Microbiology 204 Background Slides on T Cell Development For Preparation for Flipped Classroom setting Art Weiss October 23, 2015

Thymic Lobule Structure From: Immunobiology, Janeway, et al., 5th edition

Summary of Thymic Development Anderson and Jenkinson, NRI, 2001

Thymic Immigration, Emmigration and Developmental Waves Immigration and Emmigration Hematopoietic precursors derived from fetal liver and bone marrow seed the thymus (in waves, as best demonstrated in the chicken) at the cortico-medullary junction. Thymic anlage produces chemotactic factors that attract hemopoietic progenitors (common lymphoid progenitor (CLP)). Seeding can be blocked by pertussis toxin -suggests the involvement of chemokines working through Gi-coupled receptors. Teck and SDF-1 have been implicated. Thymocytes that survive the rigors of selection leave the thymus from venules in the medulla - also controlled by sphingosine 1-phosphate receptors (Edg1- receptors), based on blockade by pertussis toxin and FTY720 (sphingosine 1- phosphate analogue).

Seeding the Thymus from Precursors in the Blood Bhandoola, et al., Immunity, 26: , 2007 HSC Hematopoietic Stem Cell MPP Multipotent Progenitor ELP Early Lymphoid Progenitor CLP Common Lymphoid Progenitor CMP Common Myeloid Progenitor CTP Circulating T cell Progenitor TSP Thymus Settling Progenitors ETP Early Thymic Progenitor

Signals Controlling Traffic In, Around and Out of the Thymus Love and Bhandoola, Nat. Rev. Immunol., 2011

Notch (1,2,3 are in the thymus) in the context of other stromal cell factors, interacting with its ligand(s) Delta, Jagged or Serrate, plays a role in committing the CLP to T cell lineage by interfering with the transcription factor E2A (important for B cell lineage commitment). A Role for Notch in T cell Lineage Choice Lehar and Bevan, Immunity, 17:689, 2002

Notch Pre-TCR TCR  Checkpoints in Thymocyte Development Modified from Carpenter and Bosselut, Nature Immunology 2010

Major Phenotypes and Subsets of T Cell Development Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

CD4 CD8 CD4 SP DP CD8 SP DN CD4 SP DP CD8 SP DN CD8 CD4 FACS Analysis of Thymocytes

Major Thymocyte Subsets CD4 - CD8 - (Double Negative, DN) cells: 3-5% of total thymocytes Contain least mature cells, considerable cell division 2/3rds are triple negative (TN) based on TCR expression Can be further divided based on CD44 and CD25 (discussed later) TCR ,  and  rearrangements occur at this stage 1/3rd are TCR  + CD4 + CD8 + (Double Positive, DP) cells: 80-85% of total thymocytes TCR  rearrangement occurs at this stage Most have rearranged TCR  genes & express low levels (10% mature level) of TCR Small subset has high levels of TCR (most mature, positively selected cells) Small subset is actively dividing (earliest DPs) Most apoptosis occurs here, very sensitive to apoptosis inducing agents, especially sensitive to glucocorticoids CD4 + CD8 - and CD4 - CD8 + (Single positive, SP) cells: 10-15% of total thymocytes Most are mature cells with high levels of CD3 and TCR  CD4:CD8 approx 2:1 ratio Most SP cells are functionally mature and are destined to leave the thymus Small subset of SP are immature (ISP) (CD8 in mouse, CD4 in human) and have low CD3 and no TCR  - transitional cells that are on the way from DN -> DP

Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

DN Cells (CD4-/CD8-)

Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003 DN DP Sequential Rearrangement of TCR  Genes

The pre-TCR is Expressed in DN cells Pre-T  functions as a surrogate for the  chain during thymic development Expressed in DN cell and heterodimerizes with a functional  chain - assists in quality control for  chain rearrangement The pre-T  chain dimer promotes increased CD3 expression and induces a ligand- independent signal, perhaps because of constitutive localization to lipid rafts or constitutive dimerization (unusual preTalpha structure), that is responsible for maturation and probably shut off RAG expression and further rearrangement, resulting in  chain allelic exclusion 

Ligand-Independent Dimerization of the pre-TCR Pang, et al, Nature 2010 Extended structure of Pre-T  compared to TCR C  Dimer of Heterodimers of pre-T  and TCR 

Sequentially making and expressing a pre-TCR and TCR requires genes to make the receptor and signaling molecules Von Boehmer, NRI, 2005

Two Lineages of Cells Expressing Distinct TCRs Develop in the Thymus: Stages of  and  T Cell Development Modified from Ciofani and Zuniga-Pflucker, Nature Rev. Immunol., 2010 (C-Kit)

