrangement of the  TCR gene. The first step is D-J joining, followed by VDJ rearrangement. Expression of  chain stops further  chain rearrangements.

20.  chain is then expressed on the surface of the thymocyte in association with a surrogate  chain (pT  ). Following this, there is rearrangement of the  TCR gene, resulting in a functional  chain, and in the expression of surface TCR, in association with other T cell-associated cell surface molecules.

22. During this process, a cell that makes an unproductive  chain rearrangement can try again until gets a good  chain, or it exhausts its possibilities:

23. Thymocytes that have a functional  rearrangement, and express  or  + the surrogate  chain (pT  ) are induced to express both CD4 and CD8 simultaneously – these are called double-positive cells. Immature T cells that do not undergo a productive rearrangement die by apoptosis.

24. Second phase of thymic development: selection of T cells that can interact with self MHC and antigen This applies only to  TCR-bearing cells (>95% of T cells).  T cells are not restricted to interactions with MHC class I or class II molecules This phase of T cell development consists of two steps: –positive selection (TCR that can interact with self-MHC) –negative selection (eliminate self-reactive cells that are stimulated by MHC + self)

25. Positive selection refers to the selection of thymocytes that are able to bind to, and interact with, self-MHC molecules In positive selection developing thymocytes continue to live if they bind MHC well enough to receive a signal through their TCR. This signal is mediated by the interactions of these cells with MHC-expressing thymic cortical epithelial cells. The ~95% of thymocytes that do not receive this signal undergo apoptosis. Positive Selection

26. Positive selection takes place in the cortex of the thymus lobules:

27. These CD4+ CD8+ TCR+ thymocytes interact with thymic epithelial cells that express both MHC class I and MHC class II molecules, complexed with self-peptides. Thymocytes that bind MHC survive; those that don’t bind to self-MHC die. TCR  chain rearrangements can continue during positive selection, allowing cells to explore alternative  chains for MHC binding. Once a T cell is positively selected, TCR rearrangement stops.

28. The expression of either CD4 or CD8 by a given T cell is determined during positive selection, leading to single-positive cells (CD4 or CD8-positive). Those cells that have a TCR that binds to MHC class II end up as CD4 single-positive cells Those that bind MHC class I as CD8 positive cells:

30. Negative selection refers to the elimination of those thymocytes that bind to self-MHC molecules + self with high affinity. In negative selection developing thymocytes die if they bind MHC + self peptides too well (strongly enough so that they would be activated by this interaction, via signaling through their TCR). Negative Selection

31. Thymocytes undergo negative selection in the medullary region:

32. There, they interact with antigen-presenting cells (dendritic cells, macrophages) that express self- antigens + MHC class I or MHC class II molecules. Thymocytes that bind to self + MHC too strongly are eliminated as possibly self-reactive cells, and undergo apoptosis. If self-reactive T cells were allowed to exit the thymus, such cells would mediate autoimmune disease.

33. Some T cells are reactive with self molecules that are not expressed in the thymus: –such cells can be eliminated in peripheral lymphoid tissues by the induction of anergy –signal 1 only - incomplete stimulation via their TCR) X anergy or apoptosis thymocyte

34. T cells that exit the thymus have undergone a series of changes that allow them to: –develop a functional TCR –interact with self-MHC –while eliminating self-reactive T cells

35. The specificity or affinity of positive selection must differ from that of negative selection:

38. Antigen-driven T cell Differentiation in Secondary Lymphoid Organs Mature T cells leave the thymus and migrate to secondary lymphoid tissues (lymph nodes, spleen, mucosa-associated lymphoid tissue), recirculating via the blood and lymph, just like mature B cells do. Mature T cells are longer lived than mature B cells, and can survive for years without antigenic stimulation.

39. Unlike B cells, which have just one type of terminally-differentiated cell (plasma cell), there are various types of effector T cells: –CD8 T cells, which can differentiate into cytotoxic T cells –CD4 T cells, which can become either TH1 or TH2 helper cells.

40. T cells interact with antigen in the T cell-rich areas of peripheral lymphoid tissues:

41. T cells (and B cells) are targeted to, and enter, secondary lymphoid organs by their expression of various adhesion molecules. These molecules interact with ligands expressed on endothelial cells, allowing these lymphocytes to bind and enter these lymphoid organs:

42. There, they can interact with antigen-presenting cells (dendritic cells, macrophages, B cells) and be stimulated on encounter with an appropriate antigen, and function as helper T cells, interacting with B cells and other lymphocytes.

45. Ligation of the T cell’s receptor for antigen results in an initial activation signal (first signal), as is true for B cells. Again, as with B cells, this first signal is not sufficient to activate the cell: –second signals (co-stimulatory signals) are necessary for activation –The principal co-stimulatory signal for T cells is delivered via ligation of CD28 by B7 on the APC

46. Ligation of the TCR without co-stimulation results in T cells becoming non-responsive or apoptotic:

47. modified from Laâbi, Y. and A. Strasser. Science 289:883, 2000 Activation, proliferation, survival

48. T cell signaling occurs via the cytoplasmic tails of the molecules that make up the CD3 complex, which is associated with the TCR.

49. These associate with protein tyrosine kinases and initiate intracellular signaling that results in altered gene expression:

52. Encounter with antigen can result in the formation of memory T cells. Some immunologists have claimed that continuing re-contact with antigen may be important for the survival of these memory T cells. One significant differences between memory T cells and memory B cells is that the TCR does not undergo isotype switching or affinity maturation by somatic mutation, unlike the BCR. However, it is clear that there are long-lived CD4 and CD8 cells that are rapidly activated on contact with antigen.

53. Memory T cells can be defined by a change in the expression of certain surface molecules: