Chapter 7 The Development and Survival of Lymphocytes

1. Chapter 7 The Development and Survival of Lymphocytes Most of this chapter is about the maturation/development of lymphocytes in the absence of foreign antigen (from pre-lymphocyte to mature naïve lymphocyte): • Gene rearrangements • Testing whether the rearrangements are productive and the receptor is functional (and replacing nonproductive rearrangements if possible) • Testing the specificity of the receptor to provide assurance that it is not anti-self (tolerance) and get rid of cells with anti-self specificity • T cells need to be MHC restricted (bind to self MHC) but not anti-self (bind to self molecules). This seems to be somewhat contradictory and introduces a technically difficult problem for T cells Most lymphocyte die before solving all these problems

2. Summary of the life of a B cell Bone marrow Periphery With T help Plasma cells mostly in bone marrow; memory B cells in lymphoid tissue and circulation (Clonal deletion) In the bone marrow, B cell rearrange their Ig genes in the absence of antigen (panel 1). Those B cell with a BCR that bind antigen (self) in the bone marrow die (panel 2). B cell that do not bind antigen in the bone marrow can leave (mature naïve). If they bind antigen in the periphery (and get T help), they are activated (proliferate and differentiate) (panel 3 and 4). Activated B cell become plasma cells or memory cells (panel 4)

3. Lymphocytes develop from a hematopoietic stem cells Not exactly as shown in chapter 1

4. B cell development is dependent on bone marrow stromal cells

5. Ig gene rearrangements occur in an orderly fashion in developing B cells (every step in this slide is antigen-independent) periphery bone marrow

6. When B cells rearrange their H chain genes they make a pre-B cell receptor using a surrogate light chain to test whether the rearrangment was productive If successful in making a functional H chain, the cell stops rearrangement at the heavy chain locus and divides several times before rearrangements on the light chains Here is why:Case 1: no division after making a functional H chain. If 20% successful at H and 20% success at L then 4% (.2X.2) of the cells will be successful (need 100 tries (cells) to get 4 successes). Case 2: division after making a functional H chain. If 20% successful at H, then the 1 in 5 (20%) successful cells divide to form ~40 cells. Then 20% of the 40 (8) are successful at L. So, 18% of the cells will be successful at both L and H (lost 4 of 5 the first round and 32 of 40 the second round so 8 of 45 (18%) cells are successful)

7. 1 Igha gene Ighb gene IgG1 IgG1 a allotype b allotype Allelic exclusion.The expression of only one of two co-dominant alleles in any given cell (important for clonal selection) All the antibodies made by one B cell (or TCR by T cell) are identical even though each cell has the genes to make up to 8 different antibodies In theory, either of 2 H chains could pair with any of 2 k and 2 l L chains. (2H X 4L = 8 Ig binding sites) a allotype b allotype Allelic and isotype exclusion provides that, in any one B cell, only one heavy chain and one light chain gene code for all the heavy and light chains. Thus, one allotype and one specificity per B cell (this is most efficient for clonal selection) (same principle for the TCR) Serum IgG1 is 50% a-allotype and 50% b-allotype b allotype a allotype 1 allotype per B cell b allotype a allotype How is allelic/isotype exclusion achieved? By stepwise, orderly gene rearrangements with testing for success at each step (see slide 10) Isotype exclusion (k vs l) andallelic exclusion for each light chain and heavy chain locus

8. Because heavy (H) chains use up all their D gene segments in an attempt to make an H chain, they get only one chance per locus for a productive rearrangement. However, in light chains multiple attempts at gene rearrangements are possible. delete The maximum number of attempts in light chains is equal to the number V gene segments or J gene segments, which ever is fewer. Usually, less than the maximum are possible because… [see figure for why this cell had only three tries, not five(even if n=40 in Vkn and 5 Jk )]. Rearranges until there is a functional Ig (heavy and light) or until it runs out of V or J segments and dies

9. Bone Marrow Periphery For simplicity, we will refer to all these stages (before the cell has a functional BCR) as Pre-B cells Pre-B cellsImmature B cells Mature naïve B cells Nomenclature for this classPre-B cell: all cells in the B cell lineage before they have a functional BCR Immature B cell: B cell in the bone marrow with a functional BCR and which, upon binding antigen, will be deleted (killed) Mature naïve B cell: B cell in the periphery that have never bound antigen and, upon binding of antigen can be activated (or deleted) (in later chapters, additional names for activated B cell)