Thymus: Developmental Waves From: Immunobiology, Janeway, et al., 5th edition

Two Lineages of T cells (cont.) Recent data suggest that  receptor expression results in stronger signal that can provides instructional cue for cell to become  lineage ( Reviewed in Ciofani, et al., Nat. Rev. Immunol. 2010)

Instructing  vs  Lineage Commitment via Strength of Signal Ciofani and Zuniga-Pflucker, Nat. Rev. Immunol. 2010

First Checkpoint : mediated by pre-TCR (also referred to as  -Selection) Second Checkpoint: mediated by mature  TCR (also referred to as positive and negative selection) Genetic inactivation of genes demonstrates the role of Src Kinases and ZAP-70 at checkpoints 1 and 2, respectively. ZAP-70

How is functional rearrangement of  -chain detected? It must mediate a signal - via CD3 and  ITAMS once it gets to the cell surface (preT , CD3 and  KOs have block at checkpoint #1) What stimulates the pre-TCR to signal? No ligand known Dimerization or Oligomerization has been reported pre-TCR can mediate signal without EC domains of pre-T  and  -chain pre-T  is palmytoylated - localizes to the GEMs constitutively localization may be responsible for constitutive signal What is the nature of the signal - similar to mature TCR? Src (Lck or Fyn) and Syk (ZAP-70 or Syk) kinases are required Adaptors LAT and SLP-76 are required Erk activation is important Checkpoint #1 : mediated by pre-TCR (  -Selection) Purpose: Assesses whether  -chain is functionally rearranged Consequences: Proliferation and differentiation of cells that express functionally rearranged  -chain

How is specificity of the  TCR assessed? Requires peptide/MHC molecule interactions to induce a signal What happens if there is no interaction? Absence of interaction leads to apoptosis (death my neglect) - there must be timing mechanism (DP only live 3-4 days) If there is an interaction, what determines cell fate? i.e., distinct positive vs negative selection signals or CD4 vs CD8 What are the signals? Probably same requirements as pre-TCR calcium, Ras are important Ras (weak) -> -> Mek -> Erk probably important for positive selection Ras (strong) -> -> p38/Jnk probably important for negative selection Checkpoint 2: Positive and Negative Selection mediated by the Mature TCR  Receptor Purpose: Assesses 1) whether  -chain is functionally rearranged; 2) whether TCR is self-MHC restricted: and, 3) whether the TCR is auto-reactive Consequences: Maturation of thymocyte to functionally competent SP cell. Establishes a self-MHC restricted, non-autoreactive TCR repertoire with appropriately matched co-receptors and functional potential

Controlling Export of Mature T cells from the Thymus Love and Bhandoola, Nat. Rev. Immunol., 2011

Self-MHC Restriction

Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

Kisielow and von Boehmer HY transgenic mice HY transgenic mice made by isolating TCR  and  chain cDNAs from CD8+ CTL clone derived from H-2 b female mouse. This TCR recognizes a male-specific peptide bound to H-2D b. So transgenic CTL will kill H-2 b male cells but not H-2 b female cells. This CTL will not kill male cells from H-2 d because of MHC restriction. So, the thymocyte from which the CTL was derived was "educated" in an H-2 b thymus. Positive Selection as Assessed with TCR Transgenic Mice

HY transgenic mouse phenotype: 1) Allelic exclusion of the  - but not the  -chain. All transgenic T cells express only the transgenic  -chain. Endogenous  -chain genes are not rearranged. Endogenous  -chains are rearranged and can be used. Some T cells do not react with T a mAb against the TCR idiotype. Can eliminate background endogenous rearrangements by mating HY TCR tg to a mouse that can not rearrange endogenous genes (i.e., Rag-deficient or SCID mice). 2) Evidence for positive selection. The HY TCR transgene promotes the development of SP CD8 but not CD4 T cells in H-2 d/b but not in H-2 d/d mice. Very strong bias towards CD8 SP thymocytes in female mice is further increased by eliminating endogenous  -chain rearrangements. Allelic Exclusion and Positive Selection in the HY system Female H-2 d/b Female H-2 d/d “selecting” “non-selecting” Backgrounds

Negative Selection in the HY TCR Transgenic Mice Result: Massive reduction of total thymocytes in male H-2 b (selecting) background. Block in development in males that have wrong (non-selecting) MHC. CD8 CD4 What happens with male HY-TCR transgenics in H-2 b (selecting) or H-2 d (non-selecting) mice?