10. Gene rearrangements in B cells is an orderly(?) process (remember, these are diploid cells with two heavy chain loci, two k loci and two l loci) What is the test for a productive heavy chain rearrangement? What is the test for a productive light chain rearrangement? Q: Why an orderly process such as this? A: So only one heavy and one light chain per B cells (i.e. one antigen specificity per B cell). To achieve this, cells need allelic exclusion (heavy and light) and light chain isotype exclusion (see slide 7). This a achieved by the orderly rearrangements and testing for functionality after each rearrangement.

11. After gene rearrangements and production of a functional Ig molecule, the cell tests whether its specificity is anti-self Immature B cells that engage antigen (in the bone marrow) are prevented from later becoming responsive to antigen Maintenance of tolerance requires the persistence of antigen Bone marrow because self-antigens are always present but foreign antigens are transient or periphery Next slide Clonal deletion(death) (most important) Alive but not functional Ag binding is too weakly to get a response (least important) These are the cells that leave the bone marrow and constitute a normal immune response

12. Before clonal deletion of an anti-self B cell, the cell can attempt receptor editingof the light chain So, light chain can use repeated rearrangements (figure 7.9) and can receptor edit. Repeated rearrangements are to make a functional molecule whereas receptor editing is to avoid clonal deletion of anti-self specific B cells. ImmatureB cell “edits” light chain if it binds antigen (gets negative selection signal). This could rescue the cell from negative selection (i.e., death). (needs signal to edit) Later we will see that a pre-T cells continues to rearrange a chain if it does not gets positive selection signal. (will eventually die if it does not receive positive selection). (needs signal to stop “editing”; figure 7.26) Recall, pre B cells can also undergo repeated light chain locus rearrangements until a function Ig light chain is produced (slide 10, fig 7-9).

13. Summary of the life of a T cell Thymus Thymus Periphery Periphery TH1 CTL except CTLs and TH1 migrate to site of infection; most TH2 cells remain in peripheral lymphoid organs Memory T cells in lymphoid tissue and circulation T cells in the thymus are called thymocytes (negative selection is clonal deletion; positive selection is to select for MHC restriction specificity) In older individuals there is little thymus function and most T cells come from division of mature T cells in the periphery (new B cell are continually generated in the bone marrow)

14. Organization of the human thymus

15. Nude mice and DiGeorge’s syndrome humans are thymic deficient and have no T cells. Neonatally thymectomzed mice are T cell deficient, too. Before graft After graft The thymus is required for T cell development SCID* mice have a functional thymus but cannot rearrange Ig and TCR genes (RAG-/-) so they cannot make Ig or TCR and thus have no B or T cells(but they have a normal thymus) Lymphocytes in nude mice have everything needed to rearrange Ig and TCR gene but do not have a functional thymus for T cell development T cells in the thymus are called thymocytes *severe combined immune deficiency CD3- CD3+ CD3- CD3+

16. Double positive refers to the presence of both CD4 and CD8 on the cell surface Double negative refers to the absence of CD4 and CD8 on the cell surface TCR― Thymocyte differentiation correlates with expression of surface markers pTa = surrogate TCRa chain comparable in function to the surrogate Ig light chain Less than 5% of the double positives survive to become single positive. ab+ = abTCR+ Do these single positive cell express CD3 on their surface? How about a TCR? Single positive refers to the presence of either CD4 or CD8, but not both, on the cell surface

17. The fate of thymocytes in a young mouse. 100x106 - 200x106 total thymocytes. 50x106 new thymocytes each day (immigration and division). Therefore 50x106 cells must be removed each day. 1x106 - 2x106 mature T cells leave the thymus each day. 48x106 - 49x106 thymocytes die each day. That is, 96%-98% of the thymocytes never become fully functional and die in the thymus(by apoptosis). Why the carnage? Because most thymocytes either (1) fail to make functional TCR or, if they make a functional TCR, they are (2) anti-self (negative selection) or (3) not self MHC-restricted (no positive selection).

18. positive selection here negative selection here Single positive Positive selection for self MHC restriction Negative selection against cells that are anti-self

19. The sequential rearrangements at the TCR a and b loci are similar to the Ig rearrangements pTa is a surrogate a chain Double positive

20. In a TCR a locus there are about 70 V gene segments and 60 J segments. This provides for many attempts at a productive rearrangement. Rearrangements stop when there is positive selection. This is selection for self MHC restriction Q: what stops light chain rearrangements? A: productive rearrangement at a light chain locus and subsequent signaling from surface Ig (H+L)(no antigen or test for specificity) (see figure 7-9 or slide 8) Stated differently, TCRa rearranges until there is positive selection whereas Ig light chains rearrange until there is a productive rearrangement (and no more light chain receptor editing, figure 7.13, slide 12)1. stop TCRa rearrangements by positive selection for restriction to self MHC2. stop light chain by productive rearrangement3. restart light chain rearrangement if BCR is anti-self (receptor editing);for light chains, #2 and #3 until successful (productive and NOT anti-self) or out of light chain gene segments (unsuccesful) and dies

21. MHC restriction is “learned” (in the thymus) Note: The bone marrow recipient mice are called semi-allogeneic chimeras: (MHCa X MHCb) F1 MHCachimeraMHCa/bMHCa All cells, including bone marrow-derived cells (e.g., T cells) express both MHCa and MHCb on their surfaces (MHCa/b) Irradiation kills bone marrow stem cells and all lymphocytes (wipes out the immune system) MHCa=MHCa/aand MHCb=MHCb/b Thymus epithelial cells (and all other tissues) express MHCa Thymus epithelial cells (and all other tissues) express MHCb MHCa/a MHCb/b These T cells have MHCa and MHCb on their surface but are restricted to MHCa These T cells have MHCa and MHCb on their surface but are restricted to MHCb Conclusion: T cells that mature in an MHCa environment are restricted to MHCa. T cells that mature in an MHCb environment are restricted to MHCb. The T cells’ MHC genotype does not affect its restriction specificity. T cells learn their restriction specificity, they are not “born” knowing it.

22. The environment that determines the restriction specificity is the thymus Neonatal thymectomy results in no mature T cells The thymic epithelium is MHCa. All other the tissue in this chimera, including T cell, is MHCa and MHCb Inject Virus-x Results CTLs kill MHCaxb infected cellsCTLs kill MHCa infected cellsCTLs cannot kill MHCb infected cells Conclusion The CTLs express MHCa and MHCb but their TCRs are restricted to MHCa only. Thus, the thymus determines restriction specificity

23. Fully allogeneic chimera MHCaMHCb radiation chimera cannot function without MHCb APCs MHCa bone marrow into irradiated MHCb mouse (thymus:T cell education) (irradiated) APCs from the bone marrow Figure 7-28 7th edition This fully allogeneic chimeras is immune incompetent because the thymus is MHCb so T cells are restricted to MHCb. However the professional APCs (B cell, dendritic cells and macrophages) are MHCa (bone marrow derived) so APCs present antigens in association with MHCa. The MHCb restricted T cell cannot interact with the MHCa APCs and thus there are no adaptive immune responses. See next slide for an explanation of this phenomenon.

24. The Fully Allogeneic Bone Marrow Chimera:MHCa bone marrow into a MHCb recipient (previous slide) MHCaMHCb Not restricted to MHCb TMHCa Death Thymic epitheliumMHCb a a a a a a a a a a a a a a a a T MHCa b b b b MHCa BMHCa Mature naïve T cell has MHCa on its surface but its TCR is restricted to MHCb Dendritic cell MHCa T No T cell-B cell or T cell-dendritic cell interaction because the T cell is restricted to MHCb but the APCs are MHCa. Thus, no adaptive immune responses MHCb Thymus MHCa Bone marrow TCR restricted to MHCb Periphery Note: on this slide, we are examining the restriction specificity of the T cells, not the antigen specificity

25. T cells “learn” their restriction specificity by positive selection Positive selection also determines CD4 or CD8 expression and T cell function: CTL or TH (not TH1 or TH2, that choice comes later) Positive selection1. lets cells live (is required for survival)2. affects CD4 and CD8 expression3. affects function The requirement to undergo positive selection biases the repertoire toward MHC of the species and partly explains high alloreactivity

26. This mouse becomes a(MHCa x MHCb)F1 MHCa(aka MHCa/b MHCa) chimera Thymic epithelium mediates positive selection (see previous slides) but bone marrow-derived cells mediate negative selection in the thymus Figure 7.35

27. MHCa (MHCa x MHCb)F1 radiation chimera bone marrow recipient donor What is the restriction specificity of the T cells in this semi-allogeneic chimera? T and B cells express MHCa haplotype (but this is not important for the question at hand) Since the thymus is MHCa and MHCb, some T cells will be restricted to MHCa and others to MHCb (any one T cell can have only one restriction specificity so any one T cell will be restricted to MHCa or MHCb haplotype but not both) What is the MHC-type of B cells and macrophages that will mediate immune responses?