Negative selection Efficient elimination of auto-reactive T cells by inducing deletion of thymocytes expressing self reactive receptors. (Can also see elimination of thymocytes by endogenous superantigens - i.e., not limited to peptide/MHC.) Negative selection occurs via apoptosis. Apoptotic cells rapidly engulfed by macrophages (difficult to see). In TCR transgenics, negative selection often can occur in the cortex because of inappropriately early expression of TCR transgenes. However, in normal mice, the cells at cortico-medullary junction and in medulla (thymic DCs are concentrated here) play key roles. Can see deletion with antigens presented by thymic DCs, cortical epithelium, medullary epithelial cells, but not macrophages.

Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

Ligands are peptide/MHC complexes expressed on stromal and hematopoietic cells. Peptides are derived from endogenous sources in the thymus or serum components. Recent work with Thymic Proteasome suggests positively selecting peptides are different It is important to delete thymocytes expressing TCRs that would react with self peptides presented by MHC molecules on BM-derived APCs in the periphery. Therefore, presentation of self-endogenous peptides from hematopoietic cells in the thymus plays an important role in shaping the repertoire. Negative selection may not be perfect – T regulatory cells may play important role also. What about peripheral tissue specific antigens? Aire (transcriptional regulation) protein plays a key role in regulating the expression of some tissue specific antigens in thymic medullary epithelial cells. Absence of Aire results in specific types of autoimmunity in man and mouse. The nature of the peptides presented shapes the resulting TCR repertoire. In experimental models where peptide diversity can be limited, i.e., TAP-KO or  2-microglobulin KO mice for class I MHC, or in H-2 DM KO or MHC class II with covalently attached peptide, peptide diversity presented by MHC can be limited and manipulated. TCR repertoires with such limited selecting ligands are still diverse but much more limited than normal. Ligands for Positive and Negative Selection

Use of Fetal Thymic Organ Cultures to Study Selection Abbas & Lichtman. Cellular and Molecular Immunology, 5th ed. W. B. Saunders 2003

What determines the outcome? Both outcomes can occur at DP stage (especially in TCR transgenic systems - a transgene artifact). So, discrimination between positive and negative selection is not a cell-stage specific event. Different outcomes can occur for the same cell which uses the TCR and coreceptor (CD4 or CD8) to engage ligand. With experimental manipulation, i.e. in FTOCs, it is possible to convert positive selection to negative selection or to convert non-selecting system to selecting system (i.e., add more peptide to FTOC) or change the signaling threshold (i.e., manipulate the number of ITAMs in the receptor, the level of adaptor proteins or the level of the TCR). So, the outcome of selection is not peptide specific, but is likely dictated by the quality or strength of TCR signal that is generated. As a general rule, high affinity translates to slow off-rate and this favors negative selection. Lower affinity peptides have shorter off-rate and favor positive selection. Positive vs Negative Selection

Avidity Model: Avidity depends on the affinity of the TCR-peptide/MHC interaction and the density of the peptide/MHC on the thymic epithelial cell. Avidity determines the strength of signal delivered which dictates the outcome. Stronger signals may mean longer signaling or additional signaling. Positive vs Negative Selection

Positive selection: In bone marrow chimeras, radioresistant stromal cells were implicated rather than radiosensitive hematopoietic cells. Cortical epithelial cells promote positive selection. Key experiment: Differential expression of MHC class II molecules using different promoter transgenics. Class II MHC expression pattern in transgenic MHC class II - wtcortex onlymedulla only CD4+ T cells develop yes yes no Sites of Positive and Negative Selection

Negative selection: Can occur in cortex or medulla, depending on where high avidity interaction occurs. But, normally negative selection is likely to be concentrated at cortico-medullary junction and in medulla. See deletion very early in TCR transgenics (a transgenic artifact). See increased "autoreactive" CD4 T cells if medulla lacks class II MHC molecules. Tissue specific autoimmunity develops if peripheral antigens are not expressed in thymic medullary epithelial cells (Aire mutation). Cell types that can induce negative selection: thymic DCs cortical epithelial cells medullary epithelial cells Cell types that can not induce negative selection: thymic macrophages Sites of Positive and Negative Selection (cont.)

Lineage Commitment Models DeFranco, Locksley, & Robertson, Immunity, NSP, 2007)

Newer Lineage Commitment Models DeFranco, Locksley, & Robertson, Immunity, NSP, 2007)

Lineage Choice Decision

Transcription Factors Involved in Lineage Commitment Ho, et al., Nat. Rev. Immuno., 2009