28. One specificity per T cell (one antigen and one restriction specificity combination).For example, let’s look at MHC class I restricted T cells Class I b2 microglobulin is not encoded in MHC Pairs of chromosomes in each cell so each cell has two MHC loci. Within each locus are B, C and A genes encoding MHC class I proteins (polygeneic). Because MHC is polymorphic, it is likely that the alleles for B, C and A are different. Anti-X restricted to Ba Ba Ca Aa Anti-X restricted to Bb MHCa (HLAa) Bb Cb Ab Anti-X restricted to Ca Anti-X restricted to Cb MHCb (HLAb) Anti-X restricted to Aa Anti-X restricted to Ab 6 mutually exclusive restriction specificities per individual X represents an antigen (e.g., a virus) however, it is not likely that all the T cells recognize the same peptide derived from X haplotype haplotype Within an individual there will be many different T cell clones, each with one of the six restriction specificities, responding to various peptides derived from the same antigen (or pathogen). The more common HLA nomenclature is B*0702 Cw*0203 A*0209

29. The example on the previous slide is for MHC class I. However, the principle of one restriction specificity and one antigen specificity per T cell is exactly the same for MHC class II restricted T cells. Also, recall the T cells probably do not recognize the antigen and the allele-specific determinants of MHC as separate entities but recognizes a new antigenic determinant formed by the combination of antigen (peptide) and MHC.

30. How can cells be positively selected for MHC restriction and not killed by negative selection that removes anti-self reactive T cells? Positive and negative selection can be successful if each is governed by different avidities (e.g., low or high avidity for positive selection; only high avidity for negative selection) Positive selection No positive selection No positive selection High or low avidity No negative selection Negative selection Low avidity High avidity Positive and negative selection occur in the presence of self-peptides but in the absence of foreign-peptides. Changing from self-peptide to foreign will change binding affinity of the TCR for MHC + peptide. Thus, a TCR with low avidity binding for self peptide + MHC might have high avidity binding for some foreign peptide + MHC. Avidity hypothesis Not differential signaling T cells with low avidity for self-MHC plus self peptides survive (positive selection without negative selection) nothing here so this cannot be correct Figure 7-36 7th edition

31. (Interdigitating) dendritic cells are found in the paracortical areas of lymphoid organs (T cell zone). They are bone marrow derived and internalize, process and present antigen in association with MHC class II to TH cells (?and with class I for CTL?; cross-presentation). Follicular dendritic cells are found in lymphoid follicles and germinal centers (B cell zone). They bind ab-ag and ag-complement complexes on their surfaces and present the antigen (native form of ag) to B cells. They are not bone marrow-derived, do not internalize antigens and do not express MHC class II. Survival of B and T cells requires that they circulate through lymphoid organs.

32. Organization of a lymphoid organ Stromal cells (Interdigitating) dendritic cells T cells B cells Follicular dendritic cells High endothelial venules (HEV)

33. Survival of B and T cells requires that they circulate through lymphoid organs. B cell survival signal is probably not received via the antigen receptor. Without antigenic stimulation, most B cells die shortly after leaving the bone marrow; some will survive several weeks (3-8 weeks). B cell will not divide in the periphery without antigenic stimulation. Memory B cells can survive for many years but must circulate through lymphoid organ and receive survival signals. Mature naïve T cells can divide in the periphery (probably get stimulation via weak binding to self peptide and MHC, similar to positive selection in the thymus). Memory T cells can divide without MHC + peptide stimulation.

34. B and T cell tumors have many normal cell phenotypes and have been instrumental in understanding lymphocyte development. They often mimic a stage in lymphocyte development that is normally transient but is permanent in the tumor cell lineage.

35. Normal Burkitt’s lymphoma Productive rearrangement at IgH locus